U.S. patent application number 11/776747 was filed with the patent office on 2009-03-19 for nematode resistant transgenic plants.
This patent application is currently assigned to University of Georgia Research Foundation. Invention is credited to Guozhong Huang, Richard S. Hussey.
Application Number | 20090077687 11/776747 |
Document ID | / |
Family ID | 36203490 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090077687 |
Kind Code |
A1 |
Hussey; Richard S. ; et
al. |
March 19, 2009 |
NEMATODE RESISTANT TRANSGENIC PLANTS
Abstract
Compositions and methods for providing nematode resistance are
provided. One aspect provides transgenic plants or cells comprising
an inhibitory nucleic acid specific for one or more nematode
esophageal polypeptides. Other aspects provide transgenic plants or
cells resistant to at least two different root-knot nematode
species.
Inventors: |
Hussey; Richard S.; (Athens,
GA) ; Huang; Guozhong; (Athens, GA) |
Correspondence
Address: |
Pabst Patent Group LLP
1545 PEACHTREE STREET NE, SUITE 320
ATLANTA
GA
30309
US
|
Assignee: |
University of Georgia Research
Foundation
|
Family ID: |
36203490 |
Appl. No.: |
11/776747 |
Filed: |
July 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11249919 |
Oct 13, 2005 |
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11776747 |
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60618097 |
Oct 13, 2004 |
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60704560 |
Aug 2, 2005 |
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Current U.S.
Class: |
800/279 ;
435/320.1; 435/411; 435/412; 435/414; 435/415; 435/417; 435/419;
530/350; 536/23.5; 800/298; 800/305; 800/307; 800/309; 800/312;
800/313; 800/314; 800/316; 800/317; 800/317.1; 800/317.2;
800/317.3; 800/317.4; 800/318; 800/320; 800/320.1 |
Current CPC
Class: |
C12N 15/8285 20130101;
C12N 15/8218 20130101; C07K 14/43563 20130101; Y02A 40/146
20180101 |
Class at
Publication: |
800/279 ;
435/419; 800/298; 536/23.5; 530/350; 800/317.4; 800/317; 800/317.2;
800/309; 800/307; 800/305; 800/318; 800/320; 800/320.1; 800/313;
800/312; 800/314; 800/317.3; 800/316; 800/317.1; 435/411; 435/412;
435/414; 435/415; 435/417; 435/320.1 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 5/10 20060101 C12N005/10; A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; C07K 14/00 20060101
C07K014/00; C12N 15/63 20060101 C12N015/63 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Aspects of the work disclosed herein were supported, in
part, by Grant Number 2003-35302-13804 awarded by the United States
Department of Agriculture. The US government may have certain
rights in the claimed subject matter.
Claims
1.-44. (canceled)
45. A transgenic plant or cell comprising: at least a portion of a
nucleic acid encoding an esophageal gland cell secretory
polypeptide of a parasitic nematode.
46. The transgenic plant or cell of claim 45, wherein the
esophageal gland cell secretory polypeptide enhances root growth of
the transgenic plant compared to a control plant or cell.
47. The transgenic plant or cell of claim 45, wherein the
esophageal gland cell secretory polypeptide is expressed in an
amount effective to enhance root growth of the transgenic plant or
cell compared to a control plant or cell.
48. The transgenic plant or cell of claim 45, wherein the
esophageal gland cell secretory polypeptide is expressed in a root
cell.
49. The transgenic plant or cell of claim 45, wherein the nematode
is a member of Meloidogyne spp.
50. The transgenic plant or cell of claim 45, wherein the nematode
is a not a member of Heterodera spp.
51. The transgenic plant or cell of claim 45, wherein the
transgenic plant or cell is a monocot or dicot.
52. The transgenic plant or cell of claim 45, wherein the
transgenic plant or cell is a member of the family selected from
the group consisting of Rosaceae, Fabaceae, Passifloraceae,
Cucurbitaceae, Malvaceae, Euphorbiaceae, Vitaceae, Solanaceae,
Convolvulaceae, Rubiaceae, Leguminosae, and Brassicaceae.
53. The transgenic plant or cell of claim 52, wherein the plant is
a tomato, eggplant, potato, melon, cucumber, carrot, lettuce,
artichoke, celery, cucurbits, barley, corn, peanut, soybean, sugar
beet, cotton, cowpea, beans, alfalfa, tobacco, citrus, clover,
pepper, grape, coffee, olive, or tea.
54. The transgenic plant or cell of claim 45, wherein the nucleic
acid comprises at least a portion of SEQ ID NOs 1, 2, or 5-51.
55. The transgenic plant or cell of claim 45, wherein the
transgenic plant or cell comprises two or more nucleic acids
encoding different nematode secretory polypeptides.
56. A composition comprising: a nucleic acid comprising SEQ ID NOs.
1, 2 or 5-51, a complement, a fragment, or homologue thereof, in an
amount sufficient to enhance or promote root growth when expressed
in a plant.
57. A composition comprising: a polypeptide encoded by SEQ ID NOs.
1, 2 or 5-51, a complement, a fragment, or homologue thereof, in an
amount sufficient to enhance or promote root growth.
58. The composition of claim 57, wherein the nematode is a member
of Meloidogyne spp.
59. The composition of claim 57, wherein the nematode is a member
of Heterodera spp.
60. The composition of claim 57, wherein the transgenic plant or
cell is a monocot or dicot.
61. The composition of claim 57, wherein the plant or cell is a
member of the family selected from the group consisting of
Rosaceae, Fabaceae, Passifloraceae, Cucurbitaceae, Malvaceae,
Euphorbiaceae, Vitaceae, Solanaceae, Convolvulaceae, Rubiaceae,
Leguminosae, and Brassicaceae.
62. The composition of claim 61, wherein the plant or cell is a
tomato, eggplant, potato, melon, cucumber, carrot, lettuce,
artichoke, celery, cucurbits, barley, corn, peanut, soybean, sugar
beet, cotton, cowpea, beans, alfalfa, tobacco, citrus, clover,
pepper, grape, coffee, olive, or tea.
63. A cell comprising a nucleic acid encoding a nematode esophageal
gland cell secretory polypeptide that stimulates root growth.
64. A vector comprising a promoter operably linked to a nucleic
acid encoding a nematode esophageal gland cell secretory protein
that stimulates plant root growth.
65. (canceled)
66. A method for stimulating root growth or for enhancing the
formation of lateral or adventitious roots comprising delivering a
nematode esophageal gland cell secretory polypeptide to the
interior of a root cell.
67. A method for the producing an altered plant, plant cell or
plant tissue comprising introducing one or more nematode esophageal
gland cell secretory polypeptides into a cell, tissue or organ of
said plant.
68. A seed produced by the transgenic plant or cell of claim
45.
69. A recombinant plant exhibiting increased root growth as
compared to the corresponding wild-type plant, wherein said
recombinant plant comprises a recombinant nucleic acid encoding a
nematode esophageal gland cell secretory protein or fragment
thereof operably associated with a regulatory sequence.
70. A recombinant plant of claim 69, wherein the nematode
esophageal gland cell secretory protein is encoded by one or more
of SEQ ID NOs. 1, 2, or 5-51.
71. (canceled)
72. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of and priority to U.S.
provisional application No. 60/618,097 filed on Oct. 13, 2004, and
US provisional application No. 60/704,560 filed on Aug. 2, 2005,
and where permissible each is incorporated by reference in its
entirety.
BACKGROUND
[0003] 1. Technical Field
[0004] The present disclosure generally relates to compositions for
controlling plant parasites and compositions for increasing root
growth, more particularly to nucleic acid compositions for
controlling nematode disease or increasing root growth.
[0005] 2. Related Art
[0006] Nematodes are a very large group of invertebrate animals
generally referred to as roundworms, threadworms, eelworms, or
nema. Some nematodes are plant parasites and can feed on stems,
buds, leaves, and in particular on roots. One important genus of
plant parasitic nematodes is the root-knot nematode (Meloidogyne
spp.). These parasitic nematodes infect a wide range of important
field, vegetable, fruit and ornamental plants. In 2001 the
root-knot nematode was responsible for a loss of US$200.5 million
in cotton alone.
[0007] Existing methods for treating or preventing root-knot
nematode disease include the use of chemicals, pesticides, and
fumigants. The use of pre-plant soil fumigants is highly effective
in controlling root-knot and other plant-parasitic nematodes.
However, the majority of the fumigant-type nematicides are no
longer available and are also costly and difficult to apply
properly under the prevailing conditions.
[0008] Crop rotation has also been used to control nematode
disease. Rotating onion, carrot, or lettuce with a nonhost crop
such as sweet corn and other grain crops, if economically possible,
can be effective in controlling the northern root-knot nematode.
Unfortunately, current crop rotations on organic soils are of
limited value as most crops grown, including potatoes, beans,
celery, lettuce, onion, and carrot are susceptible to disease.
[0009] The use of cover crops has also been attempted to control
nematode disease. Cover crops grown between the main crops may
provide an alternative management strategy. Ryegrain, barley, oats,
sudangrass, tall fescue, annual ryegrass, and wheat have been shown
to be non- or poor hosts to this nematode. Using cover crops,
however, can be costly because the cover crops occupy space that
could be used to grow more valuable crops.
[0010] Biological control organisms have also been used to try to
control nematode disease in crops. Commercially available
preparations of biological control organisms are limited in their
use to regions that can support the growth of the control organism.
Moreover, the outcome of using one organism to control another is
unpredictable and subject to a variety of a factors such as weather
and climate.
[0011] Additionally, the root-knot nematode (RKN) is a leading
cause of crop loss due to plant parasitic nematodes. The most
important species (M. incognita, M. javanica, M. arenaria, M.
hapla, M. chitwoodi) have wide host ranges that limit nonhost
rotation options. While several examples of host resistance genes
in diverse crops exist, the availability of host plant resistance
is substantially limited with appropriate resistance loci lacking
for the majority of our crops (Roberts, P. A. 1992. Journal of
Nematology 24:213-227). In addition, the resistance is limited to
only a few RKN species or populations and some resistance genes are
heat-sensitive and thus unsuitable for hot production areas.
Another limitation of natural resistance genes is the durability of
resistance since resistance-breaking populations of RKN can develop
after continuous exposure to resistant cultivars, e.g. root-knot
resistant tomatoes.
[0012] Accordingly, there is need for compositions and methods for
controlling, preventing, or reducing nematode disease in
plants.
[0013] Still other problems affecting crops relate to poorly
developed root systems. Root systems of plants are an important
part of a plant, and provide many functions that are vital to plant
survival. For example, root systems store nutrients for the plant,
filter out toxins, help regulate plant growth, provide an
absorptive network for water and nutrients, and provide mechanical
structures that support the plant and strengthen the soil. Plants
with larger roots have increased growth and increased stress
tolerance. Increased or enhanced root growth in crop plants would
be particularly advantageous because the increased root growth
would increase crop yield.
[0014] In perennial crops, increased root growth would increase the
regrowth rate, increase the yield potential, and increase the
likelihood that plants will survive winter. In annuals, increased
root size would ensure yield potential under varying environmental
conditions. In root crops, enhanced root growth would mean larger
yields.
[0015] Existing root stimulators typically include fertilizers or
plant hormones that must be mixed or formulated in specific
concentrations when applied to the plant or soil near the plant.
Over application of such simulators can have adverse effects on the
plants, and under application will not achieve the desired outcome.
Additionally, application of plant hormones can have undesired
consequences. For example, one plant hormone used as a root
initiator is auxin or indole-3-acetic acid (IAA). IAA plays
important roles in a number of plant activities, including:
development of the embryo, leaf formation, phototropism,
gravitropism, apical dominance, fruit development, abscission as
well as root initiation.
[0016] Thus there is a need for new compositions and methods for
stimulating or enhancing root growth or development.
SUMMARY
[0017] Aspects of the present disclosure generally provide nucleic
acid constructs that inhibit the expression of proteins secreted by
plant parasites. In some aspects, the proteins are secreted by a
nematode and, optionally, modulate: gene expression of the plant or
cell, formation of a giant cell, nematode migration through root
tissue of the plant, cell metabolism of the plant, elicits signal
transduction in the plant cell, or forms a feeding tube that
enables the nematode to feed from giant-cells formed in the plant.
One aspect provides inhibitory nucleic acids specific for
esophageal gland cell proteins secreted by nematodes, in particular
root knot nematodes. Other aspects provide transgenic cells or
plants expressing or containing one or more inhibitory nucleic
acids, for example inhibitory double or single stranded RNA, that
inhibit or reduce the expression of nematode esophageal gland cell
proteins.
[0018] Another aspect provides a transgenic plant that comprises
inhibitory RNA that down regulates a target nematode parasitism
gene transcript in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, or more nematode species, for example RKN species.
Thus, the present disclosure provides transgenic plants that are
resistant to disease caused by multiple RKN species.
[0019] Representative esophageal gland cell proteins that are
targeted by the disclosed inhibitory nucleic acids include one or
more of the proteins encoded by SEQ ID NOs. 1, 2, and 5-51. In
certain aspects, one or more inhibitory nucleic acids are delivered
to a parasitic nematode when the nematode enters the transgenic
plant or transgenic plant cell, feeds on the transgenic plant or
transgenic plant cell, or comes into physical contact with the
transgenic plant or transgenic plant cell. Once the inhibitory
nucleic acid is internalized by the parasitic nematode, the
inhibitory nucleic acid interferes with, reduces, or inhibits the
expression of a target esophageal gland cell protein, for example,
by directly or indirectly interfering, reducing, or inhibiting the
translation of one or more mRNAs coding for one or more esophageal
gland cell proteins.
[0020] Yet another aspect provides a plant cell transfected with
heterologous nucleic acid encoding an inhibitory nucleic acid
specific for one or more nematode esophageal gland cell proteins,
wherein the heterologous nucleic acid is expressed in an amount
sufficient to reduce or prevent nematode disease. In one aspect,
the transgenic plant expresses the inhibitory nucleic acid, and the
inhibitory nucleic acid is delivered to a nematode feeding or
attempting to feed on the transgenic plant. Generally, the
inhibitory nucleic acid is internalized by a nematode. Exemplary
methods of internalizing the inhibitory nucleic acid include
ingesting the nucleic acid or absorbing the nucleic acid.
[0021] Still another aspect provides a transgenic plant comprising
an inhibitory nucleic acid specific for one or more nematode
parasitism polypeptides, wherein the inhibitory nucleic acid
provides resistance to two or more nematode species, for example
two or more root-knot nematode species.
[0022] Further aspects provide compositions for stimulating,
promoting, or enhancing root growth or development in plants or
trees. Certain aspects provide nucleic acid constructs encoding
proteins secreted by nematode esophageal gland cells, wherein the
proteins or fragments thereof stimulate or enhance root development
when delivered to or in contact with a plant. Other aspects provide
compositions containing one more nematode esophageal gland cell
proteins or fragments thereof that stimulate root growth when in
contract with a plant or plant cell. Still other aspects provide
transgenic plants comprising one or more nematode esophageal gland
cell proteins or fragments thereof or nucleic acids encoding one
more nematode esophageal gland cell proteins or fragments thereof
sufficient to stimulate, enhance, or promote root growth compared
to non-transgenic or control plants.
[0023] Representative nematode esophageal gland cell proteins (also
referred to as esophageal proteins) include one or more of the
proteins encoded by SEQ ID NOs. 1, 2, and 5-51 or combinations
thereof.
[0024] Yet another aspect provides a plant cell transfected with
heterologous nucleic acid encoding one or more nematode esophageal
gland cell proteins, wherein the heterologous nucleic acid is
expressed in an amount sufficient to stimulate, enhance, or promote
root growth or development.
[0025] The details of one or more embodiments are set forth in the
accompanying drawings and the description below. Other features,
objects, and advantages of the disclosure will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1A shows A. thaliana expressing 16D10 dsRNA inoculated
with M. incognita. Note that no root knot disease (galls) on roots
of A. thaliana expressing 16D10 dsRNA.
[0027] FIG. 1B shows control plants inoculated with M.
incognita.
[0028] FIG. 2 shows a photograph of a transgenic A. thaliana plant
expressing 16D10 and having enhanced root growth compared to a
control plant (empty vector).
[0029] FIG. 3 shows a bar graph indicating enhanced root growth of
four transgenic Arabidopsis T.sub.2 homozygous lines L7, L10, L11,
L17 compared to control lines (L2, L3).
[0030] FIG. 4 shows RT-PCR analysis of 16D10 dsRNA (RNA1 and RNA2)
treated second-stage juveniles of root-knot nematode showing a
significant reduction of transcripts of parasitism gene 16D10 in
the treated nematodes. Resorcinol (Res) was used to help stimulate
uptake of the dsRNA. No reduction of transcripts with dsRNA or res
alone. Mi-act--internal transcript control.
[0031] FIG. 5 shows a photograph of a gel indicating that RNAi
directed to 8H11 or 31H06 down-regulates expression of parasitism
genes 8H11 or 31H06 in nematodes.
[0032] FIG. 6 shows DNA blot hybridization of restriction
endonuclease-digested genomic DNA from four Meloidogyne species
with a DIG-labeled 16D10 probe. Mi, M. incognita; Mj, M. javanica;
Ma, M. arenaria; Mh, M. hapla. E, EcoRI; B, BamHI. M, 80 ng
DIG-labeled molecular weight marker in kb.
[0033] FIG. 7 shows a bar graph indicating reproduction (eggs per
gram root) of four Meloidogyne species (Mi, M. incognita; MJ, M.
javanica; Ma, M. arenadia; Mh, M. hapla) on transgenic A. thaliana
expressing 16D10 dsRNA was decreased compared with control
plants.
DETAILED DESCRIPTION
1. Definitions
[0034] Before explaining the various embodiments of the disclosure,
it is to be understood that the invention is not limited in its
application to the details of construction and the arrangement of
the components set forth in the following description. Other
embodiments can be practiced or carried out in various ways. Also,
it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.
[0035] Throughout this disclosure, various publications, patents
and published patent specifications are referenced. Where
permissible, the disclosures of these publications, patents and
published patent specifications are hereby incorporated by
reference in their entirety into the present disclosure to more
fully describe the state of the art. Unless otherwise indicated,
the disclosure encompasses conventional techniques of plant
breeding, immunology, molecular biology, microbiology, cell biology
and recombinant DNA, which are within the skill of the art. See,
e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual,
3rd edition (2001); Current Protocols In Molecular Biology [(F. M.
Ausubel, et al. eds., (1987)]; Plant Breeding. Principles and
Prospects (Plant Breeding, Vol 1) M. D. Hayward, N, O. Bosemark, I.
Romagosa; Chapman & Hall, (1993.); Coligan, Dunn, Ploegh,
Speicher and Wingfeld, eds. (1995) CURRENT Protocols in Protein
Science (John Wiley & Sons, Inc.); the series Methods in
Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.
J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995), Harlow and
Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell
Culture [R. I. Freshney, ed. (1987)].
[0036] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Lewin, Genes VII, published by Oxford
University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of
Molecular Biology, published by Wiley-Interscience., 1999; and
Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a
Comprehensive Desk Reference, published by VCH Publishers, Inc.,
1995; Ausubel et al. (1987) Current Protocols in Molecular Biology,
Green Publishing; Sambrook and Russell. (2001) Molecular Cloning: A
Laboratory Manual 3rd. edition.
[0037] In order to facilitate understanding of the disclosure, the
following definitions are provided:
[0038] To "alter" the expression of a target gene in a plant cell
means that the level of expression of the target gene in a plant
cell after applying a method of the present invention is different
from its expression in the cell before applying the method. To
alter gene expression preferably means that the expression of the
target gene in the plant is reduced, preferably strongly reduced,
more preferably the expression of the gene is not detectable. The
alteration of the expression of an essential gene may result in a
knockout mutant phenotype in plant cells or plants derived
therefrom. Alternatively, altered expression can included
upregulating expression of plant genes.
[0039] "Antisense RNA" is an RNA strand having a sequence
complementary to a target gene mRNA, and thought to induce RNAi by
binding to the target gene mRNA. "Sense RNA" has a sequence
complementary to the antisense RNA, and annealed to its
complementary antisense RNA to form siRNA. These antisense and
sense RNAs have been conventionally synthesized with an RNA
synthesizer. In the present invention, these RNAs are
intracellularly expressed from DNAs coding for antisense and sense
RNAs (antisense and sense code DNAs) respectively using the siRNA
expression system.
[0040] The term "biological sample" refers to a body sample from
any animal, such as a mammal, for example, a human. The biological
sample can be obtained from vascular, diabetic, or cancer patients,
for example. A biological sample can be biological fluids such as
serum, plasma, vitreous fluid, lymph fluid, synovial fluid,
follicular fluid, seminal fluid, amniotic fluid, milk, whole blood,
urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration,
mucus, and tissue culture medium, as well as tissue extracts such
as homogenized tissue, cellular extracts, or whole cells or tissue.
The biological sample can be, for example, serum, plasma, or
urine.
[0041] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components.
[0042] When referring to expression, "control sequences" means DNA
sequences necessary for the expression of an operably linked coding
sequence in a particular host organism. Control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, a ribosome binding site, and the
like. Eukaryotic cells are known to utilize promoters,
polyadenylation signals, and enhancers.
[0043] The term "cell" refers to a membrane-bound biological unit
capable of replication or division.
[0044] The term "construct" refers to a recombinant genetic
molecule comprising one or more isolated polynucleotide sequences
of the invention.
[0045] Genetic constructs used for transgene expression in a host
organism comprise in the 5'-3' direction, a promoter sequence; a
sequence encoding an inhibitory nucleic acid disclosed herein; and
a termination sequence. The open reading frame may be orientated in
either a sense or anti-sense direction. The construct may also
comprise selectable marker gene(s) and other regulatory elements
for expression.
[0046] As used herein, the term "control element" or "regulatory
element" are used interchangeably herein to mean sequences
positioned within or adjacent to a promoter sequence so as to
influence promoter activity. Control elements may be positive or
negative control elements. Positive control elements require
binding of a regulatory element for initiation of transcription.
Many such positive and negative control elements are known. Where
heterologous control elements are added to promoters to alter
promoter activity as described herein, they are positioned within
or adjacent the promoter sequence so as to aid the promoter's
regulated activity in expressing an operationally linked
polynucleotide sequence.
[0047] The term "heterologous" refers to elements occurring where
they are not normally found. For example, a promoter may be linked
to a heterologous nucleic acid sequence, e.g., a sequence that is
not normally found operably linked to the promoter. When used
herein to describe a promoter element, heterologous means a
promoter element that differs from that normally found in the
native promoter, either in sequence, species, or number. For
example, a heterologous control element in a promoter sequence may
be a control/regulatory element of a different promoter added to
enhance promoter control, or an additional control element of the
same promoter.
[0048] As used herein, the term "homologues" is generic to
"orthologues" and "paralogues".
[0049] The term "host plant" refers to a plant subject to nematode
disease.
[0050] As used herein, the phrase "induce expression" means to
increase the amount or rate of transcription and/or translation
from specific genes by exposure of the cells containing such genes
to an effector or inducer reagent or condition.
[0051] An "inducer" is a chemical or physical agent which, when
applied to a population of cells, will increase the amount of
transcription from specific genes. These are usually small
molecules whose effects are specific to particular operons or
groups of genes, and can include sugars, phosphate, alcohol, metal
ions, hormones, heat, cold, and the like. For example, isopropyl
(beta)-D-thiogalactopyranoside (IPTG) and lactose are inducers of
the tacII promoter, and L-arabinose is a suitable inducer of the
arabinose promoter.
[0052] The term "isolated," when used to describe the various
compositions disclosed herein, means a substance that has been
identified and separated and/or recovered from a component of its
natural environment. For example an isolated polypeptide or
polynucleotide is free of association with at least one component
with which it is naturally associated. Contaminant components of
its natural environment are materials that would typically
interfere with diagnostic or therapeutic uses for the polypeptide
or polynucleotide and may include enzymes, and other proteinaceous
or non-proteinaceous solutes. An isolated substance includes the
substance in situ within recombinant cells. Ordinarily, however, an
isolated substance will be prepared by at least one purification
step.
[0053] An "isolated" nucleic acid molecule or polynucleotide is a
nucleic acid molecule that is identified and separated from at
least one contaminant nucleic acid molecule with which it is
ordinarily associated in the natural source. The isolated nucleic
can be, for example, free of association with all components with
which it is naturally associated. An isolated nucleic acid molecule
is other than in the form or setting in which it is found in
nature.
[0054] "IPTG" is the compound "isopropyl
(beta)-D-thiogalactopyranoside", and is used herein as an inducer
of lac operon. IPTG binds to a lac repressor effecting a
conformational change in the lac repressor that results in
dissociation of the lac repressor from the lac operator. With the
lac repressor unbound, an operably linked promoter is activated and
downstream genes are transcribed.
[0055] The term "lac operator" refers to a nucleic acid sequence
that can be bound by a lac repressor, lacI, as described, for
example, in Jacob et al, 1961, J. Mol. Biol., 3: 318-356. A
promoter is not activated when the lac repressor is bound to the
lac operator. When the lac repressor is induced to dissociate from
the operator, the promoter is activated.
[0056] The term "leader sequence" refers to a nucleic acid sequence
positioned upstream of a coding sequence of interest. Leader
sequences described herein contain specific sequences known to bind
efficiently to ribosomes, thus delivering a greater efficiency of
translation initiation of some polynucleotides.
[0057] As used herein, the term "mammal" refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. The mammal can be, for example, human.
[0058] The term "nematode esophageal glands or nematode esophageal
gland cell" refers to three large, transcriptionally active gland
cells, one dorsal and two subventral, located in the esophagus of a
nematode and that are the principal sources of secretions
(parasitism proteins) involved in infection and parasitism of
plants by plant-parasitic nematodes in the orders Tylenchida and
Aphelenchida.
[0059] A nucleic acid sequence or polynucleotide is "operably
linked" when it is placed into a functional relationship with
another nucleic acid sequence. For example, DNA for a presequence
or secretory leader is operably linked to DNA for a polypeptide if
it is expressed as a preprotein that participates in the secretion
of the polypeptide; a promoter or enhancer is operably linked to a
coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if
it is positioned so as to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are
contiguous and, in the case of a secretory leader, contiguous and
in reading frame. Linking can be accomplished by ligation at
convenient restriction sites. If such sites do not exist, synthetic
oligonucleotide adaptors or linkers are used in accordance with
conventional practice.
[0060] The term "orthologues" refers to separate occurrences of the
same gene in multiple species, The separate occurrences have
similar, albeit nonidentical, amino acid sequences, the degree of
sequence similarity depending, in part, upon the evolutionary
distance of the species from a common ancestor having the same
gene.
[0061] As used herein, the term "paralogues" indicates separate
occurrences of a gene in one species. The separate occurrences have
similar, albeit nonidentical, amino acid sequences, the degree of
sequence similarity depending, in part, upon the evolutionary
distance from the gene duplication event giving rise to the
separate occurrences.
[0062] The term "parasitism proteins, parasitism polypeptides,
esophageal polypeptides, or nematode esophageal gland cell
secretory polypeptide" refers to the principal molecules involved
in nematode parasitism of plants; products of parasitism genes
expressed in plant-parasitic nematode esophageal gland cells and
injected through their stylet into host tissues to mediate
parasitism of plants.
[0063] "Percent (%) nucleic acid sequence identity" is defined as
the percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in a reference nucleic acid
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity.
Alignment for purposes of determining percent nucleic acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign
(DNASTAR) software. Appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full-length of the sequences being compared can be determined
by known methods.
[0064] For purposes herein, the % nucleic acid sequence identity of
a given nucleic acid sequence C to, with, or against a given
nucleic acid sequence D (which can alternatively be phrased as a
given nucleic acid sequence C that has or comprises a certain %
nucleic acid sequence identity to, with, or against a given nucleic
acid sequence D) is calculated as follows:
100 times the fraction W/Z,
where W is the number of nucleotides scored as identical matches by
the sequence alignment program in that program's alignment of C and
D, and where Z is the total number of nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not
equal to the length of nucleic acid sequence D, the % nucleic acid
sequence identity of C to D will not equal the % nucleic acid
sequence identity of D to C.
[0065] The term "plant" is used in it broadest sense. It includes,
but is not limited to, any species of woody, ornamental or
decorative, crop or cereal, fruit or vegetable plant, and
photosynthetic green algae (e.g., Chiamydomonas reinhardtii). It
also refers to a plurality of plant cells that are largely
differentiated into a structure that is present at any stage of a
plant's development. Such structures include, but are not limited
to, a fruit, shoot, stem, leaf, flower petal, etc. The term "plant
tissue" includes differentiated and undifferentiated tissues of
plants including those present in roots, shoots, leaves, pollen,
seeds and tumors, as well as cells in culture (e.g., single cells,
protoplasts, embryos, callus, etc.). Plant tissue may be in planta,
in organ culture, tissue culture, or cell culture. The term "plant
part" as used herein refers to a plant structure, a plant organ, or
a plant tissue.
[0066] A non-naturally occurring plant refers to a plant that does
not occur in nature without human intervention. Non-naturally
occurring plants include transgenic plants and plants produced by
non-transgenic means such as plant breeding.
[0067] The term "plant cell" refers to a structural and
physiological unit of a plant, comprising a protoplast and a cell
wall. The plant cell may be in form of an isolated single cell or a
cultured cell, or as a part of higher organized unit such as, for
example, a plant tissue, a plant organ, or a whole plant.
[0068] The term "plant cell culture" refers to cultures of plant
units such as, for example, protoplasts, cell culture cells, cells
in plant tissues, pollen, pollen tubes, ovules, embryo sacs,
zygotes and embryos at various stages of development.
[0069] The term "plant material" refers to leaves, stems, roots,
flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds,
cuttings, cell or tissue cultures, or any other part or product of
a plant.
[0070] A "plant organ" refers to a distinct and visibly structured
and differentiated part of a plant such as a root, stem, leaf,
flower bud, or embryo.
[0071] "Plant tissue" refers to a group of plant cells organized
into a structural and functional unit. Any tissue of a plant
whether in a plant or in culture is included. This term includes,
but is not limited to, whole plants, plant organs, plant seeds,
tissue culture and any groups of plant cells organized into
structural and/or functional units. The use of this term in
conjunction with, or in the absence of, any specific type of plant
tissue as listed above or otherwise embraced by this definition is
not intended to be exclusive of any other type of plant tissue.
[0072] "Plasmids" are designated by a lower case "p" preceded
and/or followed by capital letters and/or numbers. The starting
plasmids herein are either commercially available, publicly
available on an unrestricted basis, or can be constructed from
available plasmids in accord with published procedures. In
addition, equivalent plasmids to those described are known in the
art and will be apparent to the ordinarily skilled artisan.
[0073] As used herein, "polypeptide" refers generally to peptides
and proteins having more than about ten amino acids. The
polypeptides can be "exogenous," meaning that they are
"heterologous," i.e., foreign to the host cell being utilized, such
as human polypeptide produced by a bacterial cell.
[0074] "Primate" is construed to mean any of an order of mammals
comprising humans, apes, monkeys, and related forms, such as lemurs
and tarsiers.
[0075] The term "promoter" refers to a regulatory nucleic acid
sequence, typically located upstream (5') of a gene or protein
coding sequence that, in conjunction with various elements, is
responsible for regulating the expression of the gene or protein
coding sequence. The promoters suitable for use in the constructs
of this disclosure are functional in plants and in host organisms
used for expressing the inventive polynucleotides. Many plant
promoters are publicly known. These include constitutive promoters,
inducible promoters, tissue- and cell-specific promoters and
developmentally-regulated promoters. Exemplary promoters and fusion
promoters are described, e.g., in U.S. Pat. No. 6,717,034, which is
herein incorporated by reference in its entirety.
[0076] "Purifying" means increasing the degree of purity of a
substance in a composition by removing (completely or partially) at
least one contaminant from the composition. A "purification step"
may be part of an overall purification process resulting in an
"essentially pure" composition. An essentially pure composition
contains at least about 90% by weight of the substance of interest,
based on total weight of the composition, and can contain at least
about 95% by weight.
[0077] The term "regulatory element" or "control element" refers to
DNA sequences controlling initiation of transcription. Examples of
control or regulatory elements include, but are not limited to, a
TATA box, operators, enhancers, and the like. Regulatory or control
elements include negative control elements and positive control
elements. A negative control element is one that is removed for
activation. Many such negative control elements are known, for
example operator/repressor systems. For example, binding of IPTG to
the lac repressor dissociates from the lac operator to activate and
permit transcription. Other negative elements include the E. coli
trp and lambda systems. A positive control element is one that is
added for activation. Many such positive control elements are
known.
[0078] Promoters naturally containing both positive and negative
regulatory elements are rare. The metE promoter is one example.
See, for example, Neidhardt, Ed., 1996, Escherishia coli and
Salmonella, Second Ed., pages 1300-1309. Descriptions of known
positive and negative control elements can be found, for example,
in this reference. Positioning of a positive or negative control
element within or adjacent to the promoter to achieve added
regulation of the promoter is known, and is described, for example,
in Escherishia coli and Salmonella (Supra) at pages 1232-1245.
[0079] Small RNA molecules are single stranded or double stranded
RNA molecules generally less than 200 nucleotides in length. Such
molecules are generally less than 100 nucleotides and usually vary
from 10 to 100 nucleotides in length. In a preferred format, small
RNA molecules have 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Small RNAs include
microRNAs (miRNA) and small interfering RNAs (siRNAs). mRNAs are
produced by the cleavage of short stem-loop precursors by
Dicer-like enzymes; whereas, siRNAs are produced by the cleavage of
long double-stranded RNA molecules. mRNAs are single-stranded,
whereas siRNAs are double-stranded.
[0080] The term "siRNA" means a small interfering RNA that is a
short-length double-stranded RNA that is not toxic. Generally,
there is no particular limitation in the length of siRNA as long as
it does not show toxicity. "siRNAs" can be, for example, 15 to 49
bp, preferably 15 to 35 bp, and more preferably 21 to 30 bp long.
Alternatively, the double-stranded RNA portion of a final
transcription product of siRNA to be expressed can be, for example,
15 to 49 bp, preferably 15 to 35 bp, and more preferably 21 to 30
bp long. The double-stranded RNA portions of siRNAs in which two
RNA strands pair up are not limited to the completely paired ones,
and may contain nonpairing portions due to mismatch (the
corresponding nucleotides are not complementary), bulge (lacking in
the corresponding complementary nucleotide on one strand), and the
like. Nonpairing portions can be contained to the extent that they
do not interfere with siRNA formation. The "bulge" used herein
preferably comprise 1 to 2 nonpairing nucleotides, and the
double-stranded RNA region of siRNAs in which two RNA strands pair
up contains preferably 1 to 7, more preferably 1 to 5 bulges. In
addition, the "mismatch" used herein is contained in the
double-stranded RNA region of siRNAs in which two RNA strands pair
up, preferably 1 to 7, more preferably 1 to 5, in number. In a
preferable mismatch, one of the nucleotides is guanine, and the
other is uracil. Such a mismatch is due to a mutation from C to T,
G to A, or mixtures thereof in DNA coding for sense RNA, but not
particularly limited to them. Furthermore, in the present
invention, the double-stranded RNA region of siRNAs in which two
RNA strands pair up may contain both bulge and mismatched, which
sum up to, preferably 1 to 7, more preferably 1 to 5 in number.
[0081] The terminal structure of siRNA may be either blunt or
cohesive (overhanging) as long as siRNA can silence, reduce, or
inhibit the target gene expression due to its RNAi effect. The
cohesive (overhanging) end structure is not limited only to the 3'
overhang, and the 5' overhanging structure may be included as long
as it is capable of inducing the RNAi effect. In addition, the
number of overhanging nucleotide is not limited to the already
reported 2 or 3, but can be any numbers as long as the overhang is
capable of inducing the RNAi effect. For example, the overhang
consists of 1 to 8, preferably 2 to 4 nucleotides. Herein, the
total length of siRNA having cohesive end structure is expressed as
the sum of the length of the paired double-stranded portion and
that of a pair comprising overhanging single-strands at both ends.
For example, in the case of 19 bp double-stranded RNA portion with
4 nucleotide overhangs at both ends, the total length is expressed
as 23 bp. Furthermore, since this overhanging sequence has low
specificity to a target gene, it is not necessarily complementary
(antisense) or identical (sense) to the target gene sequence.
Furthermore, as long as siRNA is able to maintain its gene
silencing effect on the target gene, siRNA may contain a low
molecular weight RNA (which may be a natural RNA molecule such as
tRNA, rRNA or viral RNA, or an artificial RNA molecule), for
example, in the overhanging portion at its one end.
[0082] In addition, the terminal structure of the "siRNA" is not
necessarily the cut off structure at both ends as described above,
and may have a stem-loop structure in which ends of one side of
double-stranded RNA are connected by a linker RNA. The length of
the double-stranded RNA region (stem-loop portion) can be, for
example, 15 to 49 bp, preferably 15 to 35 bp, and more preferably
21 to 30 bp long. Alternatively, the length of the double-stranded
RNA region that is a final transcription product of siRNAs to be
expressed is, for example, 15 to 49 bp, preferably 15 to 35 bp, and
more preferably 21 to 30 bp long. Furthermore, there is no
particular limitation in the length of the linker as long as it has
a length so as not to hinder the pairing of the stem portion. For
example, for stable pairing of the stem portion and suppression of
the recombination between DNAs coding for the portion, the linker
portion may have a clover-leaf tRNA structure. Even though the
linker has a length that hinders pairing of the stem portion, it is
possible, for example, to construct the linker portion to include
introns so that the introns are excised during processing of
precursor RNA into mature RNA, thereby allowing pairing of the stem
portion. In the case of a stem-loop siRNA, either end (head or
tail) of RNA with no loop structure may have a low molecular weight
RNA. As described above, this low molecular weight RNA may be a
natural RNA molecule such as tRNA, rRNA or viral RNA, or an
artificial RNA molecule.
[0083] "Signal peptide" refers to a short (15-60 amino acids long)
peptide chain that directs the post translational transport of a
protein; usually directs the peptide to the secretory pathway of
the cell.
[0084] "Transformed," "transgenic," "transfected" and "recombinant"
refer to a host organism such as a bacterium or a plant into which
a heterologous nucleic acid molecule has been introduced. The
nucleic acid molecule can be stably integrated into the genome of
the host or the nucleic acid molecule can also be present as an
extrachromosomal molecule. Such an extrachromosomal molecule can be
auto-replicating. Transformed cells, tissues, or plants are
understood to encompass not only the end product of a
transformation process, but also transgenic progeny thereof. A
"non-transformed," "non-transgenic," or "non-recombinant" host
refers to a wild-type organism, e.g., a bacterium or plant, which
does not contain the heterologous nucleic acid molecule.
[0085] A "transformed cell" refers to a cell into which has been
introduced a nucleic acid molecule, for example by molecular
biology techniques. As used herein, the term transformation
encompasses all techniques by which a nucleic acid molecule might
be introduced into such a cell, plant or animal cell, including
transfection with viral vectors, transformation by Agrobacterium,
with plasmid vectors, and introduction of naked DNA by
electroporation, lipofection, and particle gun acceleration and
includes transient as well as stable transformants.
[0086] The term "transgenic plant" refers to a plant or tree that
contains recombinant genetic material not normally found in plants
or trees of this type and which has been introduced into the plant
in question (or into progenitors of the plant) by human
manipulation. Thus, a plant that is grown from a plant cell into
which recombinant DNA is introduced by transformation is a
transgenic plant, as are all offspring of that plant that contain
the introduced transgene (whether produced sexually or asexually).
It is understood that the term transgenic plant encompasses the
entire plant or tree and parts of the plant or tree, for instance
grains, seeds, flowers, leaves, roots, fruit, pollen, stems
etc.
[0087] The term "translation initiation enhancer sequence", as used
herein, refers to a nucleic acid sequence that can determining a
site and efficiency of initiation of translation of a gene (See,
for example, McCarthy et al., 1990, Trends in Genetics, 6: 78-85).
A translation initiation enhancer sequence can extend to include
sequences 5' and 3' to the ribosome binding site. The ribosome
binding site is defined to include, minimally, the Shine-Dalgarno
region and the start codon, in addition to any bases in between. In
addition, the translation initiation enhancer sequence can include
an untranslated leader or the end of an upstream cistron, and thus
a translational stop codon. See, for example, U.S. Pat. No.
5,840,523.
[0088] The term "vector" refers to a nucleic acid molecule which is
used to introduce a polynucleotide sequence into a host cell,
thereby producing a transformed host cell. A "vector" may comprise
genetic material in addition to the above-described genetic
construct, e.g., one or more nucleic acid sequences that permit it
to replicate in one or more host cells, such as origin(s) of
replication, selectable marker genes and other genetic elements
known in the art (e.g., sequences for integrating the genetic
material into the genome of the host cell, and so on).
2. Exemplary Embodiments
Nematode Resistant Transgenic Plants
[0089] It has been discovered that interrupting the feeding cycle
of nematodes by down-regulating one or more nematode parasitism
genes is an effective method for reducing, preventing, or treating
nematode disease in plants. Nematode parasitism genes refers to
genes expressed in the esophageal gland cells encoding for
secretory proteins exported from the gland cell to be released
through the nematode's stylet into host tissue. In particular, it
has been discovered that interfering with the expression of
proteins secreted by nematodes related to the formation of
specialized feeding cells in host plants is an effective method for
reducing, treating, or preventing nematode disease in plants.
Representative parasitism genes encoding secreted proteins that can
be targeted, for example with inhibitory RNA include, include but
are not limited to those genes listed in Table 2, or a fragment
thereof.
[0090] Nematode disease results in substantial losses of valuable
crops. Root-knot nematodes, Meloidogyne species, are among nature's
most successful parasites. They parasitize more than 2,000 plant
species from diverse plant families and represent a tremendous
threat to crop production world-wide. These biotrophic pathogens
have evolved highly specialized and complex feeding relationships
with their hosts. A successful nematode-host interaction requires
molecular signals from the parasite to modify, directly or
indirectly, plant root cells into elaborate feeding cells, called
giant-cells, which are the sole source of nutrients needed for
nematode development and reproduction. Plant-parasitic nematodes
release proteinaceous secretions through a hollow protrusible
stylet into plant cells when feeding. These secretions,
collectively called the parasitome are encoded by parasitism genes
expressed in large and transcriptionally active esophageal gland
cells (Davis, E. L., R. Allen, and R. S. Hussey. 1994.
Developmental expression of esophageal gland antigens and their
detection in stylet secretions of Meloidogyne incognita. Fundam,
Appl. Nematol. 17:255-262.; Hussey, R. S., E. L. Davis, and T. J.
Baum. 2002. Secrets in secretions: genes that control nematode
parasitism of plants. Braz. J. Plant Physiol. 14:183-194.). The
profound cellular modifications induced by Meloidogyne species to
form the giant-cells are the result of an alteration in host root
cell gene expression and phenotype that is driven by the molecular
signals secreted through the nematode's stylet.
[0091] One embodiment provides a plant or cell comprising one or
more inhibitory RNAs specific for one or more mRNAs of one or more
nematode parasitism genes. For example, the present disclosure
provides transgenic plants that express one or more inhibitory RNAs
that down regulate nematode parasitism gene expression when the one
or more inhibitory RNAs are absorbed or ingested by a nematode. The
transgenic plant can be designed to express inhibitory RNA that
down-regulates the target parasitism gene transcript in at least
two different nematode species, for example two different RKN
species. Another embodiment provides a transgenic plant that
comprises inhibitory RNA that down regulates the target parasitism
gene transcript in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, or more nematode species, for example RKN species.
Thus, the present disclosure provides transgenic plants that are
resistant to disease caused by multiple RKN species.
[0092] Another embodiment, provides a transgenic plant comprising
inhibitory RNA specific for one or more nematode parasitism genes
in an amount effective to provide the plant with resistance to all
RKN species, for example those RKN species referenced in Jepson, S.
B. 1987. Identification of root-knot nematodes (Meloidogyne
species). C. A. B. International, Oxford, United Kingdom. 1-265
pages, which, where permissible, is incorporated by reference in
its entirety.
[0093] Another embodiment provides a transgenic plant or transgenic
cell containing or expressing one or more inhibitory nucleic acids
specific for at least a portion of a nucleic acid encoding one or
more secretory polypeptides of a parasitic nematode. The inhibitory
nucleic acid is typically a small inhibitory RNA or microRNA that
is specific for mRNA encoding a nematode esophageal gland cell
protein or polypeptide. It will be appreciated by one of skill in
the art that the inhibitory nucleic acid can be RNA, DNA, or a
combination thereof. Additionally, the inhibitory nucleic acid may
be single or multi-stranded and may be anti-sense or enzymatic. In
one embodiment, the inhibitory nucleic acid interferes, inhibits,
or reduces the translation of a target mRNA. For example, the
inhibitory nucleic acid can bind to a target mRNA and induce or
promote the degradation of the target mRNA or physically prevent
the cellular translational machinery from translating the target
mRNA into a functional protein. Inhibition of the secretory
polypeptide can be compared to controls, for example plants or
cells that do not contain or express the inhibitory nucleic acid. A
"control" refers to a sample of material which is known to be
identical to a sample containing the disclosed inhibitory nucleic
acid in every regard, except that the control sample does not
contain or express the inhibitory nucleic acid.
[0094] The term "esophageal gland cell protein or polypeptide"
refers to a secretory polypeptide encoded by a nematode parasitism
gene. In one embodiment, the esophageal gland cell protein or
polypeptide to be down-regulated generally is a secreted protein
that modulates expression of at least one host plant gene.
Exemplary nematode polypeptides that are down-regulated in the
disclosed compositions and methods include, but are not limited to
polypeptides or fragments thereof encoded by SEQ ID NOs 1, 2, or
5-51, or fragments thereof. The secretory polypeptide can increase
or decrease expression of host plant genes either directly or
indirectly. For example, direct modulation can occur when the
esophageal gland cell protein or polypeptide binds to a host plant
nucleic acid, including genomic DNA, RNA, and mRNA. Indirect
modulation can occur for example when the polypeptide binds with
one or more other proteins or factors to form a complex. The
complex can then bind to a host plant nucleic acid to either
promote or suppress transcription or translation. Down-regulation
of the secretory protein alleviates or reduces at least one symptom
associated with nematode disease. Exemplary symptoms of nematode
disease include, but are not limited to the formation of galls,
giant cells, lesions, stunting, nutrient and water deficiencies,
dieback, and numbers of nematodes infecting a plant. Levels of
reduction or inhibition of nematode disease in transgenic plants or
cells can be compared to levels of nematode disease in control
plants or cells. In one embodiment, the inhibitory nucleic acid
reduces, inhibits, alleviates, treats or prevents nematode
disease.
[0095] In another embodiment, the esophageal gland cell protein or
polypeptide to be down-regulated is encoded by a parasitism gene
involved in the formation of a giant cell. In still other
embodiments, the targeted parasitism gene encodes a polypeptide or
nucleic acid involved in nematode migration through root tissue,
alters cell metabolism, elicits signal transduction in the
recipient cell, or forms a feeding tube that enables the nematode
to feed from the giant-cells. Additionally, the esophageal gland
cell protein or polypeptide can cause cell wall modifications and
potentially interact with signal transduction receptors in the
extracellular space, influence cellular metabolism, cell cycle,
selective protein degradation, localized defense response, and
regulatory activity within the plant cell nucleus.
[0096] Exemplary plant genes that are modulated by the esophageal
gland cell protein or polypeptide include, but are not limited to
genes involved in the formation of specialized nematode feeding
cells also known as giant cells. For example, nematode parasitism
gene 16D10 encodes a protein that binds to a scarecrowlike
transcription regulator. Representative plant genes that can be
modulated by nematode esophageal gland cell polypeptides include,
but are not limited to WUN1, POX, CAT, GST, Mia-1, Mia-2, Mia-3,
Mia-4, CHS1-CHS3, LOX, Chitinase, Trypsin inhibitor, Miraculin,
HMGR, TSW12, LEA14, LEMMI9, C6-19, C27-45, TAS14, UBC DB#103, RPE,
ISDGh, IPPP, LPPL, mUCp, endomembrane protein, 20s proteasome, DAP
decarboxylase, GRP, ENOD40, ATAO1 or combinations thereof (Gheysen,
G. and Fenoll, C. 2002. Annual Review of Phytopathology 40:191,
which, where permissible, is incorporated by reference in its
entirety). Generally, the plant gene is directly or indirectly
involved in root cell growth, root cell division or the production
of specific nutrients ingested by the parasitic nematode. The gene
can be one expressed in a root cell or any other cell of the
plant.
[0097] In one embodiment expression of a targeted nematode
secretory protein is reduced, inhibited, or blocked, as compared to
a control, when the inhibitory nucleic acid is delivered to the
nematode. Delivery of the inhibitory nucleic acid can be achieved,
for example, when the nematode comes into contact with the
inhibitory nucleic acid as the nematode feeds on the transgenic
plant or cell. The nematode can ingest the inhibitory nucleic acid
during feeding, or the nucleic acid can be transported across a
cellular membrane of the nematode by active transport or passive
diffusion. It will be appreciated that the inhibitory nucleic acid
can be delivered to the nematode in combination or alternation with
an agent that induces or promotes the uptake of the inhibitory
nucleic acid by the nematode. An exemplary inducing agent includes,
but is not limited to resorcinol (3-hydroxyphenol).
[0098] In one embodiment, the transgenic plant or transgenic cell
expresses the inhibitory nucleic in an amount effective to modulate
the expression of a nematode esophageal gland cell polypeptide or
protein in a nematode when the inhibitory nucleic acid is delivered
to the nematode. Levels of expression of the inhibitory nucleic
acid in a transgenic plant or cell can be controlled using methods
known in the art, for example using vectors with strong promoters
or constitutively active promoters, high copy number vectors, etc.
The plant or cell can be stably or transiently transfected.
[0099] An exemplary parasitic nematode includes, but is not limited
to members of Meloidogyne spp, also referred to as root-knot
nematodes. Representative species include, but are not limited to
M. arenaria, M. incognita, M. javanica, M. hapla, M. chitwoodi and
M. naasi.
[0100] Representative phylogenetic families of host plants include
Acanthaceae, Aceraceae, Actinidiaceae, Agavaceae, Aizoaceae,
Amaranthaceae, Annonaceae, Apiaceae, Apocynaceae, Araceae,
Araliaceae, Arecaceae, Aristolochiaceae, Balsaminaceae,
Barringtoniaceae, Basellaceae, Berberidaceae, Betulaceae,
Bignoniaceae, Bixaceae, Bombacaceae, Boraginaceae, Buxaceae,
Byttneriaceae, Cactaceae, Caesalpiniaceae, Cannaceae, Capparaceae,
Caprifoliaceae, Caricaceae, Caryophyllaceae, Casuarinaceae,
Casuarinaceae, Celastraceae, Chenopodiaceae, Chenopodiaceae,
Chloranthaceae, Commelinaceae, Convolvulaceae, Comaceae,
Corylaceae, Crassulaceae, Cucurbitaceae, Cupressaceae, Cyatheaceae,
Cyperaceae, Datiscaceae, Dilleniaceae, Dioscoreaceae, Dipsacaceae,
Ebenaceae, Ericaceae, Euphorbiaceae, Fabaceae, Flacourtiaceae,
Fumariaceae, Gentianaceae, Geraniaceae, Gesneriaceae, Ginkgoaceae,
Goodeniaceae, Guttiferae, Haemodoraceae, Hamamelidaceae,
Heliconiaceae, Hydrophyllaceae, Hypericaceae, Iridaceae,
Juglandaceae, Juncaceae, Labiatae, Lamiaceae, Lauraceae, Liliaceae,
Linaceae, Lobeliaceae, Loganiaceae, Lythraceae, Magnoliaceae,
Malpighiaceae, Malvaceae, Marantaceae, Melastomataceae, Meliaceae,
Menispermaceae, Mimosaceae, Moraceae, Musaceae, Myoporaceae,
Myricaceae, Myristicaceae, Myrtaceae, Nyctaginaceae, Oleaceae,
Onagraceae, Orchidaceae, Othnaceae, Oxalidaceae, Paeoniaceae,
Pandanaceae, Papaveraceae, Pedaliaceae, Phytolaccaceae, Pinaceae,
Piperaceae, Pittosporaceae, Plantaginaceae, Platanaceae,
Plumbaginaceae, Poaceae, Podostemaceae, Polemoniaceae,
Polygalaceae, Portulacaceae, Primulaceae, Proteaceae, Punicaceae,
Ranunculaceae, Resedaceae, Rhamnaceae, Rosaceae, Rubiaceae,
Rutaceae, Salicaceae, Santalaceae, Sapindaceae, Sarraceniaceae,
Saxifragaceae, Scrophulariaceae, Smilacaceae, Solanaceae,
Sterculiaceae, Styracaceae, Tamaricaceae, Taxodiaceae,
Tetragoniaceae, Theaceae, Theophrastaceae, Thymelaeaceae,
Tiliaceae, Tropaeolaceae, Turneraceae, Typhaceae, Ulmaceae,
Urticaceae, Valerianaceae, Verbenaceae, Violaceae, Vitaceae,
Zamiaceae, Zingiberaceae, or Zygophyllaceae.
[0101] Common names of host plants that can be transfected with an
inhibitory nucleic acid according the present disclosure include,
but are not limited to tomato, eggplant, potato, melon, cucumber,
carrot, lettuce, artichoke, celery, cucurbits (melon, watermelon,
etc.), barley, corn, peanut, soybean, sugar beet, cotton, cowpea,
beans, alfalfa, tobacco, citrus, clover, pepper, grape, coffee,
olive, or tea.
[0102] It will be appreciated by one of skill in the art that the
present disclosure encompasses any of the fifty or more known
root-knot nematode species.
[0103] Another embodiment provides a composition having an
inhibitory nucleic acid specific for an mRNA or fragment thereof
encoding a polypeptide encoded by one or more of SEQ ID NOs. 1, 2
or 5-51 or a fragment or homologues thereof, in an amount
sufficient to inhibit expression of the polypeptide encoded by one
or more of SEQ ID NOs 1, 2 or 5-51 or homologues thereof when
delivered to a nematode, for example when the nematode is feeding
on a plant or cell expressing or containing the inhibitory nucleic
acid. The composition can contain one or more nematicides,
pesticides, fungicides, or combinations thereof. Representative
nematicides include, but are not limited to chloropicrin, methyl
bromide, 1,3-dichloropropene, sodium methyl dithiocarbamate, sodium
tetrathiocarbonate; and carbamates such as
2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime
(aldicarb), 2,3-Dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate
(carbofuran), methyl
2-(dimethylamino)-N-[[(methylamino)carbonyl]oxy]-2-oxoethanimidoth-
ioate (oxamyl), 2-methyl-2-(methylsulfonyl)propanal
O-[(methylamino)carbonyl]oxime (aldoxycarb), O,O-diethyl
O-[4-(methylsulfinyl)phenyl] phosphorothioate (fensulfothion),
O-Ethyl S,S-dipropylphosphorodithioate (ethoprop), and
Ethyl-3-methyl-4-(methylthio)phenyl(1-methylethyl)phosphoramidate
(phenamiphos).
[0104] Another embodiment provides a cell containing a nucleic acid
encoding an inhibitory nucleic acid specific for an mRNA or
fragment thereof, wherein the mRNA encodes a esophageal gland cell
protein or polypeptide that directly or indirectly modulates: root
cell gene expression, nematode migration through root tissue, cell
metabolism, signal transduction, or is involved in the formation of
a feeding tube that enables the nematode to feed from the
giant-cells of at least one plant gene involved in the formation of
a giant cell. Additionally, the esophageal gland cell protein or
polypeptide or esophageal polypeptide can cause cell wall
modifications and potentially interact with signal transduction
receptors in the extracellular space, influence cellular
metabolism, cell cycle, selective protein degradation, localized
defense response, and regulatory activity within the plant cell
nucleus. The cell can be prokaryotic or eukaryotic, and generally
is a plant cell, particularly a root cell.
[0105] Still another embodiment provides a method for providing
nematode resistance to a plant by contacting the plant with one or
more inhibitory nucleic acids specific for one or more nematode
esophageal gland cell proteins in an amount sufficient to reduce
nematode disease, wherein the one or more nematode esophageal gland
cell proteins modulate: gene expression of the plant or cell,
formation of a giant cell, nematode migration through root tissue
of the plant, cell metabolism of the plant, elicits signal
transduction in the plant cell, or forms a feeding tube that
enables the nematode to feed from giant-cells formed in the plant.
One aspect provides inhibitory nucleic acids specific for
esophageal gland cell proteins secreted by nematodes, in particular
root knot nematodes. The inhibitory nucleic acid can be sprayed
onto the plant or otherwise delivered to the plant so that the
inhibitory nucleic acid comes into contact with a parasitic
nematode.
[0106] Yet another embodiment provides transgenic plants or plant
cells containing an inhibitory nucleic acid, for example siRNA or
microRNA, that down regulates root-knot nematode esophageal gland
cell proteins when delivered to a nematode feeding on the plant or
plant cell. RNA interference is known in the art. See for example,
Kreutzer et al., International PCT Publication No. WO 00/44895;
Zernicka-Goetz et al, International PCT Publication No. WO
01/36646; Fire, International PCT Publication No. WO 99/32619;
Plaetinck et al., International PCT Publication No. WO 00/01846;
Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO
99/07409; Li et al., International PCT Publication No. WO 00/44914;
and Trick et al., US20040098761.
[0107] In one embodiment, the nematode is not a soybean cyst
nematode.
[0108] In another embodiment, the inhibitory nucleic acid is not
directly lethal to embryonic or adult nematodes or is not involved
in nematode fertility, but instead inhibits the ability of the
nematode to feed on or obtain nutrients from the transgenic plant
or plant cell.
[0109] In some embodiments, inhibitory double stranded RNA (dsRNA)
is derived from an "exogenous template". Such a template may be all
or part of a plant or nematode nucleotide sequence; it may be a DNA
gene sequence or a cDNA produced from an mRNA isolated from a
parasitic nematode, for example by reverse transcriptase. When the
template is all or a part of a DNA gene sequence, it is preferred
if it is from one or more or all exons of the gene. While the dsRNA
is derived from an endogenous or exogenous template, there is no
limitation on the manner in which it could be synthesized. For
example, the siRNA can be chemically synthesized, produced by in
vitro transcription; produced by digestion of long dsRNA by an
RNase III family enzyme (e.g., Dicer, RNase III); expressed in
cells from an siRNA expression plasmid or viral vector; or
expressed in cells from a PCR-derived siRNA expression cassette
[0110] SiRNA prepared in vitro is then introduced directly into
cells by transfection, electroporation, or by another method.
Alternatively, transfection of DNA-based vectors and cassettes that
express siRNAs within the cells can be used. RNAi may be
synthesized in vitro or in vivo, using manual and/or automated
procedures. In vitro synthesis may be chemical or enzymatic, for
example using cloned RNA polymerase (e.g., T3, T7, SP6) for
transcription of the endogenous DNA (or cDNA) template, or a
mixture of both.
[0111] In viva, the dsRNA may be synthesised using recombinant
techniques well known in the art (see e.g., Sambrook, et al.,
Molecular Cloning; A Laboratory Manual, Third Edition (2001). For
example, bacterial cells can be transformed with an expression
vector which comprises the DNA template from which the dsRNA is to
be derived. Alternatively, the cells, of a plant for example, in
which inhibition of gene expression is required may be transformed
with an expression vector or by other means. Bidirectional
transcription of one or more copies of the template may be by
endogenous RNA polymerase of the transformed cell or by a cloned
RNA polymerase (e.g., T3, T7, SP6) coded for by the expression
vector or a different expression vector. The use and production of
an expression construct are known in the art (see WO98/32016; U.S.
Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and
5,804,693). Inhibition of gene expression may be targeted by
specific transcription in an organ, tissue, or cell type; an
environmental condition (e.g. temperature, chemical); and/or
engineering transcription at a developmental stage or age,
especially when the dsRNA is synthesized in vivo in the plant cell
for example. dsRNA may also be delivered to specific tissues or
cell types using known gene delivery systems. Components of these
systems include the seed-specific lectin promoter and the flower
specific promoter from APETALA3. These vectors are listed solely by
way of illustration of the many commercially available and well
known vectors that are available to those of skill in the art.
[0112] If synthesized outside the cell, the RNA may be purified
prior to introduction into the cell. Purification may be by
extraction with a solvent (such as phenol/chloroform) or resin,
precipitation (for example in ethanol), electrophoresis,
chromatography, or a combination thereof. However, purification may
result in loss of dsRNA and may therefore be minimal or not carried
out at all. The RNA may be dried for storage or dissolved in an
aqueous solution, which may contain buffers or salts to promote
annealing, and/or stabilization of the RNA strands.
[0113] Suitable dsRNA can also contain one or more modified bases,
or have a modified a backbone to increase stability or for other
reasons. For example, the phosphodiester linkages of natural RNA
may be modified to include at least one of a nitrogen or sulfur
heteroatom. Moreover, dsRNA comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name just
two examples, can be used. It will be appreciated that a great
variety of modifications have been made to RNA that serve many
useful purposes known to those of skill in the art. The term dsRNA
as it is employed herein embraces such chemically, enzymatically or
metabolically modified forms of dsRNA, provided that it is derived
from an endogenous template.
[0114] The double-stranded structure may be formed by a single
self-complementary RNA strand or two separate complementary RNA
strands. RNA duplex formation may be initiated either inside or
outside the plant cell.
[0115] The sequence of at least one strand of the dsRNA contains a
region complementary to at least a part of the target mRNA
sufficient for the dsRNA to specifically hybridize to the target
mRNA. In one embodiment, the siRNA is substantially identical to at
least a portion of the target mRNA. "Identity", as known in the
art, is the relationship between two or more polynucleotide (or
polypeptide) sequences, as determined by comparing the sequences.
In the art, identity also means the degree of sequence relatedness
between polynucleotide sequences, as determined by the match
between strings of such sequences. Identity can be readily
calculated (Computational Molecular Biology, Lesk, A. M., ed.,
Oxford University Press, New York, 1988; Biocomputing: Informatics
and Genome Projects, Smith, D. W., ed., Academic Press, New York,
1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,
and Griffin, H. G., eds., Humana Press, N.J., 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991). While there exist a number
of methods to measure identity between two polynucleotide
sequences, the term is well known to skilled artisans (Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991; and Carillo, H., and
Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods
commonly employed to determine identity between sequences include,
but are not limited to those disclosed in Carillo, H., and Lipman,
D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods to
determine identity are designed to give the largest match between
the sequences tested. Methods to determine identity are codified in
computer programs. Computer program methods to determine identity
between two sequences include, but are not limited to, GCG program
package (Devereux, J., et al., Nucleic Acids Research 12(1): 387
(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J.
Molec. Biol. 215: 403 (1990)). Another software package well known
in the art for carrying out this procedure is the CLUSTAL program.
It compares the sequences of two polynucleotides and finds the
optimal alignment by inserting spaces in either sequence as
appropriate. The identity for an optimal alignment can also be
calculated using a software package such as BLASTx. This program
aligns the largest stretch of similar sequence and assigns a value
to the fit. For any one pattern comparison several regions of
similarity may be found, each having a different score. One skilled
in the art will appreciate that two polynucleotides of different
lengths may be compared over the entire length of the longer
fragment. Alternatively small regions may be compared. Normally
sequences of the same length are compared for a useful comparison
to be made.
[0116] In one embodiment, the inhibitory nucleic acid has 100%
sequence identity with at least a part of the target mRNA. However,
inhibitory nucleic acids having 70%, 80% or greater than 90% or 95%
sequence identity may be used. Thus sequence variations that might
be expected due to genetic mutation, strain polymorphism, or
evolutionary divergence can be tolerated.
[0117] The duplex region of the RNA may have a nucleotide sequence
that is capable of hybridizing with a portion of the target gene
transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA,
50.degree. C. or 70.degree. C. hybridization for 12-16 hours;
followed by washing).
[0118] While the optimum length of the dsRNA may vary according to
the target gene and experimental conditions, the duplex region of
the RNA may be at least 19, 20, 21-23, 25, 50, 100, 200, 300, 400
or more bases long.
[0119] Target genes are nematode genes encoding secreted proteins,
in particular secreted proteins that modulate: gene expression of
the plant or cell, formation of a giant cell, nematode migration
through root tissue of the plant, cell metabolism of the plant,
elicits signal transduction in the plant cell, or forms a feeding
tube that enables the nematode to feed from giant-cells formed in
the plant. One aspect provides inhibitory nucleic acids specific
for esophageal gland cell proteins secreted by nematodes, in
particular root knot nematodes. Typically, the dsRNA or inhibitory
nucleic acid is substantially identical to the whole of the target
gene, i.e. the coding portion of the gene. However, the dsRNA or
inhibitory nucleic acid can be substantially identical to a part of
the target gene. The size of this part depends on the particular
target gene and can be determined by those skilled in the art by
varying the size of the dsRNA and observing whether expression of
the gene has been inhibited.
Plants with Enhanced Root Growth
[0120] One embodiment provides a transgenic plant or transgenic
cell containing or expressing one or more nucleic acids encoding
one or more nematode esophageal gland cell polypeptides or
fragments thereof of a parasitic nematode. Expression of the one or
more nematode esophageal gland cell polypeptides or fragments
thereof in a plant or plant cell promotes, stimulates, or enhances
root growth of the transgenic plant compared to non-transgenic
plants or control plants. A root includes a seminal root,
adventitious root, first order lateral root, second order laterals,
etc., feeder roots primary roots, secondary roots, and coarse
roots.
[0121] The nematode esophageal gland cell polypeptides or fragments
used with the disclosed embodiments can increase the size of roots,
the number of roots, the surface area of roots, and the overall
quality of a root system. Root crops can be produced with the
disclosed compositions and methods that are larger than root crops
produced in the absence of the disclosed compositions and methods.
Other crops produced using the disclosed compositions and methods
can be resistant to drought, erosion, or increased environmental
stress. Environmental stress includes changes in climate such as
rainfall, temperature, and humidity.
[0122] Exemplary nematode esophageal gland cell polypeptides or
fragments thereof include, but are not limited to polypeptides
encoded by SEQ ID NOs 1, 2, or 5-51, fragments thereof, or
combinations thereof. The nematode esophageal gland cell
polypeptides or fragments can increase, stimulate, or enhance root
growth directly or indirectly. For example, direct modulation can
occur when the nematode secretory polypeptide binds to a host plant
nucleic acid, including genomic DNA, RNA, and mRNA. Indirect
modulation can occur for example when the polypeptide binds with
one or more other proteins or factors to form a complex. The
complex can then bind to a host plant nucleic acid to either
promote or suppress transcription or translation.
[0123] In one embodiment, the transgenic plant or transgenic cell
expresses the nematode esophageal gland cell polypeptide or
fragment thereof in an amount effective to stimulate, enhance or
promote root growth or development. Alternatively, the nematode
esophageal gland cell polypeptide or fragment thereof can be
delivered directly to the plant. Levels of nematode esophageal
gland cell polypeptide or fragment thereof expression in a
transgenic plant or cell can be controlled using methods known in
the art, for example using vectors with strong promoters or
constitutively active promoters, high copy number vectors, etc. The
plant or cell can be stably or transiently transfected.
[0124] An exemplary nematode includes, but is not limited to
members of Meloidogyne spp. also referred to as root-knot
nematodes. Representative species include, but are not limited to
M. arenaria, M. incognita, M. javanica, M. hapla, and M. naasi.
[0125] Representative phylogenetic families of host plants include
Acanthaceae, Aceraceae, Actinidiaceae, Agavaceae, Aizoaceae,
Amaranthaceae, Annonaceae, Apiaceae, Apocynaceae, Araceae,
Araliaceae, Arecaceae, Aristolochiaceae, Balsaminaceae,
Barringtoniaceae, Basellaceae, Berberidaceae, Betulaceae,
Bignoniaceae, Bixaceae, Bombacaceae, Boraginaceae, Buxaceae,
Byttneriaceae, Cactaceae, Caesalpiniaceae, Cannaceae, Capparaceae,
Caprifoliaceae, Caricaceae, Caryophyllaceae, Casuarinaceae,
Casuarinaceae, Celastraceae, Chenopodiaceae, Chenopodiaceae,
Chloranthaceae, Commelinaceae, Convolvulaceae, Comaceae,
Corylaceae, Crassulaceae, Cucurbitaceae, Cupressaceae, Cyatheaceae,
Cyperaceae, Datiscaceae, Dilleniaceae, Dioscoreaceae, Dipsacaceae,
Ebenaceae, Ericaceae, Euphorbiaceae, Fabaceae, Flacourtiaceae,
Fumariaceae, Gentianaceae, Geraniaceae, Gesneriaceae, Ginkgoaceae,
Goodeniaceae, Guttiferae, Haemodoraceae, Hamamelidaceae,
Heliconiaceae, Hydrophyllaceae, Hypericaceae, Iridaceae,
Juglandaceae, Juncaceae, Labiatae, Lamiaceae, Lauraceae, Liliaceae,
Linaceae, Lobeliaceae, Loganiaceae, Lythraceae, Magnoliaceae,
Malpighiaceae, Malvaceae, Marantaceae, Melastomataceae, Meliaceae,
Menispermaceae, Mimosaceae, Moraceae, Musaceae, Myoporaceae,
Myricaceae, Myristicaceae, Myrtaceae, Nyctaginaceae, Oleaceae,
Onagraceae, Orchidaceae, Othnaceae, Oxalidaceae, Paeoniaceae,
Pandanaceae, Papaveraceae, Pedaliaceae, Phytolaccaceae, Pinaceae,
Piperaceae, Pittosporaceae, Plantaginaceae, Platanaceae,
Plumbaginaceae, Poaceae, Podostemaceae, Polemoniaceae,
Polygalaceae, Portulacaceae, Primulaceae, Proteaceae, Punicaceae,
Ranunculaceae, Resedaceae, Rhamnaceae, Rosaceae, Rubiaceae,
Rutaceae, Salicaceae, Santalaceae, Sapindaceae, Sarraceniaceae,
Saxifragaceae, Scrophulariaceae, Smilacaceae, Solanaceab,
Sterculiaceae, Styracaceae, Tamaricaceae, Taxodiaceae,
Tetragoniaceae, Theaceae, Theophrastaceae, Thymelaeaceae,
Tiliaceae, Tropaeolaceae, Turneraceae, Typhaceae, Ulmaceae,
Urticaceae, Valerianaceae, Verbenaceae, Violaceae, Vitaceae,
Zamiaceae, Zingiberaceae, or Zygophyllaceae.
[0126] Common names of host plants that can be transfected with
nucleic acid encoding a RKN esophageal gland cell secretory
polypeptide according the present disclosure include, but are not
limited to tomato, eggplant, potato, melon, cucumber, carrot,
lettuce, artichoke, celery, cucurbits (melon, watermelon, etc.),
barley, corn, peanut, soybean, sugar beet, cotton, cowpea, beans,
alfalfa, tobacco, citrus, clover, pepper, grape, coffee, olive, or
tea.
[0127] It will be appreciated by one of skill in the art that the
present disclosure encompasses any nematode that secretes a protein
that alters the expression of a host gene. For example, one
embodiment provides a transgenic plant or cell containing a nucleic
acid encoding a protein secreted by a member of Meloidogyne spp.,
wherein the secreted protein stimulates, enhances, or promotes root
growth or development.
[0128] Another embodiment provides a composition comprising a
nucleic acid having a sequence of SEQ ID NOs. 1, 2 or 5-51 or a
fragment or homologues thereof. The composition stimulates,
promotes, or enhances root growth or development when delivered to
a plant or plant cell.
[0129] Still another embodiment provides a composition comprising a
one or more polypeptides or fragments thereof encoded by SEQ ID NOs
1, 2 or 5-51 or homologues thereof when delivered to a plant or
plant cell.
[0130] Root stimulating compositions disclosed herein can
optionally contain a growth enhancer, fertilizer, one or more
nemiticides, pesticides, fungicides, or combinations thereof.
Representative nematicides include, but are not limited to
chloropicrin, methyl bromide, 1,3-dichloropropene, sodium methyl
dithiocarbamate, sodium tetrathiocarbonate; and carbamates such as
2-methyl-2-(methylthio)propionaldehyde O-methylcarbamoyloxime
(aldicarb), 2,3-Dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate
(carbofuran), methyl
2-(dimethylamino)-N-[[(methylamino)carbonyl]oxy]-2-oxoethanimidoth-
ioate (oxamyl), 2-methyl-2-(methylsulfonyl)propanal
O-[(methylamino)carbonyl]oxime (aldoxycarb), O,O-diethyl
O-[4-(methylsulfinyl)phenyl]phosphorothioate (fensulfothion),
O-Ethyl S,S-dipropylphosphorodithioate (ethoprop), and
Ethyl-3-methyl-4-(methylthio)phenyl(1-methylethyl)phosphoramidate
(phenamiphos).
[0131] Another embodiment provides a cell, for example a plant
cell, containing one or more nucleic acids encoding a nematode
secretory polypeptide or fragment thereof wherein the nematode
secretory polypeptides directly or indirectly stimulate, enhance or
promote root growth or development. The cell can be prokaryotic or
eukaryotic, and generally is a plant cell, particularly a root
cell.
[0132] Still another embodiment provides a method for providing
drought resistance to a plant by contacting the plant with one or
more nematode esophageal proteins or nucleic acids encoding
nematode esophageal proteins in an amount sufficient to stimulate,
promote, or enhance root development. The composition can be
sprayed onto the plant, applied to the soil surrounding the plant
or otherwise delivered to the plant so that the composition
contacts the plant.
Plant Transformation Technology
[0133] DNA molecules and RNA molecules of the present disclosure
are incorporated in plant or bacterial cells using conventional
recombinant DNA technology. Generally, a DNA or an RNA molecule of
the present disclosure is comprised in a transformation vector. A
large number of such vector systems known in the art may be used,
such as plasmids. The components of the expression system are also
modified, e.g., to increase expression of the introduced RNA
fragments. For example, truncated sequences, nucleotide
substitutions or other modifications may be employed. Expression
systems known in the art may be used to transform virtually any
plant cell under suitable conditions. A transgene comprising a DNA
molecule of the present invention is preferably stably transformed
and integrated into the genome of the host cells. Transformed cells
are preferably regenerated into whole plants. Detailed description
of transformation techniques are within the knowledge of those
skilled in the art.
[0134] Reporter genes or selectable marker genes may be included in
the expression cassette. Examples of suitable reporter genes known
in the art can be found in, for example, Jefferson et al. (1991) in
Plant Molecular Biology Manual, ed. Gelvin et al. (Kluwer Academic
Publishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol.
7:725-737; Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al.
(1995) Bio Techniques 19:650-655; and Chiu et al. (1996) Current
Biology 6:325-330.
[0135] Selectable marker genes for selection of transformed cells
or tissues can include genes that confer antibiotic resistance or
resistance to herbicides. Examples of suitable selectable marker
genes include, but are not limited to, genes encoding resistance to
chloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992);
methotrexate (Herrera Estrella et al (1983) Nature 303:209-213;
Meijer et al. (1991) Plant Mol. Biol. 16:807-820); hygromycin
(Waldron et al. (1985) Plant Mol. Biol. 5:103-108; Zhijian et al.
(1995) Plant Science 108:219-227); streptomycin (Jones et al.
(1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard
et al. (1996) Transgenic Res. 5:131-137); bleomycin (Hille et al.
(1990) Plant Mol. Biol. 7:171-176); sulfonamide (Guerineau et al.
1990) Plant Mol. Biol. 15:127-136); bromoxynil (Stalker et al.
(1988) Science 242:41 9423); glyphosate (Shaw et al. (1986) Science
233:478481); phosphinothricin (DeBlock et al. (1987) EMBO J.
6:2513-2518).
[0136] Other genes that could serve utility in the recovery of
transgenic events but might not be required in the final product
would include, but are not limited to, examples such as GUS
(b-glucoronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5:387),
GFP (green florescence protein; Chalfie et al. (1994) Science
263:802), luciferase (Riggs et al. (1987) Nucleic Acids Res.
15(19):8115 and Luehrsen et al. (1992) Methods Enzymol.
216:397-414) and the maize genes encoding for anthocyanin
production (Ludwig et al. (1990) Science 247:449).
[0137] The expression cassette comprising a promoter sequence
operably linked to a heterologous nucleotide sequence of interest
can be used to transform any plant. In this manner, genetically
modified plants, plant cells, plant tissue, seed, and the like can
be obtained.
[0138] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606, Agrobacterium-mediated transformation
(Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al. WO
US98101268), direct gene transfer (Paszkowski et al. (1984) EMBO J.
3:2717-2722), and ballistic particle acceleration (see, for
example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.
(1995) "Direct DNA Transfer into Intact Plant Cells via
Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture: Fundamental Methods, ed, Gamborg and Phillips
(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology
6:923-926). Also see Weissinger et al. (1988) Ann. Rev, Genet.
22:421-477; Sanford et al. (1987) Particulate Science and
Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.
87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926
(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.
27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.
96:319-324 (soybean); Dafta et al. (1990) Biotechnology 8:736-740
(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309
(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize);
Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos.
5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer
into Intact Plant Cells via Microprojectile Bombardment," in Plant
Cell, Tissue, and Organ Culture Fundamental Methods, ed. Gamborg
(Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant
Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology
8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature
(London) 311:763-764; Bowen et alt., U.S. Pat. No. 5,736,369
(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.),
pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports
9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566
(whisker-mediated transformation); D'Halluin et al. (1992) Plant
Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell
Reports 12:250-255 and Christou and Ford (1995) Annals of Botany
75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology
14:745-750 (maize via Agrobacterium tumefaciens); all of which are
herein incorporated by reference in their entirety.
[0139] The cells that have been transformed may be grown into
plants in accordance with conventional techniques. See, for
example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These
plants may then be grown, and either pollinated with the same
transformed strain or different strains, and the resulting hybrid
having constitutive expression of the desired phenotypic
characteristic identified. Two or more generations may be grown to
ensure that constitutive expression of the desired phenotypic
characteristic is stably maintained and inherited and then seeds
harvested to ensure constitutive expression of the desired
phenotypic characteristic has been achieved.
[0140] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize 1n2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-1 a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference in their entirety.
[0141] Constitutive promoters include, for example, the core
promoter of the Rsyn7 promoter and other constitutive promoters
disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CAMV
35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin
(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin
(Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and
Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last
et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al.
(1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026), and the like. Other constitutive promoters include, for
example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142.
[0142] Where low level expression is desired, weak promoters may be
used. Generally, by "weak promoter" is intended a promoter that
drives expression of a coding sequence at a low level. By low level
is intended at levels of about 1/1000 transcripts to about
1/100,000 transcripts to about 1/500,000 transcripts.
Alternatively, it is recognized that weak promoters also
encompasses promoters that are expressed in only a few cells and
not in others to give a total low level of expression. Where a
promoter is expressed at unacceptably high levels, portions of the
promoter sequence can be deleted or modified to decrease expression
levels.
[0143] Such weak constitutive promoters include, for example, the
core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.
6,072,050), the core 35S CaMV promoter, and the like. Other
constitutive promoters include, for example, U.S. Pat. Nos.
5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463; and 5,608,142.
[0144] "Tissue-preferred" promoters can be used to target a gene
expression within a particular tissue. Tissue-preferred promoters
include Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et
al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al (1997)
Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic
Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol.
112(3):1331-1341; Van Camp et al (1996) Plant Physiol.
112(2):525-535; Canevascini et al. (1996) Plant Physiol.
112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol,
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters
can be modified, if necessary, for weak expression.
[0145] "Seed-preferred" promoters include both "seed-specified"
promoters (those promoters active during seed development such as
promoters of seed storage proteins) as well as "seed-germinating"
promoters (those promoters active during seed germination). See
Thompson et al. (1989) BioEssays 10:108, herein incorporated by
reference. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa
zein); milps (myo-inositol-1-phosphate synthase); and ce1A
(cellulose synthase). Gama-zein is a preferred endosperm-specific
promoter. Glob-1 is a preferred embryo-specific promoter. For
dicots, seed-specific promoters include, but are not limited to,
bean.beta.-phaseolin, napin, beta.-conglycinin, soybean lecfin,
cruciferin, and the like. For monocots, seed-specific promoters
include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27
kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1,
etc.
[0146] Leaf-specific promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0147] Root-preferred promoters are known and may be selected from
the many available from the literature or isolated de novo from
various compatible species. See, for example, Hire et al. (1992)
Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine
synthetase gene); Keller and Baumgartner (1991) Plant Cell
3(10):1051-1061 (root-specific control element in the GRP 1.8 gene
of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-specific promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):I 1'-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also U.S. Pat. Nos. 5,837,876; 5,750,386;
5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
[0148] Chloroplast targeting sequences are known in the art and
include the chloroplast small subunit of ribulose-1,5-bisphosphate
carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant
Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem.
266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase
(EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6):789-810);
tryptophan synthase (Zhao et al. (1995) J, Biol. Chem.
270(11):6081-6087); plastocyanin (Lawrence et at. (1997) J. Biol.
Chem. 272(33):20357-20363); chorismate synthase (Schmidt et al.
(1993) J. Biol. Chem. 268(36):27447-27457); and the light
harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.
(1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al.
(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J.
Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant
Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res.
Commun. 196:1414-1421; and Shah et al. (1986) Science
233:478-481.
[0149] Methods for transformation of chloroplasts are known in the
art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci.
USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA
90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method
relies on particle gun delivery of DNA containing a selectable
marker and targeting of the DNA to the plastid genome through
homologous recombination. Additionally, plastid transformation may
be accomplished by transactivation of a silent plastid-bome
transgene by tissue-preferred expression of a nuclear-encoded and
plastid-directed RNA polymerase. Such a system has been reported in
McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
[0150] The nucleic acids of interest to be targeted to the
chloroplast may be optimized for expression in the chloroplast to
account for differences in codon usage between the plant nucleus
and this organelle. In this manner, the nucleic acids of interest
may be synthesized using chloroplast-preferred codons. See, for
example, U.S. Pat. No. 5,380,831, herein incorporated by
reference.
[0151] Plants transformed in accordance with the present disclosure
may be monocots or dicots and include, but are not limited to, any
nematode host plant.
Requirements for Construction of Plant Expression Cassettes
[0152] Nucleic acid sequences intended for expression in transgenic
plants are first assembled in expression cassettes behind a
suitable promoter expressible in plants. The expression cassettes
may also comprise any further sequences required or selected for
the expression of the transgene. Such sequences include, but are
not restricted to, transcription terminators, extraneous sequences
to enhance expression such as introns, vital sequences, and
sequences intended for the targeting of the gene product to
specific organelles and cell compartments. These expression
cassettes can then be easily transferred to the plant
transformation vectors described infra. The following is a
description of various components of typical expression
cassettes.
[0153] Promoters
[0154] The selection of the promoter used in expression cassettes
determine the spatial and temporal expression pattern of the
transgene in the transgenic plant. Selected promoters express
transgenes in specific cell types (such as leaf epidermal cells,
mesophyll cells, root cortex cells) or in specific tissues or
organs (roots, leaves or flowers, for example) and the selection
reflects the desired location of accumulation of the gene product.
Alternatively, the selected promoter drives expression of the gene
under various inducing conditions.
[0155] Promoters vary in their strength, i.e., ability to promote
transcription. Depending upon the host cell system utilized, any
one of a number of suitable promoters known in the art may be used.
For example, for constitutive expression, the CaMV 35S promoter,
the rice actin promoter, or the ubiquitin promoter may be used. For
example, for regulatable expression, the chemically inducible PR-1
promoter from tobacco or Arabidopsis may be used (see, e.g., U.S.
Pat. No. 5,689,044).
[0156] A suitable category of promoters is that which is wound
inducible. Numerous promoters have been described which are
expressed at wound sites. Preferred promoters of this kind include
those described by Stanford et al. Mol. Gen. Genet. 215: 200-208
(1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann
et al. Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant
Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol.
22: 129-142 (1993), and Warner et al. Plant J. 3: 191-201
(1993).
[0157] Suitable tissue specific expression patterns include green
tissue specific, root specific, stem specific, and flower specific.
Promoters suitable for expression in green tissue include many
which regulate genes involved in photosynthesis, and many of these
have been cloned from both monocotyledons and dicotyledons. A
suitable promoter is the maize PEPC promoter from the phosphoenol
carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:
579-589 (1989)). A suitable promoter for root specific expression
is that described by de Framond (FEBS 290: 103-106 (1991); EP 0 452
269 and a root-specific promoter is that from the T-1 gene. A
suitable stem specific promoter is that described in U.S. Pat. No.
5,625,136 and which drives expression of the maize trpA gene.
[0158] Transcriptional Terminators
[0159] A variety of transcriptional terminators are available for
use in expression cassettes. These are responsible for the
termination of transcription beyond the transgene and its correct
polyadenylation. Appropriate transcriptional terminators are those
that are known to function in plants and include the CaMV 35S
terminator, the tm1 terminator, the nopaline synthase terminator
and the pea rbcS E9 terminator. These are used in both
monocotyledonous and dicotyledonous plants.
[0160] Sequences for the Enhancement or Regulation of
Expression
[0161] Numerous sequences have been found to enhance gene
expression from within the transcriptional unit and these sequences
can be used in conjunction with the genes to increase their
expression in transgenic plants. For example, various intron
sequences such as introns of the maize Adh1 gene have been shown to
enhance expression, particularly in monocotyledonous cells. In
addition, a number of non-translated leader sequences derived from
viruses are also known to enhance expression, and these are
particularly effective in dicotyledonous cells.
[0162] Coding Sequence Optimization
[0163] The coding sequence of the selected gene may be genetically
engineered by altering the coding sequence for optimal expression
in the crop species of interest. Methods for modifying coding
sequences to achieve optimal expression in a particular crop
species are well known (see, e.g. Perlak et al., Proc. Natl. Acad.
Sci. USA 88: 3324 (1991); and Koziel et alp Bio/technol. 11: 194
(1993)).
[0164] Another embodiment provides an RNA molecule directly
transformed into the plastid genome. Plastid transformation
technology is extensively described in U.S. Pat. Nos. 5,451,513,
5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and
in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305.
The basic technique for chloroplast transformation involves
introducing regions of cloned plastid DNA flanking a selectable
marker together with the gene of interest into a suitable target
tissue, e.g., using biolistics or protoplast transformation (e.g.,
calcium chloride or PEG mediated transformation). The 1 to 1.5 kb
flanking regions, termed targeting sequences, facilitate homologous
recombination with the plastid genome and thus allow the
replacement or modification of specific regions of the plastome.
Initially, point mutations in the chloroplast 16S rRNA and rps12
genes conferring resistance to spectinomycin and/or streptomycin
are utilized as selectable markers for transformation (Svab, Z.,
Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA
87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4,
39-45). The presence of cloning sites between these markers allowed
creation of a plastid targeting vector for introduction of foreign
DNA molecules (Staub, J. M., and Maliga, P. (1993) EMBO J. 12,
601-606). Substantial increases in transformation frequency are
obtained by replacement of the recessive rRNA or r-protein
antibiotic resistance genes with a dominant selectable marker, the
bacterial aadA gene encoding the spectinomycin-detoxifying enzyme
aminoglycoside-3'-adenyltransferase (Svab, Z., and Maliga, P.
(1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this
marker had been used successfully for high-frequency transformation
of the plastid genome of the green alga Chiamydomonas reinhardtii
(Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089).
Other selectable markers useful for plastid transformation are
known in the art and are encompassed within the scope of the
invention.
Construction of Plant Transformation Vectors
[0165] Numerous transformation vectors available for plant
transformation are known to those of ordinary skill in the plant
transformation arts, and the genes pertinent to this disclosure can
be used in conjunction with any such vectors. The selection of
vector depends upon the selected transformation technique and the
target species for transformation. For certain target species,
different antibiotic or herbicide selection markers are preferred.
Selection markers used routinely in transformation include the
npt11 gene, which confers resistance to kanamycin and related
antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan
et al., Nature 304: 184-187 (1983)), the bar gene, which confers
resistance to the herbicide phosphinothricin (White et al., Nucl.
Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet. 79;
625-631 (1990)), the hph gene, which confers resistance to the
antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol
4: 2929-2931), the manA gene, which allows for positive selection
in the presence of mannose (Miles and Guest (1984) Gene, 32: 41-48;
U.S. Pat. No. 5,767,378), and the dhfr gene, which confers
resistance to methotrexate (Bourouis et al., EMBO J. 2 (7):
1099-1104 (1983)), and the EPSPS gene, which confers resistance to
glyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642).
[0166] Many vectors are available for transformation using
Agrobacterium tumefaciens. These typically carry at least one T-DNA
border sequence and include vectors such as pBIN19 (Bevan, Nucl.
Acids Res. (1984). Typical vectors suitable for Agrobacterium
transformation include the binary vectors pCIB200 and pCIB2001, as
well as the binary vector pCIB 10 and hygromycin selection
derivatives thereof. (See, for example, U.S. Pat. No.
5,639,949).
[0167] Transformation without the use of Agrobacterium tumefaciens
circumvents the requirement for T-DNA sequences in the chosen
transformation vector and consequently vectors lacking these
sequences are utilized in addition to vectors such as the ones
described above which contain T-DNA sequences. Transformation
techniques that do not rely on Agrobacterium include transformation
via particle bombardment, protoplast uptake (e.g. PEG and
electroporation) and microinjection. The choice of vector depends
largely on the preferred selection for the species being
transformed. Typical vectors suitable for non-Agrobacterium
transformation include pCIB3064, pSOG 19, and pSOG35. (See, for
example, U.S. Pat. No. 5,639,949).
Transformation Techniques
[0168] Once the DNA sequence of interest is cloned into an
expression system, it is transformed into a plant cell. Methods for
transformation and regeneration of plants are well known in the
art. For example, Ti plasmid vectors have been utilized for the
delivery of foreign DNA, as well as direct DNA uptake, liposomes,
electroporation, micro-injection, and microprojectiles. In
addition, bacteria from the genus Agrobacterium can be utilized to
transform plant cells.
[0169] Transformation techniques for dicotyledons are well known in
the art and include Agrobacterium-based techniques and techniques
that do not require Agrobacterium. Non Agrobacterium techniques
involve the uptake of exogenous genetic material directly by
protoplasts or cells. This is accomplished by PEG or
electroporation mediated uptake, particle bombardment-mediated
delivery, or microinjection. In each case the transformed cells may
be regenerated to whole plants using standard techniques known in
the art.
[0170] Transformation of most monocotyledon species has now become
somewhat routine. Preferred techniques include direct gene transfer
into protoplasts using PEG or electroporation techniques, particle
bombardment into callus tissue or organized structures, as well as
Agrobacterium-mediated transformation.
[0171] Plants from transformation events are grown, propagated and
bred to yield progeny with the desired trait, and seeds are
obtained with the desired trait, using processes well known in the
art. The methods can result in plant cells comprising the RNA
fragments of the present invention, wherein the expression of said
target gene in said plant cell is altered by said RNA fragments, a
plant and the progeny thereof derived from the plant cell, and
seeds derived from the plant.
[0172] The disclosed inhibitory nucleic acids or RKN esophageal
gland cell secretory polypeptides may be used alone or as a
component of a kit having at least one of the reagents necessary to
carry out the in vitro or in vivo introduction of RNA to subjects.
Suitable components are the dsRNA and a vehicle that promotes
introduction of the dsRNA. Such a kit may also include instructions
to allow a user of the kit to practice the invention.
[0173] Another embodiment provides a method for providing
resistance to nematode disease by introducing into a nematode host
plant cell an RNA comprising a double stranded structure having a
nucleotide sequence which is complementary to at least a part of
the target mRNA; and optionally verifying inhibition of expression
of the target mRNA.
[0174] One embodiment provides a method for treating or preventing
nematode disease in a plant by contacting a parasitic nematode in
or on the plant with a with dsRNA having a sequence which is
complementary to at least a part of a mRNA encoding a nematode
secretory protein, for example an esophageal gland cell protein;
wherein the secretory protein modulates gene expression of
plant.
[0175] Still another embodiment provides a plant cell, for example,
containing an expression construct, the construct coding for an RNA
which forms a double stranded structure having. a nucleotide
sequence which is complementary to at least a part of a target mRNA
that encodes a nematode secretory protein, for example an
esophageal gland cell protein, as well as a transgenic plant
containing such a cell.
[0176] In another embodiment, the RNA fragments are comprised in
two different RNA molecules. In this case, the RNA fragments are
mixed before being introduced into said cell, e.g. under conditions
allowing them to form a double-stranded RNA molecule. In another
embodiment, the RNA fragments are introduced into said cell
sequentially. Preferably, the time interval between the
introduction of each of the RNA molecules is short, preferably less
than one hour.
[0177] In still another embodiment, the RNA fragments are comprised
in one RNA molecule, By using one single RNA molecule, the two
complementary RNA fragments are in close proximity such that
pairing and double strand formation is favored. In such case, the
RNA molecule is preferably capable of folding such that said RNA
fragments comprised therein form a double-stranded region. In this
case, the complementary parts of the RNA fragments recognize one
another, pair with each other and form the double-stranded RNA
molecule. In another embodiment, the RNA fragments are incubated
under conditions allowing them to form a double-stranded RNA
molecule prior to introduction into the cell. In yet another
embodiment, the RNA molecule comprises a linker between the sense
RNA fragment and the antisense RNA fragment. The linker preferably
comprises a RNA sequence encoded by an expression cassette
comprising a functional gene, e.g. a selectable marker gene. In
another embodiment, the linker comprises a RNA sequence encoded by
regulatory sequences, which e.g. comprise intron processing
signals.
[0178] Another embodiment provides a dsRNA construct having a
promoter operably linked to said dsRNA and might further comprise
said dsRNA molecule. The promoter can be a heterologous promoter,
for example a tissue specific promoter, a developmentally regulated
promoter, a constitutive promoter, divergent or an inducible
promoter. Termination signal are also optionally included in the
DNA molecules.
[0179] The single RNA molecule or the two distinct RNA molecules
are preferably capable of forming a double-stranded region, in
which the complementary parts of the RNA fragments recognize one
another, pair with each other and form the double-stranded RNA
molecule.
[0180] Another embodiment provides the disclosed transgenic plant
material in the form of feedstock, pellets, granules, flakes and
the like. The inhibitory nucleic acids disclosed here can be in
seeds and seed products derived from the transgenic plants
described above. Another embodiment provides a composition
comprising the disclosed inhibitory nucleic acids that can be
coated on seeds. The coating can be formulated so that the
inhibitory nucleic acids remain able to inhibit nematode secretory
proteins as the seed matures and develops roots.
[0181] A further embodiment provides chimeric or fusion proteins
containing the disclosed nematode esophageal gland cell proteins or
fragments thereof. As used herein, a "chimeric protein" or "fusion
protein" includes a nematode esophageal gland cell protein or
fragment thereof linked to a foreign polypeptide. A "foreign
polypeptide" is polypeptide that is not substantially homologous to
a nematode esophageal gland cell protein or fragment thereof. The
foreign polypeptide can be fused to the N-terminus or C-terminus of
the nematode esophageal gland cell protein or fragment thereof.
[0182] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a GST
fusion protein in which a nematode esophageal gland cell protein or
fragment thereof is fused to the C-terminus of GST. Such fusion
proteins can facilitate the purification of the polypeptide.
Alternatively, the fusion protein can contain a heterologous signal
sequence at its N-terminus. In certain host cells, expression,
secretion or transport of a protein can be increased through use of
a heterologous signal sequence. For example, in a plant cell, a
polypeptide of the invention may be fused with a chloroplast
transit peptide. The chloroplast transit peptide allows the
polypeptide to be transported from the cytoplasm of the plant cell
into the chloroplast, thereby increase root growth. Expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST polypeptide). A nucleic acid encoding a
nematode esophageal gland cell protein or fragment thereof can be
cloned into such an expression vector so that the fusion moiety is
linked in-frame to the polypeptide.
[0183] The following are only exemplary examples. It should be
understood that the invention is not limited to these examples.
Other important applications of disclosure would be readily
recognized by those of ordinary skills in the art. Other uses which
are potentially recognizable by those of ordinary skills in the art
are also part of the disclosure.
[0184] The references mentioned herein are incorporated in their
entirety to the fullest extent permitted by applicable law.
EXAMPLES
Example 1
Nematodes and Plants
[0185] Meloidogyne species were propagated on roots of
greenhouse-grown tomato (Lycopersicon esculentum cv. Marion or
Better-Boy). Meloidogyne eggs were collected as described (Hussey
and Barker, 1973). Pre-parasitic second-stage juveniles (pre-J2)
were collected via hatching eggs on 25-.mu.m-pore sieves in
deionized water in plastic bowls. The different parasitic stages of
M. incognita were collected by root blending and sieving (Ding et
al. 1998). Mixed parasitic stages (MS) of M. incognita for in situ
hybridizations were collected 13-15 days after inoculation of eggs
as described in De Boer et al. (1998). Similarly, pre-J2 and MS of
Heterogera glycines were collected from infected soybean (Glycine
max) roots. Caenorhabditis elegans was cultured on OP50 of E. coli
(Brenner, 1974). One-month-old host plant leaves were collected
from growth-chamber grown Nicotiana tabacum cv. Petite Havana SR1,
and Arabidopsis thaliana ecotype Col-0.
Example 2
Nucleic Acid Manipulation
[0186] Pre-J2 of packed nematodes were frozen in 1.5-ml
microcentrifuge tubes with liquid nitrogen and ground with a
smooth-end metal bar. The frozen nematode fragments were mixed with
0.5 ml extraction solution (100 mM NaCl, 100 mM Tris-HCl [pH8.0],
50 mM EDTA, 1% sodium dodecyl sulfate, 4 mg/ml proteinase K and 10
.mu.g/ml RNase) and incubated at 37.degree. C. for 1 hr. DNA was
extracted with phenol/chloroform and then precipitated with
isopropanol (Sambrook et al., 1989). The DNA was re-suspended in
H.sub.2O. Tobacco and Arabidopsis genomic DNA was extracted using
standard techniques (Dellaporta 1993).
[0187] mRNAs were extracted and purified from ground plant tissues
using Dynabeads mRNA DIRECT kit (Dynal, Lake Success, N.Y.), eluted
with 10 .mu.l diethylpyrocarbonate (DEPC)-treated water, and
converted into first-strand cDNA by reverse transcription (RT)-PCR
SMART PCR cDNA Synthesis kit (BD Biosciences, Palo Alto, Calif.),
following the manufacturer's instructions. RT-PCR reactions
contained the following components; 4.0 .mu.l of 5.times.
first-strand buffer, 2.0 .mu.l of 20 mM DTT, 2.0 .mu.l of 10 mM
50.times. dNTP, 1 .mu.l of 10 .mu.M 3'-CDS primer, 10 .mu.l of
isolated mRNA and 1 .mu.l of Superscript II reverse transcriptase
(200 units/.mu.l, Gibco BRL, Rockville, Md.). The reaction was
incubated at 42.degree. C. for 1 hr.
Example 3
Isolation of 16D10 cDNA Clone
[0188] Clone 16D10 encoding a secretory signaling peptide was
identified during random sequencing of a gland-cell specific cDNA
library of M. incognita (Huang et al., 2003) and designated as
16D10. The full-length double-strand cDNA sequences of 16D10 in
pGEM-T Easy vector were obtained by using T7 and SP6 primers in
sequencing reactions. The longest open reading frame of the 16D10
cDNA (364 bp) encoded a deduced protein of 43 aa including a 30 aa
N-terminal hydrophobic signal peptide as predicted by Signal P
(Nielsen et al., 1997). While the mature 16D10 peptide of 13 aa
(GKKPSGPNPGGNN, M.sub.r 1,223 Da) (SEQ ID NO:52) provided no
significant BLASTX similarity, it did contain 8 aa (K---PSGPNP--N)
(SEQ ID NO:53) of the conserved C-terminal 13 aa motif
(KRLVPSGPNPLHN) (SEQ ID NO:54) of the functional domain of
Arabidopsis CLV3-like proteins (Cock and McCormick, 2001) as well
as a cAMP/cGMP-dependent protein kinase phosphorylation site [KKpS]
as predicted by PROSITE (Hofmann et al, 1999).
Example 4
Genomic Clones in Meloidogyne Species
[0189] One pair of the gene-specific primers 16D10GF
(5'-GAGAAAATAAAATATAAATTATTCCTC-3') (SEQ ID NO:55) and 16D10GR
(5'-CAGATATAATTTTATTCAG-3') (SEQ ID NO:56) designed from the most
extreme 5'- and 3'-ends of the cDNA sequence of M. incognita 16D10,
were used to amplify the corresponding genomic sequences (or the
highest homologues) from 200 ng of M. incognita, M. javanica, M.
arenaria and M. hapla genomic DNA. The PCR products were cut from a
1.2% agarose gel, and purified with a QIAquick gel extraction kit
(Qiagen, Valencia, Calif.). The purified products were cloned into
pGEM-T Easy vector (Promega, Madison, Wis.) for sequencing. The
16D10 homologues from the Meloidogyne species shared over 95%
identity at the nucleotide level and the deduced proteins encoded
by putative cDNAs were identical to that of M. incognita 16D10.
Example 5
Southern Blot Analysis
[0190] For each sample, 10 .mu.g of genomic DNA was completely
digested with 50 units of EcoRI or BamHI (New England Biolabs,
Beverly, Mass.), separated on a 0.7% (w/v) agarose gel, transferred
onto a Hybond-N Nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) and blotting using a standard protocol (Sambrook
et al., 1989). 16D10 probe was generated by amplification of the
corresponding full-length cDNA from insert in pGEM-T Easy vector
with T7 and SP6 primers. Gel-purified PCR products were labeled by
PCR with a PCR-DIG probe synthesis system (Roche Applied Science,
Indianapolis, Ind.). About 15 ng of DIG-labeled probe per ml was
used for each-hybridization. Hybridizations were performed in DIG
Easy Hyb solution (Roche Applied Science, Indianapolis, Ind.) at
40.degree. C. for 16 h followed by two 5-min washes in
2.times.SSC/0.1% SDS solution at RT. The membranes were then washed
twice at 68.degree. C. with 0.5.times.SSC/0.1% SDS solution for 30
min. After incubating the membrane in 1% blocking reagent for 1 hr,
the membranes were incubated with a 1:10,000 dilution of sheep
anti-DIG alkaline phosphatase (AP) conjugate for 30 min. Unbound
antibody was removed by two 15-min washes with maleic acid washing
buffer (100 mM maleic acid, 150 mM NaCl, pH7.5, and 0.3% Tween 20).
The membrane was incubated in AP detection buffer (100 mM Tris-HCl,
pH9.5, 100 mM NaCl, and 50 mM MgCl.sub.2) for 10 min followed by a
1:50 dilution of the chemiluminescent substrate disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1.sup.3,7]-
decan}-4-yl)pheryl phosphate (CSPD) (Roche Applied Science) before
sealing the membrane in two sheets of transparency film and
exposing it to X-ray film for 1.5 hr. A blot containing genomic DNA
from M. incognita, M. javanica, M. arenaria and M. hapla hybridized
with a 16D10 cDNA probe showed that 16D10 was present in each of
the four agriculturally important Meloidogyne species with 3-4
copies or homologues (FIG. 6). No hybridization was detected with
genomic DNAs from the soybean cyst nematode H. glycines, the
non-parasitic free-living nematode Caenorhabditis elegans, and
plants (tobacco and Arabidopsis).
Example 6
Sequence Analyses
[0191] Sequence similarity searches were carried out using the
BLAST programs PSI-BLASTP and BLASTX at the National Center for
Biotechnology Information (NCBI) (Altschul et al., 1998). Multiple
sequence alignments of Meloidogyne 16D10 genomic DNA sequences were
generated using ClustalW1.8 (Jeanmougin et al., 1994). Prediction
of a signal peptide for secretion and the cleavage site was
performed via the SignalP program (Nielsen et al., 1997).
Example 7
In Situ Hybridization
[0192] Specific forward and reverse primers for 16D10 cDNA clone
were used to synthesize digoxigenin (DIG)-labeled sense and
antisense cDNA probes (Roche Applied Science, Indianapolis, Ind.)
by asymmetric PCR (Huang et al., 2003). In situ hybridization was
performed using formalin-fixed, permeabilized pre-parasitic
juveniles and mixed parasitic stages of M. incognita (De Boer et
al., 1998; Huang et al., 2003). cDNA probes that hybridized within
the nematode were detected with alkaline phosphatase-conjugated
anti-DIG antibody and substrate, and specimens were observed with a
compound light microscope (De Boer et al., 1998). In situ mRNA
hybridization revealed that 16D10 was strongly expressed in the two
subventral esophageal gland cells of M. incognita at the early
parasitic stages.
Example 8
Immunofluorescence Assay
[0193] The purified 16D10 polyclonal antiserum was used to localize
16D10 expression in sections of pre-parasitic J2, mixed parasitic
stages of M. incognita with indirect immunofluorescence as
described previously by Goverse et al. (1994). Following fixation
in freshly prepared 2% paraformaldehyde in PBS buffer (80 mM
Na.sub.2HPO.sub.4, 20 mM NaH.sub.2PO.sub.4, 100 mM NaCl, pH7.4) for
5 days at 4.degree. C., the nematodes were washed three times in
PBS buffer and once in deionized water. The fixed nematodes were
cut into sections and incubated in 0.6 mg of proteinase K (Roche
Applied Science, Indianapolis, Ind.) per ml in phosphate buffered
saline (PBS) buffer at 37.degree. C. for 1 hr. After washed once
with PBS, the partially digested nematodes were placed in a
-80.degree. C. freezer for 20 min, incubated in dry-ice cold
methanol for 3 min, and then incubated in dry-ice cold acetone for
15 min. The nematodes were washed once with blocking solution (10%
goat serum, 0.02% NaN.sub.3, 1 mM phenylmethylsulfonyl fluoride,
1.times.PBS) amended with protease inhibitors as previously
described (Goverse et al., 1994), incubated at 4.degree. C. for 3
days and then used immediately for immunofluorescence. The blocked
nematodes were aliquoted to wells of a 96-well MultiScreen plate
(Millipore, Bedford, Mass.), and agitated in a 1:250 dilution of
the 16D10 purified polyclonal antibody in ELISA diluent (0.05%
Tween, 0.02% NaN.sub.3, 1% BSA, 1.times.PBS) in a moisture chamber
overnight at RT. Nematode sections were washed three times for 5
min each with PBST (1.times.PBS, 0.5% Triton X-100) and agitated in
a 1:1000 dilution of fluorescein isothiocyanate (FITC)-conjugated
goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, Mo.) in
Tris-Saline-BSA (0.15M NaCl, 0.01M Tris, pH7.2, 0.2% Triton X-100,
3% BSA) in the dark for 3 h at RT. Sections were washed twice in
PBST and once with distilled water. Treated sections were
transferred in a 15-.mu.l drop of water to individual wells on
Multitest slides (ICN-Flow, Horsham, Pa.) that previously coated
with 5 .mu.l of 0.1% poly-L-lysine (Sigma Chemical). Sections were
airy dried on slides, covered with a 3 .mu.l drop of antiquenching
agent (0.02 mg/ml phenylenediamine in 500 mM carbonate buffer,
pH8.6, mixed with nonfluorescent glycerol), and a coverslip was
applied. Specimens were observed on an Olympus fluorescence
microscope. Negative control consisted of pre-immune rabbit serum.
The purified 16D10 antiserum bound to secretory granules within the
subventral gland cells of pre-parasitic and parasitic J2 and their
cytoplasmic extensions and expanded ampullae, which are located
posterior to the pump chamber at the metacarpus. No specific
labeling with the rabbit preimmune serum was observed in any
nematode specimens.
Example 9
Protein Extraction
[0194] Nematode proteins were extracted by grinding pre-parasitic
J2 and mixed parasitic stages of M. incognita and H. glycines in
200 .mu.l of extraction buffer [100 mM Tris-HCl, pH7.0, 150 mM NaCl
and 1.times. complete protease inhibitors (Roche Applied Science,
Indianapolis, Ind.)] in microcentrifuge tubes in liquid nitrogen.
Plant proteins (0.5 g) were extracted by grinding transgenic
seedlings or root tissues in 200 .mu.l of extraction buffer [50 mM
Tris-HCl, pH7.0, 150 mM NaCl, 1.times. complete protease inhibitors
(Roche Applied Science)] in microcentrifuge tubes in liquid
nitrogen. Supernatant was recovered from homogenates after
centrifugation at 13,000 rpm for 10 min. All protein concentrations
were estimated (with a Bio-Rad Protein Assay Kit II) with BSA as a
standard. As the positive control, the 16D10 peptide
(GKKPSGPNPGGNN, >95% purity) (SEQ ID NO:52) was synthesized from
Sigma-Genosys, TX for immunodetection assays (see examples
12-13).
Example 10
Collection of Stylet Secretions
[0195] Stylet secretions from M. incognita J2 were produced and
collected in vitro as described by Davis et al. (1994).
Pre-parasitic J2 were incubated in 0.4% resorcinol (Sigma-Aldrich,
St. Louis, Mo.) for 6 hr at room temperature in a humid chamber.
Stylet secretions were solubilized via adding an equal volume of
0.1M Tris-NaOH, pH11.0. Solubilized stylet secretions were
concentrated with StrataClean (Stratagene, La Jolla, Calif.).
Briefly, soluble secretory proteins were trapped via suspending 1.5
ml of beads in the supernatant of induction mixture (460 ml) and
incubating it for 1 hr under constant mixing. The beads were
centrifuged, re-suspended in 2.times.SDS-PAGE sample buffer, and
boiled for 3 min to release the absorbed proteins. The concentrated
stylet secretions were used in enzyme-linked immunosorbent assay
(ELISA) and immunoblotting analyses using the purified 16D10
antiserum (see Examples 11-13). Both assays identified 16D10
peptide in the stylet secretions as well as total extracts of J2
and mixed parasitic stages of M. incognita.
Example 11
Production of Antisera
[0196] Polyclonal antiserum to 16D10 was produced by immunizing two
rabbits with a synthetic mature (i.e., without the N-terminal
signal peptide) 16D10 peptide (GKKPSGPNPGGNN) (SEQ ID NO:52) from
Eurogentec, Inc. (Herstal, Belgium). The antiserum was
affinity-purified from 15 ml of last crude sera with the peptide
antigen. Peptide affinity-purified 16D10 polyclonal antiserum was
used to localize 16D10 expression in specimens of M. incognita
using immunofluorescence microscopy (Goverse et al, 1994), and for
immunodetection of 16D10 in stylet secretions and transgenic
plant-expressed or in vitro translated 16D10.
Example 12
Western Dot-Blot Analysis
[0197] Protein samples (2 .mu.l) were spotted onto Hybond ECL
nitrocellulose. The nitrocellulose membrane was allowed to air dry
for 20 min. The membrane was incubated in a blocking solution (2%
nonfat dry milk, 1.times. Tris-buffered-saline-Tween [TBS-T: 20 mM
Tris-HCl, pH7.4, 0.8% NaCl, 0.1% Tween 20] overnight at 37.degree.
C. and then treated with the purified 16D10 polyclonal antiserum
(1:2,000), followed by anti-rabbit IgG (whole molecule) alkaline
phosphatase conjugate (1:30,000) (Sigma). The membrane was washed
three times in 1.times.TBS-T buffer at room temperature, and
incubated in the substrate solution (45 .mu.l of nitroblue
tetrazolium [NBT] solution and 35 .mu.l of
5-bromo-4-chloro-3-indolyl-phosphate toluidinium [BCIP] solution in
10 ml of AP buffer [100 mM Tris-HCl, pH9.5, 100 mM NaCl, 5 mM
MgCl]) at room temperature until color develops.
Example 13
ELISA Assay
[0198] ELISA was modified from Pratt et al. (1986). Dynatech
Immulon plate wells were coated overnight at 4.degree. C. with
proteins diluted in borate saline (0.2M sodium borate, 75 mM NaCl,
pH8.5) from the following sources: 2 .mu.l of 1000.times.
concentrated stylet secretions of M. incognita J2, 10 .mu.g of
total extracted proteins of pre-J2 of M. incognita, MS of M.
incognita, pre-J2 and MS of H. glycines, or 10 .mu.g of BSA (Sigma
Chemical) as a negative control. As a positive control, wells were
coated with 100 ng of synthetic 16D10 peptide (>95% purity,
Sigma-Genosys, TX). Wells were rinsed three times with wash buffers
(10 mM Tris.HCl, pH8.0, 0.5M NaCl) and blocked with 1% BSA in PBS
(32.9 mM Na.sub.2HPO.sub.4, 1.77 mM NaH.sub.2PO.sub.4, 0.14M NaCl,
pH7.4) for 30 min at room temperature. After being rinsed once with
wash buffer, each coated well was incubated with 16D10 purified
polyclonal antisera diluted 1:1,000 with 0.5% BSA in PBS for 1 hr
at room temperature. Negative controls included omitting incubation
with the primary polyclonal antibody, and incubation with the
rabbit pre-immune serum. The wells were washed three times,
incubated with alkaline phosphatase-conjugated goat anti-rabbit
antibody (Sigma Chemical) at 1:5,000 dilution for 1 hr at room
temperature, washed three times before phosphate calorimetric
substrate was added. The substrate, p-nitrophenyl phosphate was
prepared according to manufacturer's directions in alkaline
phosphatase buffer (1M diethanolamine, 0.5 mM MgCl.sub.2, pH 9.8)
and incubated in the treated wells 30 min at room temperature
before the reaction was stopped with 3 N NaOH. Absorbance was
measured at 405 nm and 490 nm on an ELISA reader.
Example 14
Plasmid Construction
[0199] The coding regions of 16D10 with or without a signal peptide
sequence were amplified from the full-length cDNA clone with
primers 16D10SF (5'-CGGGGTACCTAGATGTTTACTAATTCAATTAA-3') (SEQ ID
NO:57) or 16D10F (5'-CGGGGTACCTAGATGGGCAAAAAGCCTAGTG-3') (SEQ ID
NO:58) and 16D10R (5'-GCTCTAGATCAATTATTTCCTCCAGG-3') (SEQ ID NO:59)
that introduced KpnI or XbaI restriction sites (underlined) and the
stop/start codons (in italics), cloned into the KpnI and XbaI sites
of binary vector pBIX under the control of CaMV 35S promoter to
generate pBIX(16D10S) and pBIX(16D10), respectively, and confirmed
by sequencing. pBIX was derived from pBI101 (BD Biosciences, Palo
Alto, Calif.) and contains a nos promoter-nptII-nos terminator
cassette, a 35S promoter-gusA-nos terminator, and a second 35S
promoter with a polylinker having KpnI and XbaI sites. The hybrid
expressed sequence of clv3 and 16D10 was generated by PCR
amplifications from Arabidopsis genomic DNA using primers C3K
(5'-GGGGTACCATGGATTCTAAAAGCTTTG-3') (SEQ ID NO:60) that introduced
KpnI restriction site (underlined) and C3R
(5'-CCACTAGGCTTTTTGCCAAGGAACAAGAAGCAG-3') (SEQ ID NO:61) for signal
sequence, and from 16D10 cDNA using primers C3F
(5'-CTTCTGCTTCTTGTTCCTTGGCAAAAAGCCTAGTGG-3') (SEQ ID NO:62) and
16D10X (5'-GCTCTAGATCAATTATTTCCTCCAGG-3') (SEQ ID NO:63) that
introduced XbaI restriction site (underlined) for mature peptide
coding sequence using Vent polymerase (New England Biolabs,
Beverly, Mass.). The two products were then used to prime each
other in a fusion PCR reaction. The resulting fragment was cloned
into pBIX to generate pBIX(C35-16D10) and verified by
sequencing.
Example 15
Tobacco Hairy-Root Transformation
[0200] The plasmids pBIX(16D10), pBIX(16D10S) and the empty vector
pBIX as a control were transferred into Agrobacterium rhizogenes
ATCC 15834 by electroporation (Shen and Forde, 1989) and
transformed into tobacco (Nicotiana tabacum cv Petite Havana SR1)
using the A. rhyzogenes-mediated cotyledon transformation
(Christey, 1997). Transformed hairy roots were generated from
inoculated tobacco cotyledons on Gamborg's B-5 plates containing
0.8% Noble agar with 100 mg/L kanamycin and timentins (230.8 mg/L
ticarcillin disodium plus 7.69 mg/L clavulanate potassium).
Individual hairy root tips (about 0.5 cm) were cultured for 3 weeks
at 24.degree. C. in the dark, and 2 to 3 roots from individual
hairy root system were subjected to GUS-staining (Jefferson et al.,
1987). The kanamycin-resistant and GUS-positive root lines with no
bacterial contamination, confirmed by PCR analyses, were used to
establish hairy root lines. The root-tips were sub-cultured for
root growth assay on Gamborg's B-5 plates without hormones every 2
weeks and the cut roots were kept in culture on the old plates at
24.degree. C. in the dark for assays. For root-growth assays,
plates were cultured horizontally in the dark and 5 hairy roots
from each transgenic line in each of the three repeats were
investigated. Relative RT-PCR and immunoblotting analyses of
transgenic hairy roots or calli with a single transgenic copy
identified as described (Does et al, 1991) were carried out using
the same procedures as in those of transgenic Arabidopsis.
Expression of 16D10 in the cytoplasm of hairy root cells increased
root growth at the rate of approximately 65% [mean root length
after 2 weeks of 5.20.+-.0.61 cm (n=90) in 16D10 transgenic lines,
compared to 3.15.+-.0.34 cm (n=90) in control lines], generated
extensive lateral roots and led to the formation of calli where
roots were cut for subculturing at 5 weeks. RT-PCR analysis of
16D10 expression showed that the steady-state mRNA levels in calli
were higher than in the hairy roots. Immunoblotting analysis with
the purified 16D10 antiserum revealed that 16D10 was produced in
both hairy roots and calli. No expression of 16D10 was detected in
the control vector-transformed hairy roots.
Example 16
Arabidopsis Floral-Dip Transformation
[0201] The plasmids pBIX(16D10), pBIX(C3S-16D10) and the empty
vector pBIX as a control were introduced into Agrobacterium
tumefaciens C58C1 by electroporation (Shen and Forde, 1989) and
transformed into A. thaliana wild-type Col-0 plants by the floral
dip method (Clough and Bent, 1998). Segregation of kanamycin
resistance, GUS-straining (Jefferson et al., 1987), and 16D10
expression coupled to PCR analyses confirmed generation of the
transgenic homogenous T.sub.2 lines. Inverse PCR (Does et al.,
1991) identified the homogenous lines with a single transgenic copy
in the genome for molecular and root growth assays. Thirty plants
from each transgenic line in each of the three repeats were in
vitro cultured on MS plates with 3% sucrose with 16 h light
(24.degree. C.)/8 h dark (20.degree. C.) cycles and the plates were
kept vertically for root growth assay.
[0202] Four transgenic Arabidopsis T.sub.2 homozygous lines
containing a single-copy of 16D10 without a signal peptide under
the control of the 35S promoter were generated. Two transgenic
lines originating from the blank transformation vector were also
generated as controls. RT-PCR and immunoblotting analyses confirmed
that 16D10 was expressed in all of the 16D10 transgenic lines, but
not in the control lines. Compared to controls, expression of 16D10
in the cytoplasm of Arabidopsis cells increased the length of
primary roots 85% [mean 54.01.+-.8.75 mm in four 16D10 transgenic
lines (n=90/line), and 29.20.+-.4.50 mm in 2 control lines
(n=90/line)] and the number of lateral branches and adventitious
roots increased 1.4-fold and 2.08-fold, respectively (FIG. 2 and
FIG. 3). Increased primary root growth was closely correlated with
increased lateral root number and increased adventitious root
number. Measurements of the root tip growth rate over 3 days
revealed an increase (20%) in length only in the meristematic zone
of 16D10 roots, indicating increase in cell number and not cell
size contributed to the enhanced root growth.
Example 17
Complementation Tests
[0203] Since the mature 16D10 peptide of 13 aa (GKKPSGPNPGGNN) (SEQ
ID NO:52) contained 8 aa (K---PSGPNP--N) (SEQ ID NO:53) of the
conserved C-terminal 13 aa motif (KRLVPSGPNPLHN) (SEQ ID NO:54) of
the functional domain of Arabidopsis CLV3-like proteins (Cock and
McCormick, 2001), the plasmid pBIX(Clv3S-16D10) encoding M.
incognita 16D10 with A. thaliana CLAVATA3 signal peptide was
transferred into the A. thaliana clv3 mutants clv3-1
(intermediate), clv3-2 and clv3-6 via A. tumefaciens C58C1-mediated
floral-dip transformation (Clough and Bent, 1998) for functional
complementation tests. As controls, the plasmids pBIX(16D10) and
pBIX were also introduced into the clv3 mutants. Three transgenic
T.sub.2 homozygous lines for each construct were also generated.
The phenotypes (flower and shoot apical meristem) of
16D10-transformed clv3 lines were investigated and compared with
those of vector-transformed lines, A. thaliana wild-type ecotype
Col-0 and the clv3 mutant progeny as described in Fletcher et al.
(1999). While 16D10 contained the functional domain of Arabidopsis
CLV3-like proteins, expression of 16D10 in the apoplast or
cytoplasm of Arabidopsis clv3 mutants did not restore wild type
phenotype, indicating 16D10 does not function as CLV3-like
proteins.
Example 18
Histological Analysis
[0204] Primary root tissues of A. thaliana were fixed and
dehydrated (Dolan et al, 1993), and embedded in Spurrs resin using
Low Viscosity Embedding kit (Electron Microscopy Sciences,
Hatfield, Pa.) according to the manufacturers instructions. Thin
sections (0.4 .mu.M) were made on a Reichert-Jung Ultracut E and
stained with 1% toluidine blue. Transverse root sections in and
above the root meristem and longitudinal sections at the root-tip
revealed that the average cell-size and number of cell types and
cell-layer did not differ in the transgenic lines, compared to wild
type. Root morphology was also not altered in our transgenic
plants, and increased growth was accompanied by accelerated
development of the root system. Thus ectopic 16D10 expression
enhanced root growth rate and induced lateral root initiation,
possibly by stimulation of cell division in meristems, increasing
the rate of cell production without altering meristem
organization.
Example 19
Relative RT-PCR
[0205] Reverse transcription (RT)-PCR was conducted on mRNA
extracted from equivalent amounts of plant tissue. The 16D10
gene-specific primers 16D10F and 16D10R as described above were
used in subsequent PCR amplifications. In controls, the primers
UBQ1 (5'-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3') (SEQ ID NO:64) and
UBQ2 (5'-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3') (SEQ ID NO:65)
designed from the uniformly expressed UBQ10 gene (GenBank accession
no. NM.sub.--202787) of A. thaliana wild-type ecotype Col-0, were
used to amplify a 483 bp unique sequence of UBQ10 from transgenic
Arabidopsis lines. The primers ActF (5'-CCGGTCGTGGTCTTACTGAT-3')
(SEQ ID NO:66) and ActR (5'-GCACCGATTGTGATGACTTG-3') (SEQ ID NO:67)
designed from the uniformly expressed actin gene (GenBank accession
no. U60494) of N. tabacum cv Petite Havana SR1 were used to amplify
a 271 bp unique sequence of the tobacco actin (Tob104) gene from
transgenic tobacco hairy roots. PCRs containing the following
components: 5 .mu.l of 10.times.BD Advantage 2 PCR buffer, 1.0
.mu.l of 10 mM dNTP mix, 1.5 .mu.l of 5' primer, 1.5 .mu.l of 3'
primer, 2 .mu.l of cDNA, 38 .mu.l of water, and 1.0 .mu.l of
50.times.BD Advantage 2 Polymerase Mix (BD Biosciences, Palo Alto,
Calif.). PCR cycles consisted of an initial denaturation step at
94.degree. C. for 2 min, followed by 35 cycles of 94.degree. C. for
1 min, 55.degree. C. for 30 seconds, 72.degree. C. for 40 seconds,
and a final 10-min elongation step at 72.degree. C. Ten-microliter
aliquots of each RT-PCR reaction were electrophoresed on a 2%
agarose gel, transferred to nylon membranes, and hybridized with
corresponding DIG-labeled DNA probes. RT-PCR analysis revealed that
16D10 transcripts were steadily present in the 16D10 transgenic
tobacco hairy roots and Arabidopsis lines, but absent in the
vector-transformed control lines.
Example 20
Yeast Two-Hybrid Screens
[0206] The MATCHMAKER yeast two-hybrid system II (BD Biosciences,
Palo Alto, Calif.) was used in the yeast two-hybrid screening. The
cDNA encoding the mature peptide of 16D10 was cloned in frame into
the GAL4-binding domain (BD) of pGBKT7 to generate pGBKT7(16D10)
and expressed as bait to screen a tomato root cDNA library
constructed from mRNA from tomato root tissues in the GAL4
activation domain (AD) of pGADT7. Twelve full-length SCL-encoding
cDNAs (AtSCL1, AtSCL3, AtSCL5, AtSCL6, AtSCL9, AtSCL13, AtSCL14,
AtSCL21, AtSCR, AtSHR, AtRGA, AtGAI) were amplified from a root
cDNA pool made from mRNA from A. thaliana root tissues with
specific primers of each gene based on the corresponding sequences
in GenBank databases (Bolle, 2004), and cloned in frame into
pGADT7. Each of the constructs was introduced with pGBKT7(16D10)
into the yeast strain AH109. cDNAs encoding the specific regions of
AtSCL6 and AtSCL21 were cloned into pGADT7, and then co-transformed
with pGBKT7(16D10) into the strain AH109. All procedures including
cDNA library screening, selection of positive clones and the assay
of .beta.-galactosidase activity, were performed by following the
protocol of MATCHMAKER yeast two-hybrid system II (BD Biosciences,
Palo Alto, Calif.). Two Arabidopsis SCL proteins, AtSCL6 and
AtSCL21, interacted with 16D10 in yeast. Domain analysis revealed
the specific interaction of 16D10 with the SAW domain of AtSCL6 and
AtSCL21, and no interaction of 16D10 with the rest of the domains
of the SCL proteins, and indicated that the SCL transcription
factor(s) was a putative target of the secreted 16D10 during RKN
parasitism of plants.
Example 21
RNAi by Soaking
[0207] Forty-two bp and 271 bp sequences of 16D10 were respectively
amplified from the full-length cDNA clone using the primers
16D10T7F1 (5'-TAATACGACTCACTATAGGGCCTCAAAAATACCATAAAG-3') (SEQ ID
NO:68) and 16D10T7R1
(5'-TAATACGACTCACTATAGGGGAAATTAACAAAGGAAACC-3') (SEQ ID NO:69), and
16D10T7F2 (5'-TAATACGACTCACTATAGGGGGCAAAAAGCCTAGTGGGC-3) (SEQ ID
NO:70) and 16D10T7R2 (5'-TAATACGACTCACTATAGGGTCAATTATTTCCTCCAGG-3')
(SEQ ID NO:71) each of that incorporates the RNA primer site T7
(underlined). The gel-purified PCR products were used as templates
for synthesis of sense and antisense 16D10 RNAs in a single
reaction in vitro using the MEGAscript RNAi kit (Ambion, Austin,
Tex.) according to manufacturer's instructions, except that the
reactions were incubated for 16 hr to increase RNA yield. The
amount and quality of generated double-strand (ds) RNA were
estimated and quantitated by standard procedures (Sambrook et al.,
1989). The dsRNA products were ethanol precipitated and
re-suspended in nuclease-free water to 10-15 .mu.g/.mu.l.
[0208] Approximately 10,000 freshly hatched J2s of M. incognita
were soaked in 1/4 M9 buffer (10.9 mM Na.sub.2HPO.sub.4, 5 mM
KH.sub.2PO.sub.4, 4.7 mM NH.sub.4Cl, and 2.2 mM NaCl) containing 1
mg/ml of 16D10 dsRNA, 1% resorcinol, 0.13 mg/ml FITC isomer I,
0.05% gelatin and 3 mM spermidine, and incubated for 4 hr in the
dark at RT on a rotator. Resorcinol (Res) was used to help
stimulate uptake of the dsRNA. Control samples were incubated in
the same solution but without resorcinol or dsRNA. After soaking,
nematodes were thoroughly washed five times with nuclease-free
water by centrifugation and about 100% of treated nematodes were
observed with an Olympus fluorescence microscope to take up FITC, a
marker for uptake of dsRNA. The FITC-labeled transgenic J2 were
assayed to determine silencing of the 16D10 transcripts by relative
RT-PCR analysis, using first-strand cDNAs synthesized from mRNA of
equivalent number of treated J2 as templates and a 284 bp amplified
fragment of the M. incognita constitutively expressed actin gene
(GenBank accession no. BE225475) as a control. The ingestion of
short or full-length 16D10 dsRNA by second-stage juveniles of
root-knot nematode caused a significant reduction of 16D10
transcripts in the treated nematodes (FIG. 4), providing direct
evidence for in vivo targeting of 16D10 in root-knot nematodes by
RNAi.
[0209] J2s of M. incognita were also soaked as described above with
1 mg/ml of dsRNA specific for 8H11 (SEQ ID NO:17) or 31H06 (SEQ ID
NO:33). Relative RT-PCR analysis revealed that ingestion of 8H11
and 31H06 dsRNA by second-stage juveniles of root-knot nematode
caused a significant reduction of transcripts of these two
additional parasitism genes in the treated nematodes (FIG. 5).
Example 22
In Planta Delivery of RNAi
[0210] The sense and anti-sense cDNAs (42 bp or 271 bp) of 16D10
were amplified from the full-length cDNA clone with the
gene-specific primers 16D10Xho1
(5'-CCGCTCGAGGGCAAAAAGCCTAGTGGGC-3') (SEQ ID NO:72) and 16D10 Kpn1
(5'-CGGGGTACCTCAATTATTTCCTCCAGG-3') (SEQ ID NO:73), 16D10Cla1
(5'-CCATCGATTCAATTATTTCCTCCAGG-3') (SEQ ID NO:74) and 16D10Xba1
(5'-GCTCTAGAGGCAAAAAGCCTAGTGGGC-3') (SEQ ID NO:75), 16D10Xho3
(5'-CCGCTCGAGCCTCAAAAATACCATAAAG-3'(SEQ ID NO:76) and 16D10Kpn2
(5'-CGGGGTACCGAAATTAACAAAGGAAACC-3') (SEQ ID NO:77), 16D10Cla2
(5'-CCATCGATGAAATTAACAAAGGAAACC-3') (SEQ ID NO:78) and 16D10Xba3
(5'GCTCTAGACCTCAAAAATACCATAAAG-3') (SEQ ID NO:79) that introduced
XhoI, KpnI, ClaI or XbaI restriction sites (underlined),
respectively. The PCR products were gel-purified, and digested with
the restriction enzymes XhoI and KpnI, or ClaI and XbaI,
respectively. The digested-PCR products were cloned into the
Xho-KpnI sites, and the ClaI-XbaI sites of pHANNIBAL to generate
pHANNIBAL(16D10#1) and pHANNIBAL(16D10#2), respectively. The sense
and antisense 16D10 cDNAs of pHANNIBAL-derived plasmids were
subcloned as NotI fragments into the binary vector pART27 (Gleave,
1992) to produce highly effective intron-containing "hairpin" RNA
(ihpRNA) silencing constructs (Wesley et al., 2001). The
pART27-derived constructs were electroporation transformed into A.
tumefaciens C58C1. The transformants were selected on LB media
containing rifampicin (50 mg/L), gentamycin (25 mg/L) and
spectinomycin (100 mg/L), and then introduced into A. thaliana
ecotype Col-0 via floral-dip transformation as described above.
Transgenic homologous T2 lines constitutively transcribing the
specific ihpRNA of 16D10 under the CaMV35S promoter were generated
for resistance assays to the root-knot nematodes, Meloidogyne
species.
Example 23
Resistance Assays
[0211] Seeds from the A. thaliana transgenic lines generated from
transformation of pART27-derived constructs were surface sterilized
in 70% (v/v) ethanol for 1 min and 3% (v/v) sodium hypochloride for
5 min, and then rinsed 5 times in sterile distilled water. The
sterilized seeds were geminated and grown on Gamborg's B-5 medium
for 3 weeks. M. incognita eggs were sterilized and then inoculated
about 500 eggs for each plant near to the roots as described
(Sijmons et al., 1991). The number and size of galls on the
infected roots were analyzed after inoculation of 3 weeks, and the
infected roots were stained red with acid fuschin as described
(Hwang et al., 2000) and assayed by the number of eggs per gram of
roots after inoculation of 8 weeks. Transgenic Arabidopsis lines
expressing 16D10 dsRNA were resistant to the four major Meloidogyne
species--M. incognita, M. javanica, M. arenaria, and M. hapla. Root
galling assay showed a 63-90% reduction in the number (and size) of
galls on the 16D10 dsRNA transgenic Arabidopsis lines, compared to
galls on the vector-transformed line (FIGS. 1A, 1B and Table 1).
Nematode reproduction assay revealed a 70-97% reduction in the
number of RKN eggs per gram root in the 16D10 dsRNA transgenic
lines when compared to the control plants (FIG. 7).
TABLE-US-00001 TABLE 1 Gall production on transgenic A. thaliana
expressing 16D10 dsRNA and inoculated with four Meloidogyne species
(M. incognita, M. javanica, M. arenaria, M. hapla) compared with
control plants. Galling No. CK 16D10i-1 16D10i-2 (Mean value) T L M
S T L M S T L M S M. incognita 13.50 4.83 7.67 1.00 1.50 0 0.25
1.25 3.40 0 0.80 2.60 M. javanica 14.38 7.41 5.47 1.50 3.29 0.14
1.00 2.15 2.50 0.13 1.25 1.12 M. araneria 11.75 6.75 3.25 1.75 3.00
0.17 1.50 1.33 3.50 0.25 0.75 2.50 M hapla 10.21 3.46 6.25 0.50
3.63 0.25 2.33 1.05 3.78 0.50 1.67 1.61 T: total L: large (>2
mm) M: medium (1-2 mm) S: small (<1 mm) n: 8-16
Example 24
16d10 Sequence Data
[0212] 16D10 Genomic DNA sequence (840 bp)
TABLE-US-00002 (SEQ ID NO:1)
GAGAAAATAAAATATAAATTATTCCTCAAAAATACCATAAAGGTTAGCCA
ATATTAATTCTTTTGAAATTTTCTTTGCTTCCATAAATTAAAAAAAATTG
TTTTTAAGTGAGGGAATGTGGATTAAGCATCTTTCTTATTTTTAAAATTT
TTGATAGAGTGTAGCGACAGTCAATCAAAATATTTTGATTTTTTTAAAGT
TAAAAATTAAGGATGATAAAGAAGTTTAAAATGTAGGTGGAAATATAAGT
ATACCGAAAAACATCTTTTATTTTTAAGTTTAAACAAGCAGTAAAACTTT
GTCTGGTTTTATCACCGGGCAACTGTAAGGGAAGCTTTAATAAAAATTTT
GTAAGATACGAAAATCATTGTCCCCAGTAGCTTGAGTGATCGAAGCGCCT
GGTTGCCATTAAGTTTTTTGCTTGAGACTTATATAACAAGTATATATCAA
ACCGGATTATAAAGTTAAAGAACAGAAAAAATTTCACGGAATAAATATTG
GCTAACCACTCAATTTATTTAATTATTCTTCAATCAAAAAATGTTTACTA
ATTCAATTAAAAATTTAATTATTTATTTAATGCCTTTAATGGTTACTTTA
ATGCTTTTGTCTGTCTCATTTGTGGATGCAGGCAAAAAGCCTAGTGGGCC
AAATCCTGGAGGAAATAATGAAGAAAAATGATTGAAGAAAAACGTTTAAA
TTAAACGATAAATGGGAAATAATGGAATTTAAATTAAGCTAATTTTGATG
GTTTCCTTTGTTAATTTCAACATAAAATTAATTGAATTTACTGAATAAAA
TTATATCTGAAAAAAA
(One 476-bp intron sequence is bolded).
[0213] 16D10 cDNA sequence (364 bp)
TABLE-US-00003 (SEQ ID NO:2)
GAGAAAATAAAATATAAATTATTCCTCAAAAATACCATAAAGTTAATTAT
TCTTCAATCAAAAAAATGTTTACTAATTCAATTAAAAATTTAATTATTTA
TTTAATGCCTTTAATGGTTACTTTAATGCTTTTGTCTGTCTCATTTGTGG
ATGCAGGCAAAAAGCCTAGTGGGCCAAATCCTGGAGGAAATAATTGAAGA
AAAATGATTGAAGAAAAACGTTTAAATTAAACGATAAATGGGAAATAATG
GAATTTAAATTAAGCTAATTTTGATGGTTTCCTTTGTTAATTTCAACATA
AAATTAATTGAATTTACTGAATAAAATTATATCTGAAAAAAAAAAAAAAA
AAAAAAAAAAAAAA
[0214] 16D10 cDNA sequence region used for making 16D 0 RNAi
constructs
TABLE-US-00004 (SEQ ID NO:2)
GAGAAAATAAAATATAAATTATTCCTCAAAAATACCATAAAGTTAATTAT
TCTTCAATCAAAAAATGTTTACTAATTCAATTAAAAATTTAATTATTTAT
TTAATGCCTTTAATGGTTACTTTAATGCTTTTGTCTGTCTCATTTGTGGA
TGCAGGCAAAAAGCCTAGTGGGCCAAATCCTGGAGGAAATAATTGAAGAA
AAATGATTGAAGAAAAACGTTTAAATTAAACGATAAATGGGAAATAATGG
AATTTAAATTAAGCTAATTTTGATGGTTTCCTTTGTTAATTTCAACATAA
AATTAATTGAATTTACTGAATAAAATTATATCTGAAAAAAAAAAAAAAAA
AAAAAAAAAAAAA
[The bold 42-bp sequence was used for constructing
pHANNIBAL(16D10#1), and the underlined 271-bp sequence was used for
constructing pHANNIBAL(16D10#2)] pHANNIBAL(16D10#1):
[0215] (a) Construct: (XhoI+42 bp 16D10
sense-strand-sequence+KpnI=54 bp)+Pdk intron+(ClaI+42 bp 16D10
antisense-strand-sequence+XbaI=54 bp)
TABLE-US-00005 SEQ ID NO:3) XhoI
CTCGAGGGCAAAAAGCCTAGTGGGCCAAATCCTGGAGGAAATAATTGAGGTACC--------
------- KpnI ----------Pdk
intron------------------------------------------ ----- ClaI
ATCGATTCAATTATTTCCTCCAGGATTTGGCCCACTAGGCTTTTTGCCTCTAGA XbaI
[0216] (b) PCR detection: primers H1F1 & H1R1 (234bp PCR
product) Primers H1F2 & H1R2 (273 bp PCR product)
pHANNIBAL(16D10#2) (1). Construct #2: (XhoI+271 bp 16D10
sense-strand-sequence+KpnI=283 bp)+Pdk intron+(ClaI+271 bp 16D10
antisense-strand-sequence+XbaI=283 bp)
TABLE-US-00006 (SEQ ID NO:4) XhoI
CTCGAGCCTCAAAAATACCATAAAGTTAATTATTCTTCAATCAAAAAAAT
GTTTACTAATTCAATTAAAAATTTAATTATTTATTTAATGCCTTTAATGG
TTACTTTAATGCTTTTGTCTGTCTCATTTGTGGATGCAOGCAAAAAGCCT
AGTGGGCCAAATCCTGGAGGAAATAATTGAAGAAAAATGATTGAAGAAAA
ACGTTTAAATTAAACGATAAATGGGAAATAATGGAATTTAAATTAAGCTA
ATTTTGATGGTTTCCTTTGTTAATTTCGGTACC KpnI-----------------Pdk intron
----- ClaI ATGGATGAAATTAACAAAGGAAACCATCAAAATTAGCTTAATTTAAATTC
CATTATTTCCCATTTATCGTTTAATTTAAAOGTTTTTCTTCAATCATTTT
TCTTCAATTATTTCCTCCAGGATTTGGCCCACTAGGCTTTTTGCCTGCAT
CCACAAATGAGACAGACAAMGCATTAAAGTAACCATTAAAGGCATTAAAT
AAATAATTAAATTTTTAATTGAATTAGTMACATTTTTTTGATTGAAGAAT
AATTAACTTTATGGTATTTTTGAGGTCTAGA XaI
TABLE-US-00007 TABLE 2
>2E07>msp1>>bankit482031>>>AF531160
GATCAAACAATCTCCTCAACAACTAAAAAAACTCAAAAAACACCCCAAAA
CCAAACTAAAAAATCAAAAATGTCCATCTTCCTCACTTCTGCTCTTCTAA
TCATTTCATTAATCGCTATGACCGAGGGAGCAGGCGATCGAAGCGCTTCA
ACCTCTACTGGTTGTACAACCTATTTTGGAATGCTAGATCATGCGGATAC
CAAGGAAAATAACAAAAGAAAAACTTTCAAACCCAACGATAAAACCATAT
CCAACACTTTGCAAGTGATTGGTGGGACAAAGTTCAGCAATACCTCGGTG
GCGTTGGTTGTCGGTGATGAGGTGTTATGTATGGCTAAGACAGGAGGTTC
AGGCGATTGCGGAATGCGCTACGATGCGTTGACTGGATCAATGAAATTTA
TCATTTCTGATAATATTATTGTTGAGGTTCCATTTGAAGGCGTTTTTTTC
TTCACCGACAACAAGTGTGTCATCCAGCTTGTAGGCTACGATATTAAAAC
TAATATAACTCTTCTCAAAATTAATGATGTCGACTTCAAAATTGTCCCTA
CTGATAAGAAAATTTCCCCGAAGGCTTGTACTATGAAAATGTGAGGGAAA
AAAGTAAAGAAAATGTGTAAATATGGAAGGATAAAAACTAAACAAAAAAG
AATGTGAAGTAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:5)
>2G02>msp2>>bankit478474>>>AF531161
GGATTTAAAAAATTAATTTAAAAAAAGTGAAAAATTCAATTAAAATTAAA
AAATATTTTTCAATGAATTTATTTTCTATTTTTTTATTTTTATTTCCAAT
CGGGTTTATTTGGGCTGAATGTAGCGGAGATTGTTCTATAGAGAACCAAT
ATAATTATAAATGTGAGGATAGAAGTGAATTTTGTGAAGAATGGGGAAAA
TACTGCGAAAATGTCTTTCTTCACAAATGTGTAAGAAAGGCTTGTCCAAA
GAAATGTAAAGTTTGTCATAGTTCTGGTGAAGAACCTAAACCAAATCCTA
CAACTATAACAACGGCATCAACAATAACAACACCATTAGCAACAACACCT
CAAAACTCAGCAGTTACTTCGGCAACCTCAAAAAGTGCTACTCCATCAAA
AACTTATTCAACCGAGACAACCGAATGTGCTAACACAACTACTGAGGAAT
ATGAAGCAACTATTGAGGAATATCAAACAACTACAGAAGAATATGAAGAG
GTAACAACCCCTATAATTACAACCACCAATCCAACAACTTATTTATTAAT
GACTACAATAGTTGAAGAAATTAGTGACGACGAATTCAAAGACGCAAAGA
AGATGAAATGTAAATCATGTAATGCAAAAAGGAAGAAATTGGCTGAAATT
TATGACAAATATTATCCGAAAGTTAAGATTCATGCTAAATTGTAAATTAT
GATGGAAAATGTTTTTGAATTGTGAAAATAAAAATTTAATTAACCCAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:6)
>2G10>msp27>>bankit482965>>>AY135363
GATAGCACAGATCTATTTTTAGATTTTTTTAGCTTTTTAGAAAATTTTAA
TTTAAAAATTATGTTTATTCTTCCAAAACCTTTTTATCTTTTAATTTTAC
TAATTATTTCAACAATTTTCCTTTTATTTTTAATTCTTCGTTTGCCTTCA
ATTTCTTACCAAACAAATCAATGTCAACATTTATGGGACTCTTCCGAGTG
CAAAAATTTAAATAATTCTTTAAATTGGCATCCAATAACTTGTTTTATTG
ACGGAAAGCAAAAAAGAGTTCCCTGTCTTCAACAAAATGATTTAAAAGAA
GTTTATCTTCCATTCAATTCATTTTTGAAAAAACAATTTGATTTGTATGG
AGAGACTGACAAAAGCATTTTTTTAATTTTAAAATTTATTTTTAACAAAA
TTTGCGGACAGATAAAATTTGTCCGCAAAATATTTGCGGACAAATAATTT
TTTTTGTTGTTAGGCTTTTTTAGGGTATTTTTTTCTACAATTTTTTGGAG
TTTTTTTCTACAATTTTTTGGAGTTAATTCTAAATTATAATTTTATTATA
TATTTTATAATTTTAAAATTTTTTTCTTTTTAAGAAAACAATTCTTTTGA
TTATTTTACTTCAAACATTCCTCCACGTCTTTTTAAAAATAAAAATAAAA
TGGTGGCTGCAAATCCAATTGAACAATTTAGCAATGTGGCTATTCGTCAA
AGAATAAAATGTTTAAAACCTGAAAATGGATTACCAATGAGCGTTCAATG
GAGTCCAATTCCCTACTTCTATCCTGTTCAAATACTCCAATTTGGCTTTG
ATTATTTTATGAGAAATCGAACAGAACAGAGGAAATTAATTGAAAGAAGG
TTATCAAACAAAGATGATTTCTTGGTACTAAAAAGTGGAGAGAAAGTTAG
CGAATTTTCAACTTTTTTCTGATTTGCCATTTTACCTTTTCTGCAAAATT
GAATCAATGGATGCTTCCTTGTAATATTTTTGAGAAGATTGGGGGAATT (SEQ ID NO:7)
>4D01>msp3>>bankit478504>AF531162
GACAATAAACGATCCAATTTCCTAAAATTTTTTAAAAATTTTTAAAATTT
ATTTTATGCCCCTTTTTGTTTATTTAAACAAATTTGCTTGATTATTAATG
CCAAAATTAATTTTATTATTTTATTTAATTATTTATGGAATTTTATTGTT
AATAAGTTTAAGTGAAGCATTTGGGTTTGGTGGAGGATGTGGATGCCCTT
GTATGCCGCAACCATGTATTCCACAACCACCTCCAATTGCTTTACCTTCT
CTATGTTTCCCTCAAATCCAATTGCCCTGTCCCCCTCCATCTTGTGGATG
TTGTGGTAGAAGAAAAAGAGAAAGTGGAGCTTCAGCATTATTAACAGCAG
TTTCAACAAAGTCGGGAATTAAAAGAATTGGAGAAGAAAAAAATCATTGT
AATAATCCACACATTAAAAGAATTATTTTAAAGAATTTAATTATTGGAGA
TTGGGTTGGTACAAGAAATGCAATATATTCAGAATTAAGAGCTAAATTAG
GGGGGAATTATATAATTAATTGTGCTCATGCCCCCTCATTTGCGTATTCT
GGTGATTCTGTGATTGATTATTGTGTGGATGGACATCAGGCAATAACTTG
TGCAGTCTTCAAAATTCAATGAGAATAAAATCAGAATGAATTCTATTTTT
TTAATAAATATAAAAATTTTTATAATATATTTTGAGCATTATAAATATTT
ATAAATTAGTTTTTTTTGATAAATTAATTTGAAAATGTATAAATTAGTTT
TTACTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:8)
>4D03>msp28>>bankit479214>>>AY135364
GACGCAATTCCATTTTGCGTTCAATCAATTTAGAAAAAGGCTGGAAATAA
TGATTCATCAACAACTTTATTATTAGCGCTTAGTGTTCCGGGCTTTCATT
TCAACAGAGAAATTTCAAATTCACCTATTTGGAGCTTGGATCAGTTCTTC
TATCGACTTGTAGTCCTAACCTATACTAAAAATTTTTTAAATTAACCACA
ATGGAACTTGCTATTAACAGTCGATTGTTATCATTTTTGTCTTTATTCCT
ATTCATATTTCCTTTAAATGTTGTTGCTCAACGGCATCGTTACCCACACA
ATCAAGGAAATTATTTCAGCAGACAAAAGCTGCAAGAAATACAGAAGGAG
GAAAATGAGGCTGAAAATTCTTTACCAAAAATCTTTTGCGCGCATGGAGC
TTCAGTAGCCGGCCGTTGCGTATGTGATCATGGTTGGGCCGGTACTAATT
GCCAGCGGGAAATGCATTGTGCTACTTTTGAGCGAAATGCTAATGGAAGC
TGCCCAGTCTGTCAGCCCAATTTTCAAGGGGATAAGTGCGAATATATTGA
ATGCCAAAATGGAGGCCAAGAATCATTGGAAACTCAGAATTGTAACTGCC
CAAAGCCTTATTCTGGCCGTTTTTGTGATGAATTACTCACAGGAAATGTC
TACTACTACTATAACTCTAAAGTAGCAACCCTTGGTCCTCTTGGACTTAT
TTCTGTTATACCAATGATTTGTCTTTATGTTTTATGTGAAAAGATTTGCA
AGGAAAAGACAAGTGAGACGGATTGAGAAAACTTGGAATTTACAGAGCAG
TAAAACTGTGAATCCTGCTCATATTGAATTTCTATTAAGGGAAAAAAAAA AAAAAAAAAAAAAA
(SEQ ID NO:9)
>5G05>msp26>>bankit478498>>>AY135362
GACCTTAATCAATAAAAAATATTTTTTACATAAAAATGTTTTATTTATTT
TATTTTAAATTATTTTTATTTTCCTTAATTTCTTTAAATAAAGTTAATGG
ATTTTGTATGAAGACTATTTGTTCTGCGGACACCGATTCTCGACACCCTG
TAAATCGAGTAATCGGTATTGGTTCTGATGGAATTAGTGGGGAATATAAA
GCTTTGAGACGAAATGATCAAATTGTTGAAGCTGTAGATTTAAGTTGTAG
AGAAGGAAGTTTTGTTTATTCTCCCTTTGAAGGGGAAATTTCTGCTTGGA
GACCTTTTGATGGAAATGGGCAAGATGAAATTAGAACGGATGAAAATAAG
AAGAATACAGATGGATGCAGACCTGACCGAGGAGTTAGAATTGATGGAAA
AGGACAATGGCAGGGATATCACGTCCTTATTGGCTCTGTTCGTTTATTCC
GTTACAGTGGACATGTTAATGCTGGACAAAAAATTGGTGTATCTTTGGAT
ATTGAATGTGAATTGAAATTAAATAAACAAAAGATGAATAAACGTCCTCG
AAAAGAAGAAAATTTTGTCAGAGTTTATTTACACAAGGAAGGACGTCCAA
TTGATCCAACACATCATTTAATTGATTGTATGTGTATAAACCAAGTCTGT
GAGACAAACAGAATTAATGCTTTGGAAGGACCGTTATTTAAATTTGACAG
TCGTTTTAACGGTGTTAGAGGATGGGAAATTAAATGTCCAGATATTCAAC
AAATTGAAGAAGAAAATTCTTCAGAAGAAGAGGAAGAAAAGAAAAAAGAA
GAAAATAATTTAAATGAAGAATGGGGAACTCCAAAATTTATTCACCTATA
GAAGGGGAATTGGTTGGAAGAATTAGAGTTAATAGTGAACCTGGGGCACA
GACTTATACTGGATGTACTAATGAAGGAATATTTATGGTTGGGGCTGGAA
AGTGGAATGATTATGAAGTTCGAATTT (SEQ ID NO:10)
>6F06>msp4>>bankit482257>>>AF531163
GTTCATTTAAAAATTTTTTCCTAAAAAACTTCAAAAAAGCAACTTTTATT
GCGTAAATGAAAGAAAATCTGTTTAAAAAGAGCCTTATAGGCCTATTTTT
GTTGTTAGCATTCAATTTTACTGAAGCTAAGGACTCTGGAGAGAATACTA
GTCTTGAAGCTAGTTTGAAACCAACTAAAAGTATTGAAAATGCTTCCCTA
GAAGAAAAGAATCAAAAAGAAGAAAATGGAGTAACATTCCCGGCAGAAGG
TCATGAAATTGTCGAAACAAAAAAAGAAATCAACTCACCAGAAGAGGTGA
CAGATTCAACTAAAGGACAGGAAAATTCCGAGGATCGTAAAGTGACAATG
AATGGTGATGAGTCTGAGGCCGATAAATTAAACAATGAAAATGTTGAGGG
TGAAGAAAAGAAAGCAACTGAAAACAAGAATGAAGTTGAGGAAAAAGAAG
TTTTAGAGGATGAGAAGACAAAAGAAGAGGAAGATAAAATTAGCGATGAG
CCTGTGAAGACAAAGGAAATGAAATCAACAAACAATGATAAGGAAGTTGA
AGATTTGAAAGAAGAGGAAGAGAAAGTCGAGGTAAAAGGTAACAAGGATG
AAGAAGAAAATAAGGAAGAGAAGAAGGAAGATAAGAAGACAAAGGATGAA
AAAAAGGTTCCAGAGGTTATTGAGGGAGAGAAGAAAACACCCAAGGAAAA
GGAACACAAAAGCCATTGGTTTATGGACAAATTTAAACATGCTTTCTGTT
TCATAACTCATTACTTCTTTTGTCCATCTAACTCTGCAGAAAAAGGCAAA
GAATCCCATCATGAAGGAAAAGAATCACACCGTGGAAAGCGTCTTAACTC
TGATTTTAGTTCTTTAAGCAGTGATGAGGAAATGATTGAGAATTTTGAAA
ATGCCCACGAATTTAGTGAAGAAATTGAAGAAAATGGGGAATTTAAAGCT
AAAATGAATGTTGGTGCAACATACTTCAAAGCTGAGACAGATAATTCTGG
AAAGATGCGCGGCAAAATTGAAAAATTTAATGCTGAAATGCATAATTGAA
AAGATTGTAAGGATGGTGGGTGTGCTGATGAGTAAAACAAAAAAAAGCAA
TCCGATTTTATTCTAAATTTTATTTTTTAAAGTGATTCCAACAAGTGATT
CCATTAACCCCTCAAATTTATTTAAAAAAACGAAATTTTAAAAGTTCTGG
ATTTATGTCCCAAAAATTGTACAAATTATTCAAACAAACTCAATGGTTTT
GGACATTATATTTTTTTATTATTTTCTAACAATTTTTATTAATGTTGAAG
TAAAAGATTAATTCAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:11)
>6G07>msp5>>bankit482261>>>AF531164
ATTCTTAATTTATTTAAAGAATTTATTCTGCATGATGAAATTAATTAATA
TTTTATTTTTATTTTTTGTTATTTTACTGAATTCTATGGCTTTCGGAAGG
TTTTCTTTATTTTTGGAAAAATCAAAATTTCAATTGAATATTTTGTTTTC
ATTCCAAGATTTTCTCACACAGATCCCGCTTAGTGTGAGATATCGGATAA
AGCTTCATAACCTCTACAATTTTAAGATATCCACATTACTCCGTCCAAAT
TCCCTTATATCTCCTTTGATCCCTACAAATCTACCGATATCCCCTCCACC
GATATTTTCCTTTTCCGAACCTTAACTTCCGATCAATCCGCTATCTGGAC
AAATCGTTATTCCTCTAAACAAGAATTTATGCTTTTAAATGTATAAAACC
AATCTTTAATATTCTTCAAAAAAATTTTCAGTCCTTCTCTCAATTCAGTG
CGTGCTAAACGTCAAGGCTGGGGAGGATGGGGTTGGAACCCTCAAGTTCA
AACAGATATTGATCGTCTTCGTATTGATAAAGACAAACTGCGATTAGATA
TGGACCGTTTAAGACTAGATCAGGATAGCTCTTGGGGATGGGGAAAATGA
GAGAATCAAACGACTAATTTAAGTGTAACGATTTTTAATTAACGATTTAT
AAATTAATAAATACTTGATTGATACACAATTTAGATAATTTAAAATAAAT
TTTATTAAATGATAAAATTAAATTGCCGTTTTAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA
(SEQ ID NO:12)
>7A01>msp6>>bankit482263>>>AF531165
GACCATCAAATCATCTCCTCATCAACTAAAAATCCCTAAAAACACCCCAA
AACATCCATAAAAACAACCACGAAAATGGCCACCTTTTTCACTTTTACCC
TTCTAATCATTTCAATTATTGCCACAACTGAGGGAATGAATACTAATCGA
AGTGCTTCAACCTCCGATTCTCTCAAAGCCCAAAAGGATTGTAAAGTGAT
ATATGGCATGTTTGTGCCTGTAGCAGGGTCAAAAATGCATGGAGACGCCA
AAAGCGCAATGAAGCCAAACAATCCAAGTCTCTCCAATAAATTAATTGTA
TCAGGTGGCAACTCAAAATATTCAGTGACTTTACAGGTTGAAAACCAGCC
GAAGTGTGTTGCCCAAAATGACGGAAACCCTGTAGAATGCCAAATTCAAG
GAGACAAACTTTCAGGAAAATTGATTTATGATATTGAAAACGGCCCTTCT
GTCAACGTTCCCTTCAAAGACACCCCAATCTTTGTTGGAAATAAATGCGA
AATTGTTTTTGTAGACTACGATAAGGACCACAAATTAACTCTTCTTATGA
ATAAAGTAAAGCTGATGATTGAGCCGACTGAAAAGCAAATTGTAAAGGCT
TGTGGAGTGAAAAATTAGATGGAAAAATGATATATGAATGAATGAATGTG
AGAGGGAGGGAAAGAAAAATATTTTTAAAATTGAAGAAAGCATTCAAAAA
AATTAAAAAAAAACAATTCTTCAAATAATATAACCTTAAAATTTCTGATA
AATTATGTTTTTACAAAAAAAAAAAAAA (SEQ ID NO: 13)
>7E12>msp7>>bankit478534>>>AF531166
GCCATCAAATAATCTCCTCAACAACTAAAAAACTCAAAAAAACACCCCAA
AACAACTCTAAAATGGCGGCTCTCCTCTTCACTTCTACCCTTCTAATCAT
TTCATTGGCTTTTATTGCCATAGCTGAGGGAGCAGGCGATCGAAATGCAT
CAGCTTCAAGCCCTGGTTGTATGCAGGTTGCAACCCTTATTCATATAGGG
GAAATTCGCCCAGCAAAAGCAAACAAACCAGGTGTACAAAATACTCTAAA
AATGTCTGGAAATGTTCAAACATTCAAAACTACTCAAGTGACATTACAAG
TAGCTGGGCAAGAGCCTTGTACCGTTAAAATTAATAATGGCGAAACCAAA
TGTAAAATAACCGGAGATGAATTAAATGGAAAATTAATTTTCAAAACTGA
AAAAGGAACTGAAATTTCTGCTTATTTCGAACTGGTTCCATTATTTTCTG
AAAATAAGTGTGTTATTGAACTTGACACTTATAACAAGGAAACCCATGAA
ACTAAACTTAAAATTAATGGAAATAATTTTATGATTAAAAAGAAGGAAGG
TAATGTGTCAATTAAGTGTGGTGGAAGAGCTAATACTGTTTAAATTTTAA
AAGTGTGAATTGAAAGAGGAAGAGAATAAACAAATGTGAAGATGAGAAAA
AAATATTTTGAAGAAAGCATTATAAAAATATTAAAAAAAATTAATTCTTC
AAATTTTTATTTGATTTTTGAATAAATTATTTTATTAAAAAAAAAAAAAA AAAAAAA (SEQ ID
NO:14) >7H08>msp8>>bankit482285>>>AF531168
GCTCATTAATTAGTTAAAAAATTTAAAAAATAATTTAAAAAATGAAAATT
TATTTTAATTTAATTGTTTTTCTATTTATTTTAAATTTTTATTTTGTCGA
ATTGGCAAAAAGGAAGGCAACGGATACTGAGATTCCTGAGCAAAATAAAA
AGCAAAATACAAGCAACCATGCCCATCAACAATTAACTCCTTCTTCTTCA
AATGCTGATAATGAGAAGCAAGGAAATCTTTCCTCTGAAGCTTCAAATAT
TCGAGGAAAAAATATTCTGCATGATCAGTCTGCTATTAAAAACAATTCGT
TAACTAATCAACAATTAGGAGCCTCCTCTTCTAATGCTGGGCAACAGAGA
AATAATAATTCGGATCTTTTAAAATTAACAATTATAAATCATTTGTTATC
CCATCGCCAATTTAATGCCTCTTCTTCAAATGCTGGTCAACACAAAAATA
TTCCCTCCGAAAATCTAAATTTTCATCAAAAAACTATTCCAATTGCTACT
AAAAATAATTTGTTCCCCAATCAGCAATTTATTGCATCTTCTTCAAATGA
TCTTGATTTTCAACAAAAAAATATTCCATATGGAACTAAAAAGAAGGTGT
TACATCAATTTATGCCATCTTCTTCCAATGCTAATAAACGCAAAAATAGT
TCCACGGAATATTTAAAATATGCAATTAAAAATAGATTTTTATCTAATCA
GCCATTTGATGACGACATTTATGGTAAAAAGAAAAATGTTTCCCCGGAAT
ATCAAAATATTCAACAAAAAAATCTTCCATATGTCCAATATGCTATTGAT
AATAATTTGAAATTGCCAATTCCAAAAAATCCTAAAGCACTTCCATATGA
TTTGTCTAAATACGCATTTAACTTCCCCAATATGAACAAGAAAAATATTT
ATGAAGGAGCATATGATCCTTATTATATTAATTTTCAACAATAACAGATT
TGGCTAATAAAACGTTGGAAAACGACTAAGAAGTTATACATTTGACATAA
ATTAAATAAATAAAATTAAATTACTATTATAAAATTGTTAATTATCGTAA
TAAAATTTTTTAACTCAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:15)
>8D05>msp9>>bankit478548>>>AF531169
CTAGTCAGTCATTTAAAATAATTTAATATTCCTCTAAAAATCCCTAAATT
AATTTAAATATTTCTTTAATCAATTTTTCTTCAAAAAATTTAAAGAAGGA
AATGTTTTTACAAAAACAATTATTGTTTTTGGTTGTTCTTCTATTAGCCT
TTTCTCTTGTAAAGGGAGTAACCGAGAATAAGAATAAAAGCGAAATAAAA
AATGAAACAACCACAAAAGTAATTCAAACATCAACTGGAGGTTATGATGA
TAACGAAAAAGCAGACTATGGCGATTTGGCTGCAGAATTGGCTAAACTTG
TTGAGGAGGAAGATGAATTAAATAAAAAGAAGAATGCTTTGAGTTCGGAG
AATGGAAATAAAAATAGCACAGGAAAGCCTTATATTCAAAAAGATAAAAG
TAAAAAATATTTGGAAGAAGATAAAGGAAAATATGAGGAAAGAAATTCTA
GAAATAAATATGAAAACTCGGATGAAACCCATGAAAGTGAATCAGGTTCA
AGTTCGGATGAGGATTTAGATGAAGATAATTTAGAAAGATTGCCAGGGCC
TTCGCCACACAATGAAGGAATTTCTAGGCGAAGAGTTGAAAAGGAAAAAG
GTGGAGAAGATGAGGAGGAGGAAGAAAAAGAGCAAGAAAATTCTAATGAT
AAAGAAGAAAGAAAGAAGAAAAGGAACACCAAATATAATCCAAAAGATGA
GAGTGAGGAAGATATTTCTTTTGATGGTCAAATACCTAAAAGTGTACGTA
AATTACTTAAACAATTAGCAGCTGGTGGAAAGAATCCTGTAATTATACCT
TTAATTATAAATAACAACAATATACCGAATCGAAGAGAAGATGAGTCTGA
GGAATGGAATAAAAAAAGACATGGGAGACCTCATAGATTAAATGATTGGA
ATAATCCGTTTCCTCCATTCTTTCAATCTTCAATGTTTCAACCACCAATG
TTTCAACCACCTATGTTTCCACCACAACAGCCACCTTTTGGTGGCCCTCC
AACATTTGCTCAGCACTTAATCTTCCTGGAGGGCCTCTCGGAGGAGGTCT
TGCTGGCAGTCTTCCCAACACAAATCCATTTTTATCACAACTAAATCGTG
GTGTAAGTCCTAATCAATTTCCCAATCCTCCCTCTAATCACGTTCCACCT
TTTGGGCAACAAAATCAATTCTATCCTCCTCAACAACAACAACAAAATCA
AGTCAACCCACAGGGAGCAGATGGCAATGATGTGAAAAAAGTGAATTAAA
CAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:16)
>8H11>msp10>>bankit478550>>>AF531170
GATCGTCATTCTTGTAAACTAAAAATCTTCAAACTTCAAAAAATATTCCT
TAAACTTCTTCACAAAAAGAATTGAAAAATGTTATTAAAATTCTTTCTCC
CATTATTGCTTTTGGTTACCCTTATCTATTTGGGGTGTTCTGAGGAGGAT
AAGGAAGACATTGCAAATGGTCCTCAGGAATCTGAGAATCAGGTTGATCA
AGAATTGGTTAGATTGAAAAGAGATGATGAAGAAGAGGAGGGAGAGAAGG
CTGAAGATGAAGAGAAGCCTGAAGAGGAGGGAGAAAAGGCCGAAGATGCT
GAGAATGCAGAAGGAGATGCTGATAAAGGAGATGCTGATGAGGAAGAAAA
AAAAGAAAGTGAAGATGAAGAGAAAAAGAGTGAAGGTGAAGAAGAAAAAG
CGGAAGGTGAAGAGGAAGAAAAAAAGGATGGAATTGAGGAAGAAAAGAAG
GATGAAGATGAAGAAGAGAAGAAAGATGATGATGAAGAAAAAAACGAGGA
AGAAGGAAAAAAGGATGATGAAGAAGAAAACGTAGACAAAGAAGAAAAGA
AAGATGATACGGAAGAGAAAGAGGATAAACATTCAAAGGATAAAAGTAAG
AAGGATAGTAAGTCCGTTCAAAAGGATAAAAAGGAGGAGAAGGAGAAAAA
GGATAAAAGTTCAAGTGGTGATAATTCTAAAACAGATAAATCAGATAAAT
CACATAGTAATCAAAAACAAGACAGCAAAGAACCATGTAATGGGGATACT
GCTTACAACTGTCCTAAACTATCAGGTCTTTGTGAATCAAAAATTCAAGT
ACAACAAGACTTCATGGGTGAAAAATGTTGTGCTACATGCAAAAATTCGG
TTCCTGTCGCGAAGAAAGATATACCCTTATGCACTGATTTGGCTGATAAT
TGTGATCAAATAGCATCCACCTGTGGGGAAGAGGCGTGGCAACCGACTAT
GATTTCTGATTGTGCTCAGACATGCGATAAGTGTGAATTACATTTTCAAA
TGTTGGAAAAGAAACTTGCAGCAGCTGCTGCTTAAAAATTTTGAAAGGAA
AAGAATTTTTATCAAAAATATATGTATCAAAAATATATTTTCTTTGATTT
TCACACCCTTAATACTAAAATTTCAATTTATTCATCAGTGTTTCTCGTAA
TTATATTTTATTAATTTGTTTCGAGATTTAGTAAAGATGCTTTAAACCAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:17)
>9H10>msp11>>bankit478544>>>AF531167
GGAATTTTTCAAAAAAAGTAGGCTGGAGAATAAATTTATTGAAAAACCAG
AATTCTTAAAGTTTCAACCATTTAAAAAATGTCAAACAATTTTAAAACTT
GCCCAGCTTTATTATATTTATTGCTTCTGTTGGGAAAAGCAAGTTGCAAT
TATTTTGAATCAGAATTAAGCTTAGCTAATGACAAAACTTCTATAGTTCG
CAAATGTTGTCCTAAGGAGAAGATTAGACACCATCGGAGACCGTTGCATT
GCTGCCAGGATGGGTTATTCCGTGATGAAGTTGATGGTTATTTATTAAAA
GAATGTGCAGATCAAGGTGATTCCATAGTCAAAACAATTAGATGTGCTCA
ACAAGAAATACATGGTGAAAATGCAGTGGAGATTTGCAAAGCCTATTGCT
GCGAATTATTCAGAGATAATAATTGTTCCAAAATATGCCTAACAAACATT
ACCAAAGTAAACATGTCTATTGAAATATTATTTGAGCTGTTAAAAAAATG
CAGGAATCATGAGAATTATGGGGAAGTCCATGACTGTATCCATTCAAAAA
GACCAAAAAACATGGATGCCGCAGAGTTGGAAATTTATTGTAAAAGGGCT
ATTAATATGGTTTAAATCTGGAATTTATTTTTTAATTTATTCTACTCGAT
CTCCTTTTATCTATTTAATTATTAATTTATTTTTGGCAATAAAATTTAAT
AAAAAATGTAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:18)
>10A08>msp34>>bankit487923>>>AY142117
GGTCGTCATTCTTGTAAACTAAAAATCTTCAAACTTCACAAAAATATTCC
TTAAACTTCTTCACAAAAAGAATTGAAAAATGTTATTAAAATTCTTTCTC
CCATTATTGCTTTTGGTTACCCTTATCTATTTGGGGTGTTCTGAGGAGGA
TAAGGAAGACATTGCAAATGGTCCTCAGGAATCTGAGAATCAGGTTGATC
AAGAATTGGTTAGATTGAAAAGAGATGATGAAGAAGAGGAGGGAGAGAAG
GCTGAAGATGAAGAGAAGCCTGAAGAGGAGGGAGAAAAGGCCGAAGATGC
TGAGAATGCAGAAGGAGATGCTGATAAAGGAGATGCTGATGAGGAAGAAA
AAAAAGAAAGTGAAGATGAAGAGAAAAAGAGTGAAGGTGAAGAAGAAAAA
GCGGAAGGTGAAGAGGAAGAAAAAAAGGATGGAATTGAGGAAGAAAAGAA
GGATGAAGATGAAGAAGAGAAGAAAGGTGATGATGAAGAAAAAAACGAGG
AAGAAGGAAAAAAGGATGATGAAGAAGAAAACGTAGACAAAGAAGAAAAG
AAAGATGATACGGAAGAGAAAGAGGAATAAACATTCAAAGGATAAAAGTA
AGAAGGATAGTAAGTCCGTTCAAAAGGACAAAAAAAAAAAAAAAAAAAAA AAAAAA (SEQ ID
NO:19) >10G02>msp29>>bankit479222>>>AY135355
ATATTTATTTTTTAATTTAACAAAAATATTTTTAATTAAAATTATTTATT
TAATGTTTAAATTGTTGTTTTTCATTTTGTTTGCCTTATTAAATTCTGTT
GATTGTCTTTTAAAATTACGAACACTGGATAAAGAACATCTTCTGGTTGA
GGAGAGATATGCCAAGGAAGATACGCTTTATCTTTTTGTTTTTCCTAGAA
CATCAAATGCCCCATATTTTGGAGCAATGTGTCTTTATGTTGAAGCTGTT
TTAACTTGGAAAGGAATTCCTTTTCATAGAATAAGTAACCAATTCTTTCT
TGGTTCAAAAACTGATGGAGCAATTCCTTTTGCTATTTATAACGGGAAAT
ATTTGGATGGAGCAGAAAAAATAATTGAAGAAGTTAGAAAAAAGGGAAAT
AAAAAATTGAGTGATGAACATGATGATAATATTAGAAAATTTGCAACTAG
AACCTTGCTAAAGACTCTAATTGCTGATAGAACATTTCGGAGAGATCTTC
CCCATGCAACAATTCCAAAAAATAATTCCGAAACACAAATAGCCTCTTCT
TCATTATCAAATAGTGCACCAGCAACTCCCAAGGGTGGAATCCCTACAAG
AAAGAGATTTAGTCCAATTGATATTAAAATCCCTCATACTAAAAATGAAG
AAATAATAATGGCAAAATCTGAGGGGCATTCTCCTGGAAGTTCTTTCTTT
TCTAGAACTATTGCTCATTTAAAATTACATAATAATAATTCTCCAAAGAA
AGGTCCGGGTGGTCTTGATTGGATGTTAAAAGATGAAGGAGTTCGTGAAC
AATTAATTCCAGTTATTCCAGAGGCTTTTTTAGAAGAAAGTATGAGTGAT
GAATATTTTGATTCCCCGGTAAAAGATAAAAATGAAAAGAAATCAAAAAG
AGAGGAGGAAGATGAAAGTGATGAAACAAAAATATCTAAAATTAAATATT
CCATTAAATTGACGTTAAGTCCAGAATTGTGGAAAGATTATTTTAATATT
TTAAATAAAATAAAAATAAATGGAAGGGAAAATAGAGAAGAAATTAATTT
ATTGAAAATAAATTTTCTTCAAGAATATTTCGGATTCTTAGCAAGAATTG
ATGATGATTGGGAACGTGTAAATTCTATTCTGAAAAATACAATTAACGAT
ATTTTAAAGAAATTAATTGTTGATAGCCAAATACCTTTTTGTTGGGAAAA
AAGGTTGAGAGAGATTAATGGGAAAAATATTAATGAAGTTGAAGTATTTA
ATGAATTTAAAGATAAAATAAAATCGTTGGGTATAATAAAAAGTTGACTG
AGGCAGAGACTAAAAATAATTTTTTGCATGGAAATAATCCAACTTTGGCT
GATTTTGCCCTTTTTGCTTTTCTCAATCAATTTTTTGAATTTCCTTTAAA
TATTCCAGAATTTAAAGAATTATTTACCCCAGAAAAGCTCAGTAATGAGG
AAAAAGAATTAATTGCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:20)
>11A01>msp12>>bankit478790>>>AY134431
GGCATCAAACAATCTCCTCAACAACTAATAAACTCAAAAAACACCCCAAA
ACAACCCTAAAAACAACCCAAAAATGTCCATCTTCCCTACTTCTGCTCTT
CTAGTCATTTCAATAATCGCTATGACCGAGGGTGCAGGCGATCGAAGCGC
TTCAACCTCTACTGGTTGTACAACCTATTTTGGCATGCTTGATCATGCGG
ATACCAAGGAAAATAACAAAAGAAAAACTTTCAAACCCAACGATAAAACC
AAATCCAACACCTTGCAAGTGACTGGTGGGGCAATGTTCAGCAATACCTC
GGTGGCGTTGGTTGTCGGTGATAAGGCGTTATGTATGGCTAAGACAGGAA
TTTATCATTTCTGATAATATTACTGTTGAAGTGGGTGTGGGTTTATATAA
TTGGTGCCAGACAAATGGAAAGGCCCCTGTCACTAACATACCATCCGGAG
CGTTCATGTTGCCCCGGAAGTAACTGGTGGCCCACAAAAGGGCAATCACT
ATTGATTACAACCCAAATATCTATAGGATTTTGACATTTTCTGGCATAAT
TTAGGTATTTTCTGACATTTTTCTGACATTTTTAACTAGAATTAATTCAA
TTGAAAACAAAATAATAGGATTGACCTAAATGAGCGTTTCTTGGATATCC
TTTTAACAGGAGCAGTCTCTAATTTTGTAAGAGCTCCTAATGTTTACCCT
CCTCCATCTCCCTCCCCCTCTATGCTCCTACCAATGACTGATTAAGTTAA
AAATCGTACATAAAATGGAGAGTGTATAAATCTGGGTGTATATACAATCA
GGATTCGACTTTATAACATTTGAAGGTTCCATTTGAAGACGTTTTTTTCT
TCACCGACAACAAGTGTGTCATCCAGCTTGTAAGCTACGATAATAAAACT
AATAAAACTCTTCTCAAAATTAATGATGTCGACTTCAAAATTATCCCTAC
TGATAAGAAAATTTCCCCGAAGGCTTCTACTATGAAAATGTGAGCTTGTA
CTATGAAAATGTGAGGGAAAAAAGTAAAGAAAAGAATAACAAAAAGTGTA
AATATGGAAGGATAAAAACGAAACAAAAATGAATGTGAAGTAAAAAATAA
AAAGAAATTCAAGTAGATTTAAAAAAAATGTTAAGCTTCACAATATCTGT
CTCCTTTTGTTTATGTTTTTCGAATAAATCGCATTACCAAAAAAAAAAAA AAAAAAAAAAAAAAAA
(SEQ ID NO:21)
>12H03>msp13>>bankit482577>>>AY134432
GAATCACAAAAATGGCCACCTTTTTCACTTTTACCCTTCTAATCATTTCA
ATTATTGCCACAACTGAGGGAATGAATACTAATCGAAGTGCTTCAACCTC
CGATTCTCTCAAAGACCAAAAGGATTGTAAAGTGATATATGGCATGTTTG
TGCCTGTAGCAGGGTCAAAAATGCATGGAGACGCCAAAAGCGCAATGAAG
CCAAACAATCCAAGTCTCCCCAATAAATTAATTGTATCAGGTGGCAACTC
AAAATATTCAGTGACTTTACAGGTTGAAAACCAGCCGAAGTGTGTTGCCC
AAAATGACGGAAACCCTGTAGAATGCCAAATTCAAGGAGACAAACTTTCA
GGAAAATTGATTTATGATATTGAAAACGGCCCTTCTGTCAACGTTCCCTT
CAAAGACACCCCAATCTTTGTTGGAAATAAATGCGAAATTGTTTTTGTAG
CCTACGATAAGGACCACAAATTAACTCTTCTTATGAATAAAGTAAAGCTG
ATGATTGAGCCGACAAATAAGCAAATTGTAAAGGCTTGTGGAGCGAAAAA
TTATATGGAAAAATGATGAATGAATGAATGTGGGAGGGAAGGAAATGAAA
AATATTTTTAAAATTGAAGAAAGCATTCAAAATTTAAAAAAAAAACAATT
CTTCAAATAATATATAACTTTAATATTTTTGATAAATTTTATTTCATAAA
AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:22)
>13A12>msp14>>bankit478806>>>AY134433
GGCATCAAACAATCTCCTCAACAACTAATAAACTCAAAAAACACCCCAAA
ACAACCCTAAAAACAACCCAAAAATGTCCATCTTCCTTACTTCTGCTCTT
CTAATCATTTCAATAATCGCTATGACCGAGGGTGCAGGCGATCGAAGCGC
TTCAACCTCTACTGGTTGTACAACCTATTTTGGCATGCTTGATCATGCGG
ATACCAAGGAAAATAACAAAAGAAAAACTTTCAAACCCAACGATAAAACC
AAATCCAACACCTTGCAAGTGACTGGTGGGGCAATGTTCAGCAATACCTC
GGTGGCGTTGGTTGTCGGTGATAAGGCGTTATGTATGGCTAAGACAGGAA
GTCCAGACGATTGCGGAATGCGCTACGATGCTTTGACTGGAACAATGAAA
TTTATCATTTCTGATAATATTACTGTTGAAGTTCCATTTGAAGACGTTTT
TTTCTTCACCGACAACAAGTGTGTCATCCAGCTTGTAAGCTACGATAATA
AAACTAATAAAACTCTTCTCAAAATTAATGATGTCGACTTCAAAATTATC
CCTACTGATAAGAAAATTTCCCCGAAGGCTTGTACTATGAAAATGTGAGC
TTGTACTATGAAAATGTGAGGGAAAAAAGTAAAGAAAAGAATAACAAAAA
GTGTAAATATGGAAGGATAAAAACGAAACAAAAATGAATGTGAAGTAAAA
AATAAAAAGAAATTCAAGTAGATTTAAAAAAAATGTTAAGCTTCACAATA
TCTGTCTCCTTTTGTTTATGTTTTTCGAATAAATCGCATTAGCAGCAAAA
AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:23)
>14E06>msp15>>bankit478812>>>Y134434
GAAATAATCTCCTCAACAACTAAAAAAACTCAAAAAAACACTCCAAAACA
ACTCTAAATGGCTTTCCTCTTCACTTCTACCCTTCTAATCATTTCATTGG
CTTTTATTGCCATAGCTGAGGGAGCAGGCGATCGAAATGCATCAGCTTCA
AGCCCTGGTTGTATGCAGGTTGCAACCCTTATTCATATAGGGGAAATTCG
CCCAGCAAAAGCAAACAAACCAGGTGTACAAAATACTCTAAAAATGTCTG
GAAATGTTCAAACATTCAAAACTACTCAAGTGACATTACAAGTAGCTGGG
CAAGAGCCTTGTACCGTTAAAATTAATAATGGCGAAACCAAATGTAAAAT
AACCGGAGATGAATTAAATGGAAAATTAATTTTCAAAACTGAAAAAGGAA
CTGAAATTTCTGCTTATTTCGAACTGGTTCCATTATTTTCTGAAAATAAG
TGTGTTATTGAACTTGACACTTATAACAAGGAAACCCATGAAACTAAACT
TATAATTAATGGAAATAATTTTATGATTAAAAAGAAGGAAGGTAGTGTTT
CAACTAAGTGTGGTGGAAGAGCTAATACTGTTTAAATTTTAAAAGTGTGA
ATTGAAAGAGGAAGAGAATATAAACAAATGTGAGGATGAGAAAAAAATAT
TTTTGAAGAAAGCATTACAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:24)
>16D10>msp16>>bankit478814>>>AY134435
GAGAAAATAAAATATAAATTATTCCTCAAAAATACCATAAAGTTAATTAT
TCTTCAATCAAAAAAATGTTTACTAATTCAATTAAAAATTTAATTATTTA
TTTAATGCCTTTAATGGTTACTTTAATGCTTTTGTCTGTCTCATTTGTGG
ATGCAGGCAAAAAGCCTAGTGGGCCAAATCCTGGAGGAAATAATTGAAGA
AAAATGATTGAAGAAAAACGTTTAAATTAAACGATAAATGGGAAATAATG
GAATTTAAATTAAGCTAATTTTGATGGTTTCCTTTGTTAATTTCAACATA
AAATTAATTGAATTTACTGAATAAAATTATATCTGAAAAAAAAAAAAAAA AAAAAAAAAAAAAA
(SEQ ID NO:2)
>16E05>msp17>>bankit482587>>>AY134436
GATTCAAAAAATATTATTTAAAAATTCTTTACCATTTAATTAACAAATTG
TAATAAAAGAAAGACAATTAAAAAATGAGTCCTTCCTCATTCACCTTAAC
GGCAGTACTTCTTGAGGCGATTGTTTTTCTTTACAACCGTCAAGTAGCGG
CAATGCTTTCCATGCATCCGAGCTGTTCTGGCCGTTCATCAACCATTGAG
AATAAATTGAAAATGAGCGGGGGTGGTAACGGCATCAATAAATTTACACC
GGGAAATGTTTCATTCCCGGTAGCATGCCAATACCATTCAAAGAATCTCA
AAGCAACAAATAAAAAGGAATATAAAATCTCAGAAGATTTGCCTATGAAT
CAAGAAAAGCTTACAAACAGTAAGGAAGATGATCTCATTCATAAGGTAAA
AAAGATAGATAAGGGCAATGGAGCTGCTGTTCCTTATAAAACAAACAAGA
ACAATGAAATTGGAGATGGAGCCGAGAATGGAAAAGCTGTCAAAATTAGA
GAAATTATTTTTACTGAAGAGCAAAAGAAAATGACTAGCGAAGAATTTGA
GCATTATTTGTATAGTGTTCCATATGACAAAAACAAGAAAAACAAAATTG
GAAAAAACGAAAATGGTGAAAAAGTTGATAAACCAAGCAAAGAAGGAGGA
GATACAATGTTTTATTCAAAAGCTGGGATAATTGCTAAAAAGATAAAAGA
ATATGTCCCCACTAATGGCGAATTCAAGATCCAGACTGGACTTGTATATC
GTAACAATAGTTTTAATGCTTCCCAAGATGATAGTAAAAATTTACTAAAT
ATTTCGCATATTTTAATGGCTTTAAATGAAAATGAGAGGGATTCTCAAGA
AAATTTGAGAAATGCTGCTGATTTGTTTGTGGCACTTCATGAGTGTTACC
AACTCTTTTCGGCAATTCCTCTAGTTTTTGAAGTAGAAATGGTTTTGAAA
AAACTTGAGGAAGAGGGAAACAAAGACGATCCAATAAAATTACTCGAATA
TTTCCGTTTGCCAACAATTAAATATCCATTATTGGATTTGATTAAAATTG
AGAACTCAACTGTGTCTCCAGATGAGTTGATTGAAAACGTCACTAAAACT
ATTCACAAAGCAGACAATTTTATTGCTAAAAACATCCATGCATTCTTCAT
AAATGACAACGAAACATTTTTTAATGAAATAATTTCTCGTCTTGAAACAG
CTGATATGGTTTTGGCCAGTATCAAAAAAATTCTTAAAATGTTCAATAAC
TTTAATGAGAAAATTCCCGAAAACTTTTCGATGCTAAAACGTTTAAAACC
AATTGAAATGCACGATTTATTCGAAAATTCTAAACTGCTTCAAAAGCTTC
ATGCAGCAATTTTGCCTGGAGATGAAATGAAATTTTGAAATGAGAGTTAA
ATATTTTTAAAAAAATTTTGCGAACACAAAAACAACAACAAATTAGAAGA
ATATTAAAATTATTAATGAAACAAGAGTTGCCGCGGCTGATCGGAAATAT
TAATTAAATCCAATTTAGCTGACGTTGCCTGCTCAATCACCAAATAAATC
AATTTATGATTTTGCCCATTCTCTATCATTACCTTATTTCCTATTTGTAC
ATTTTTTTTTCTTTTTTAAAAATTATTTTTAGTTTTGTTCTTGAATGTTC
GCTTAAATAAATTCTAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:25)
>17H02>msp18>>bankit482591>>>AY134437
GATCAAACAATCTCCTTAACCACTAAAAAACTCAAAAAACCCCCTAAAAG
CAGCCCAAAAAATACCCAAAAATGGCCATCCTCTTTACTTCTACCCTTCT
AATCATTTCCCTTTTGGGAATTACCGAGGGAGTGAATACAGGCATTCCGA
GCGGATCTTCTCCACCCTCTTCTGCTTGTGAGACTTACAAGGGCAAAATT
GAGCACATGCCAGAAACCGCCAGAAAAATTGAATGGAAGGAAAATACTCC
CGGAGGAAAGCATTTAATCCTTAAAAAGTCTATTCAAGGTCTAGACAAAG
TAACCCTCAAAATTGAAGGCAAAGAATGTAGTGCTTCCCTCAACAACCCT
GGAACATGTCAAGTCGATGGACAGTCCCATGCCGGTCAATTAGTCTTTGT
AACTTCAAAGGCTAAAATTGAGGTTGACTTTGGGGAAGCTCAAATCTTCT
CTGGGAACAAGTGCGAGATTGAAATTGAGAAGTATGACCGTGCTACCTAC
GTAACTCTAATCAAAATTAATGGGGGTGACTTCAAAATTACGCCTGATTC
GCCACCTATGCCGATGCCATGCAAAAATATGATGAACTAAAAAGTGAGGA
GGAGGGAAGGAAAAGTGAAGGAAGAGACCATGTAAAATTGAAATATTGAA
GAAAGCATTCAAAAATTTAATTTTTAAAAAATCTGTTTGTTTAGTAACTA
AATATAGCTTTCTATTGTTCTTATATTTTGTTTATCTTTATCAAATTAAA
ATGAAAAACTCAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:26)
>19F07>msp32>>bankit48054>>>AY142116
GACATTCATTTAAACATTCATTAATTACCTAAAATTGTTTTTCAAATTGT
TTGCCTCTGAGTTTTGCTCAACTGAGAAGAAAAATGCTTCCTTACTCAAT
TCTATTTCAATTGGGAATAGTTTCGTTGCTTCTACCTCATGCAAATGGAA
TGCAGTCTGGCAGTAGCAAAATTATGAACAAAGCATCTGAAAAGAAATAT
GCTTTGGTTGTTGCTCCAAACTTTCTTAAAGTTCATTTTAAAATGAACAG
TGTCTTTGCCAATGCGTTGACCAAAAAGTTTTTTGTGCACTTTCTAATTC
TGAACACCAAAAATGAAGAAATTGGAGATAATTTCGACTATGGAATTGAT
CTCGAAAAATTTGAAGAAGGAACGGGAAATACATATCAAGTTGTAAATTT
TCCAGATGATTATCCCGAAAAATTGAACGAAGGCGTGAAGAATTTAGAGA
ACAAATTCATTAAGAGAGGTTACGAACAGAGTAGTCAAATTCTGAAAAAT
GAAGCTTTCACCGTTTATAAAGATTTATTTGAAAACAATGGAGCTATTGT
TCATTACTTGAAGGAGGCAAAGTTTGATTTAGGGGTTTTTGACACTTGGG
ACACTGGAGCTCTCTTCATTCTCCATGCAGCAGGAATTAAAAATGTTTTT
GGCATTAACAACATTCAACTTAATGCTTATCAATTTAAATATGCTGGGAA
AGAATTTCCAAAAAATATTCCAGAAATTTAATTCGGCACAAACAGGCGAT
AATGAATTATCACCAACAAAGGAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:27)
>21E02>msp19>>bankit482599>>>AY134438
GGCATCAAACAATCTCCTCTCCTCAACAACTAAAAAACTCAAAAAACACC
CCAAAACAACTCTAAAATGTCGGCTCTCCTCTTCACTTCTACCCTTCTAA
TCATATCATTGGCTTTTATTGCCATAGCTGAGGGAACAGGCGATCGAAAT
GCATCAGCTTCAAGCCCTGGTTGTATGCAGGTTGCAACCCTTATTCATAT
AGGGGAAATTCGCCCAGCAAAAGCAAACAAACCAGGTGTACAAAATACTC
TTAAAATGTCTGGAAATGCTCAAATATTCAAAACTACTCAAGTGACATTA
CAAGTAGCTGGGCAAGAGCCTTGTACCGTTAAAATTAATAATGGTGAAAC
CAAATGTAAAATAACCGGAGATGAATTAAATGGAAAATTAATTTTCAAAA
CTGAAAAAGGAACTGAAATTTCTGCTTCTTTCGAACAGGCTAAATTGTTT
TCTGAAAATAAGTGTGTTATTGAACTTGACACTTATAACAAGGAAACCCA
TGAAACTAAACTTAAAATTAATGGAAATAATTTTATGATTAAAAAGAAGG
AAGGTAGTGTGTCAATAAAGTGTGGTGGAAGAGCTAATACTGTTTAAATT
TTAAAAATGTGAATTGAAAGAGGAAGAGAATATAAACAAATGTGAAGATG
TGAAAAAATATTTTGAAGAAAGCATTCCAAAAAAAAAAAAAAAAAAAAAA AAAA (SEQ ID
NO:28) >25B10>msp33>>bankit487909>>AY142118
GACATTCATTTAAACATTCATTAATTACCTAAAATTGTTTTTCAAATTGT
GATTTTATTTATTTCTATTTAATATCTTTAAATGCGGAGTGCTTTAAAAA
CTTTAATTGTTTTGTGGCCTCCTTTGCTTGGACATTTTATTTTGTTAATT
CCTAGTGGAGTAGCTTTTGTAGTTAAAGAGAATGTTCAAGAAGTATCGCC
TGTTATTCCTGATAAACCCGGAGTAATTGGAGGTGATGTTATTGATAAAA
GCGCAAAAACTAGTCAACTAAAAAAGGGAAGTGAAAGTCTGATTTCTGGA
ATTGAACGTAGCCATGTTGAGGAATTAAAGGAGGAAATTAAAGGAGAAGG
TAAGAAAGTACCCAAAATGAATGGACAGGATAATGAAAGCCTTGAAACTA AAATTGTTGAAAAG
(SEQ ID NO:29)
>B04>msp35>>bankit484130>>>AY142119
GATCAACCAATCCCCTCAACAACTAAAAGACTCAAAAACACCCCAAAACA
ACTCTAATATGGCTCTCCTCTTCAGTTCTACCCTTCTAATCATTTCATTT
ATTGCCATAGCTGAGGGAGCAGGCGATCGAAATGCATCAGCTTCAAGCCC
TGGTTGTATGCAGGTTGCAACCCTTATTCATATAGGGGAAATTCGCCCAG
CAAAAGCAAACAAACCAGGTGTACAAAATACTCTAAAAATGTCTGGAAAT
GCTCAAATATTCAAAACTACTCAAGTGACATTACAAGTAGCTGGGCAAGA
GCCTTGTACCGTTAAAATTAATAATGGCGAAACCAAATGTAAAATAACCG
GAGATGAATTAAATGGAAAATTAATTTTCAAAACTGAAAAGGGAACTGAA
ATTTCTGCTTCTTTCGAACAGGCTAAATTGTTTTCTGAAAATAAGTGTGT
TATTGAACTTGACACTTATAACAAGGAAACCCATGAAACTAAACTTAAAA
TTAATGGAAATAATTTTATGATTAAAAAGAAGGAAGGTAATGTGTCAATT
AAGTGTGGTGGAAGAGCTAATACTGTTTAAATTTTAAAAGAGTGAATTGA
AAGAGGAAGAAAATATAAACAAATGTGAAGATGAGAAAAAAATATTTTGA
AGAAAGCATTCCAAAAAATAAAAAAAAATTAATTCTTCAAATCCATTTAT
TTTTTGAATAAAACATTTTACTAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:30)
>30G11>msp21>>bankit482611>>>AY134440
GATTTTTATTAATTTTAAAAATTATTAACTCTCCAAAATGAAGTGTTTGC
TCCCCTTCTTTTGGATTTTATTAACAATTTTTGTTTCTTGCACTAATGGC
ACTTCAAATGAGTATAGTGAACTTGTTTTGGTTCAAGCTTTGTGGAGACA
CGGTGATCGTTCACCCACAAAAACCTTCAAAACGGATAAATATCAAGAAA
AGGATTGGCCTCAAGGATGGGGGCAATTAACACCTACAGGAATGGCTCAA
CATGTAGAGTTGGGAAGACGACTAAGACAGCGATATATAGAGGAATTGAA
ATTTGTTGGTCCTCGGTATAATAGCCATGAAATTTATGTTAGAAGTACTG
ACTGGAATAGAACATTAACTAGTGCTATATCGAATTTTATTGGCTTCTAC
GGCCCCGGAAATGATGATGAATACCCAAAGGATTTGGGCGCAAACAAATG
GCCAGGATGGTTTTTCCCAATAGCGATACATTCACTCCCTGGAAACGAAG
ATTTTATGGCTCCTGGAGAATCGGAATGTAAACGATTTGAACAAATAAAA
GAACGGATAACTTTAACAGAAGAATACAACTCGACTTTGATTAAATACAA
ATGGCTACTCGATTTTTTGAGTGAAAAGACGGGACAGAATGTCGACCCTT
TCGATATGTGGATGATTAACGATGCTTTTTATATTGAGAAATTAAAAGGC
AAAAAATTGGTAGACTGGGCAGAGGGGAACCAAACACTTTTGGATACGAT
TGCTGAACTTGACAATTTACAAGAAAGATGGATGGTTGGGTTAGATTTAA
AACCTCTGGGTGATGCCAACTTTCGCGAAGAACTTCCAAAAATTTTGGGC
GGGCCAATCTTATGGAAATTTATAACAAATATGCAGGAGAAGTTGGCTTG
TTCAAAGCGAATGAATTCTGTAAAAGAAATTGACAGGGAAATAGAGGGAA
GAAAATCGCCAATGGGGACGCCCTTGTGTAAATGGATGAACAAAATGCGC
TATTTTGCGTACTCTGCGCACGACAGCACAATTATTGCAATTTTTGCAGC
TTTGGGTTTAAACAAAACGAATTATGACGAGGATGGTTACCCGAAGTATT
CTACTTGTGTAACTTTTGAATTGTGGAGGGAGAAGAATACTGGTCAATTT
GATGTTAAGGTATTTTTATGGAGACCTAACACCAACGAGACTTCCCCTAA
AGAAATAACGACAGATATTGAAGGCTGTCAAAGCAATTCAACTCTAGAAC
AATTTGTTGAAAGATCAAAAAATTATCAAATGCTGCCTTCACCCAAAGAC
TATTGTTCACAACTTCTACAACCCCTAAATAATGCTGCACGTATGTTAAT
TCAGTGGAAATTGGAAATGCTTATTCTAATGGGAATTCCTTCAATTGTTG
CTAATGTTGTATAGAGAATTTTTTTGTTTTTGGAAATTATTTAGTTGCAC
CTATTCATCAAAAGAAGGGCAAAATAAATTTTTATCCCCTAAAAAAAAAA AAAAAAAAAAA (SEQ
ID NO:31)
>30H07>msp20>>bankit478826>>>AY134439
GATGCTTCAATTGACATTTAAAATTAAAAAGACCCAGGTTTCTTAATTAA
AGAATGTTTTTAAAAATTTTAACTTTCCTTTTAATTATAAACAAAATTAT
TGCGGATGATTCTAATAGCGGAGATAGTGGCAATGAAAATTCTAATAGTA
AGCCCAGTGATGAGCTTGCCGACTCTGTTGATGTTCGAGAGCATGATAAT
GAGCAACATCCATCCAATTCGATCGACAAGCAAAATCTTCAAGACCCACA
ATTTATTAAAGAAGATGTTACAAATGTATTGCCACTATTAAATAACGATG
AGAATAATCTCATTGATGAATACACAACAGAAAAAATTAAAGAAGATGAG
GAAGACCAACTAAATAATGAAGGATCTGGTATAGACAATGAATTTCCTGA
GGAAGATAATGATGTAAATGGATTGGATATTAATACAACTGCAAAATATG
CTAATGATGTAGATGATAATAATAACAATGAAGGTGATGGTCAATCATGC
GTTTATGAGGATGGTGTAATTAACGATAATGGTGACGAACGCACACCTAC
ATATGAAGAACAACAACAAATTGAAGAATATCTTCAAGAAATGCGTGAAT
TTGAAGAGCAAATGGTCAAAGACAGCGCTAATTTTATGAGAAATTTGGCA
CAATTTGTGATGAGTCAATTCGAAAACATTTTTGGTTCTTCTACCTCGTC
TCTATCAGGTAACAATAATAATTTGTTGGAGAAAAAACCTTTAGAAGCAC
CAACACCTCCTTGTTTATGCAAAAAATGTGACAGTATGACATTTATACAA
AATAAGCAAACCAAATATCTTAAAAATTTTGCTAATTAATTAACAAAAAT
TTGAAGAATAATTTAAATAATGTTTATCTCTTTCTTGAGATTTTCAAATT
ATTTAATCCATTTATATATAAATTTAAATTCATTTTCTTTTACAAAAAGC
TGAAGAGATTAAATTTTAATGTTTGAAAAAAAAAAAAAAAAAAA (SEQ ID NO:32)
>31H06>msp22>>bankit482615>>>AY134441
GGAATAAAAAGCGGCGAAATTTTACTTTATTCATCAAAGTACTTTAAAAT
ATTCTATAATCTAAAATGAAATTCAGCCAATTATTTGTTCTCTTAATAAT
TGCTTTACAATTTGTGGTTGCTCAAGGGTTGATTTACGATGCGAAAGCAA
TAGCCAAAGGAAAAGGAAAACCGTTCAGGGCGCTGAATATTTGGTATTTG
TCTTGGGCAACTATGTAATGAAGAAACGATATCTTTATGTAATTGATAAA
TAAAAATAACCTAAGTATCAAAAAATGTTTATGGGAAATAAAAGATTTAT
CTTCATTTAAAATCTAATAAATTTGTCAATCCCAAAAAAAAAAAAAAAAA AAAAAAAAAAAA
(SEQ ID NO:33)
>34C04>pectinase>>bankit476418>>>AF527788
GGTTTAATTACCCAAGTTTAAGGGGTAAAAAATGTTTTCAAGCAAAACTA
GCTTCAATTTCCTTCTTCTAATTTCTTCATTTGCTTTATGTAAGGCCGAC
TTTTGGCCTAAAGCAAGAAATAATATTACGGTATCCGAAACAATACAAAT
TACTAACCGTGACTGTAATTTTGATCGTTATATTCCCGATCCGAGTAAAC
TTGGAAACGGAGGTCAGAACGAGCATCAAGGCTACGTTTTTGAAATAAAA
AATGGTGGTTCTTTATCTAATTGTATAATTGGTGCTAGGCCTGGGACTAA
AGGCTCTGCTCATGGAGTTCTTTGTGATGGAGATTGCGATATAAACAATG
TTTGGTTTGAGGATGTTGGGGAAGATGCTATTAATTTTAATGGAGATTCT
GATGGTTGTGTTTATAATGTTAATGGTGGTGGTGCTAAGAATGGAGAAGA
CAAAGTTATGCAATTTGACGGAAAGGGGACACTGAATGTTAACAACTATT
ATGTAGACAATTATGTCCGTTTTTGTCGCTCCTGTGGCGACTGCGGTGAC
CAACATCAACGCCATATCGTGATTACTAATCTGACAGCGGTTCATGGCCA
AGCTGGTCAATTCGTTTGTGGAGTAAATAGCAATTATCAGGATACGTGTA
CCTTGCATGATATAAAAATGGAGAAGGGTATTCACCCCTGCAAGGTTTTT
GATGGCAATTCTGATGGATCTGAGCCAACTTCGAATAACGACGAAGAGGA
CCACGGAGACGGGAAATTTTGTATTTATAAGAAGGGCGATATTAAATATA
TTGGATCCAAACCAAAGCCGAAAAGCAAAAAGAGCGCAAAGAATTAAGTG
CCGGAAGTTAAAAAGCCTTGAAGTTAAAAACGTTTAAAGGGATAAATTGT
AGGGTTGTCGGTTCTGAACCGAACCGAGCCGAAGAACCGATGATTTTTCG
GTTCGGTTCCGGATATCCAAAGATTTTCCAAGAGCCGACAACCCTAGTAG
TATGAGTAGAATCTATTATTATTTGGAATACTAATTTAATTTTGTGAAAT
TTCTTTTTACTATATTAATCCTGTCCAATAAAATTATGAAATCGAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:34)
>34D01>msp23>>bankit482619>>>AY134442
GGCATCAAACAATCTACTCAACAAATAAAAATCCCTAAAAACACCCCAAA
ACAACCCTAAAAGCATACCAAAAATGGCCACCTTTTTCACATTTACCCTT
CTAATCATTTCAATTATTGCCACAACTGAGGGAATGCATACTAATCGAAG
TGCTTCAACCTCCGATTCTCTCAAAGCCCAAAAGGATTGTAAAGTGATAT
ATGGAATGTTTGTGCCTGTAGCAGGGTCAGAAATGCATGGAGACGCCAAA
AGCGCAATGAAGCCAAACAATCCAAGTGTCCCCAATAAATTAACTGTATC
AGGTGGCAACTCAAAATATTCAGTGACTTTACAGGTTGAAAACCAGCCGA
AGTGTGTTGCCCAAAATGACGGAAACCCTGTAGAATGCCAAATTCAAGGA
GACAAACTTTCAGGAAAATTGATTTATGATATTGAAAACGGCCCTTCTGT
CAACGTTCCCTTCAAAGACACTCCAATCTTTGTTGGAAATAAATGCGAAA
TTGTTTTTGTAGACTACGATAAGGACCACAAATTAACTCTTTTCATGAAT
AAAGTAAAGCTTATGATTTCCCCGACTGATAAGCAAATTGTAAAGGCTTG
TGGGGTGAAAAATTAGAAAGAAAAATGATGAATGAATGAAGGTGAGAGGG
AAGGAAAGAAAAAATATTTTTAAAATTGAAGAAAGCATTCAAAAATTAAA
AAAAACAATTCTTCAAGATAATATATAACGTTTAACTCTTTTTGATAAAT
TTTATTTCAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:35)
>34F06>msp24>>bankit482623>>>AY134443
ATTGAAGAAGATGATACATTCAATGTTCCTGTCATGGGAGAAGAAAATCA
TAGAGATATTCCTGTCGAAGAAGCTAATTATCAAGTTCCTCCTTCTGCTG
ATTTCACTTTTACAAGCTCTGAGCATGGAAGACGTACATCATTTACAGTT
GGAACACCGCATCATCGATACATGCAACCAGGCACTCGAGAAGCATATTT
ATTGCCCCATCCAGGAGGGGAAGGTGCAACGCTTATACGCAATGAAGTTC
GTCGAGATGGAACGCAAATTTCCCAACAAGACACACTTCAAAACATTGAA
GGAGGGAGAGGTTATGTTTATTCTTCATCGTCCCACACTCAAAACGAATC
AAGTAGTAGTTCAAGAATAACTTCGAGAATTCGTTTTGGGAATAATGAAA
GACATGGGAAAAAATGAGGAATAAAGGGAAGATTTAGGAAGACATGGAAA
AAAGTGAGGAATGGAGGAAGAGATTTATACTAATTATAAAATGATAGAAA
AATTGAAGAGATATTCTCTTATTCTTTCCTATATCTTTACTTTCACATAC
AAAATTCTATAATGGCAATTTATGATTTAACATTAAAATTGAATTTAGAA
ATATTTTTTAAAATTATTTTAGTTTTCATTTTTATCAATTTTTTGATATT
TAAATACGTCTTGTATTTATCTTCATATAATTGTTGATTAAACTTTTCTT
TATCATTCTTTTGTAGGTATTCTAAAATTAAATAATTATATGTAATATTT
TTTAATTTTCAATTTGAATAAAATTTTCTGCAAAAAAAAAAAAAAAAAAA A (SEQ ID NO:36)
>35A02>msp25>>bankit478844>>>AY134444
GACATTCCTCAGCTTCATTACCCATCCATTTTTCATAGACAACATCCCCC
TTGCCAAACATTAAAAATTGAGTAACGCTGAATGAAGCTTTTTGTCCTGT
TAATTGGAGTTTTAGCCTTCACGGTTCTAAATGTCCATGGAGGAGTCAGC
CATTCGACATTGACTCACAGAAACCCGCGAAGCAACGAAATCGAACAATT
AACTGATGTGTCGTTGGACGATACCCCATCCTCGCCTCCTCAAGCTGTGT
TGGACATTGGAATGTCAGGACAGCGAAATTTGCAACGTCGAGAAGCTCCA
ATGTCGATTGGGAAAAAAGTGGTGGCTGTAATTTTTTTATTTCTTCTGTC
TTTTACATCGTTATATCTATTGGCCGTGCCAAAACAACAAATTCAACAAG
TTGAATACAAACAATTTCCACAACCCTATAAATTTGTTCCGATTAGTAAC
ATTGTCAAGTGCGACAGAAAAACACGGCAATGTTCAATAAAGTTAGAGAA
TTTGGATCCGACAAACAACTATAGCCTCTATACTGCAAAGAATGACAAGG
GGAGAGGAGATAAAGTAAAGTTAACTAAAGTTGCAGATATAGATTTGGAC
AAATGTCAACTCGACAAGAATGTAAAACCAGAAGTAAATGGGGAAGAAAT
TTGTAATCAGATTGTCAAAGGAATTGATGATAACGCAAAAGCCGAAACTA
TTGAGGTTAACAGTGGAGAAATAGAATTTGGTTCGGAATTAGAAGGAACG
GAGGATTATGCGATAGTTGAAAAAGCAATGAATGAGAAGAATGAACATAA
AAATCAACAAGCGGTTGAGCATGTTCATATCCCTGGGCCAGGGGAACAAC
CAGTTGAACACAATCAGCCGACAATAGAATATCCAACAAATTCCAAACAA
GTTCATCCAGCTGACAAATATCAACATAAACTAGAAGAGCGCGCCAAAAA
ATTTGGGCTTAGCGACTTCAAACATGGAGATTTATATGAGGATTATCGCC
AACAAAAAACGGTCCAAGAAGATGAAAAGGATAAACGATATCAAAAGGTT
CTAGGAACACTAGGAGACCATAAACATCCATCGCTAGTTGATCAATATAA
CGAAGATAAAGGAAAATTCAATCAACGTGTTAAAAGTGACCCCACAGGCA
ATAAAGTTGAAAAGGCAAAAAATTCTGATTCTAATGGACTTGAACAAAAA
TTAGAAAAACTGGCACTGAGTGACTTCAAACATGGAGATTTATATAAAGA
TTATCAGCAACAAATCACGGTCAGAGATGATGAAAAGGATAAACGATATC
AAAAGGTTCTAGGAACACTAGGAGACCATAAACATCCATCGCTAGTTGAA
CAATATAACAGAGATAAAGGAAAATTCAATCAACGCGTTAAAAGTGACCC
CACTAGCAATTGGCATGAAGATTTATTCGGAAAGGATTACCGACGTGCTA
TGAGCGATTTCGATCATTTAAAGGCTAAACAACGTGAAAAGATCCTTGGA
ACACTAGAAGATCATAAGCATCCATCGCTAATTGATCAATATAACAAAGG
AAGCTTAAATCAACGCGCTAAAAGTGACCCCACAGGCAATAATATTGGAA
AGGCAAAAAATTCTAATTTTAATGGGTCTGAACAAAAATTAGAAAAACTG
GCACTGAGTGACTTCAAACATGGAGATTTATTAGGTCGAAAAGGAGGAAT
TAAACAACGCACTATAAATGTTCTCGCTGGCAAAAAAATAGAACATGAAA
AAGGAAGTGATTTTAATGCAAACGTTGAAGAAATGATAGGGGCAGAAAAC
GGCAAGGCTAATCAAGTGAATCCCAAATTAACTGGACGCAAACTAGCTGA
ATTTAATCATATTCCAGCTGTTGACAGAATTCTTGGTTTTAAACGTGGAG
GTCATGCGCTAGAGGAGCCTCATAAAAATTGAGATATTTTGCCTGAAGAG
TTGGATTGAACGATGTATATAAGATTTTTTAACCATGTAAATATTTTTAA
AAAAGATTTTATTAGAGCCAGGAAATTACGATACTGAATCCCGAAAAATA
TCGTAATGGCTCTTAATTTTTTATTTTTTAACTTTTCCATTGCAAAGATT
TTTTTAAAATTTTTCCCGATTGTCTGGTAAACTTGTGATGAGATAAACTG
ATTTTGATTGATAATAATCGTCCATTTTCCAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA (SEQ
ID NO:37) >35E04>>bankit487871>>>AY142121
TACCTAAAATTGTTTTTAAATTGTTTGCCTCTGAGTTTTGCTCAACTGAG
AAGAAAAATGCTTCCTTACTCAATTCTATTTCAATTGGGAATAGTTTCGT
TGCTTCTACCTCATGCAAATGGAATGCAGTCTGGCAGTAGCAAAATTATG
AACAAAGCATCTGAAAAGAAATATGCTTTGGTTGTTGCTCCAAACTTTCT
TAAAGTTCATTTTAAAATGAACAGTGTCTTTGCCAATGCGTTGACCAAAA
AGTTTTTTGTGCACTTTCTAATTCTGAACACCAAAAATGAAGAAATTGGA
GATAATTTCGACTATGGAATTGATCTCGAAAAATTTGAAGAAGGAACGGG
AAATACATATCAAGTTGTAAATTTTCCAGATGATTATCCCGAAAAATTGA
ACGAAGGCGTGAAGAATTTAGAGAACAAATTCATTAAGAGAGGTTACGAA
CAGAGTAGTCAAATTCTGAAAAATGAAGCTTTCACCGTTTATAAAGGTTA
AAATCCAAAATATTTTGCCTTCTAAAATTGTTATTTGATTAATAATATAT
AAAATATTTAAGATTTATTTGAAAACAATGGAGCTATTGTTCATTACTTG
AAGGAGGCAAAGTTTGATTTAGGGGTTTTTGACACTTGGGACACTGGAGC
TCTCTTCATTCTCCATGCAGCAGGAATTAAAAATGTTTTTGGCATTAACA
ACATTCAACTTAATGCTTATCAATTTAAATATGCTGGGAAAGAATTTCCA
AAAAATATTCCAGAAATTTATTCGGCACAAACAGGCGATAATGAATTATC
ACCACCAAGGGAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:38)
>35F03>>bankit487855>>>AY142120
GACATTTAATTTTTTAAATTTCTTAACATTAAATAAATTCAAAAAGAAAA
TTGAGAAAAAAAATCTTTTAATTTAAAAAAAAAGAAAAAAGAAAAATGTA
TCCTTGGACAATTTTTCTTTTATTAATTATTTTGTTGGCTATGGCCATTG
AAATAATTGGAGGAAAAGGTCGAAAGTTAAGGAAGAGAGACAAAGAGGAA
AAAGGTCATGCCTCAATTTTCTGTTGGGCATTCATCTAGGGAAGGTTTCG
AGGAAAAGCTTGATGAAATGGTTGAATCAACTTCAAATATGTTAATAAAT
CTTGGTAAAAAAGTAAAGAAAGGAGGGAAGAAAGTTGTAAAAGGAGTTGT
AGAAACTGCGCAGCTGATCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:39)
>42G06>cbp>>>AF049139
CAAGTTTGAGCGTCAGCAATTTTAAATTAAAAAAAGACAAACTATAAAAT
CTCTCTTATTTAAAATAAGCAGTATACCCTTCAATCTATCCACAATCCAA
TAAAACTTTCTAATAAAAATCCTCCACTAAAATGGCATCCTTTTTTTATT
TCTTATTTATTTCTGTTAGTCTTTTGATTCTAGCTAATGCTGATGATGCT
GGTAGATATCCTTCAGGAGATGATTTAGTTGAAGGTACTACTGCTGCTCG
GCTTCATTCGTCTTCTGACCTACCAGACGATGATGAAGAAGAATGCGAGT
GCGAAGATGACGACGAGACAACAGTCGCAACTCACATTTCTACACGCAGC
AATGGTTACCCTTCTAATAACGGAGCCCCCACTAGCACTAAACGCCCTTC
AAACAACGGAAGCTCAAACAATGGAGGCTCAAGCTCTGTCACAGGATCTG
TTATATTGAGAGATAAATGGGTAAATGGCGCCAATTGTATTTTAGCTTTC
AAGAATAATGGAAACGCTAGAGCATGTGGTGTCAAGTTCGAGCTGACTCT
CGGTGATAATCAAAGAATCCAAAGCATTTGGAACGTTGAGAAAGTCGGAG
ATAAAGTTTACAGAATTCCGGACTACATCCAACTTGGTCCAGGAGTCGAA
AACAGAGATATTGGAGTTGTTTACAATGATGTGCCAGAACTCTTCCACAA
TCAAGGTCTTGGACAAGAAGAAGGATGCAACATTATTGAATAAAAAATAT
GGATATAAAAATATTTAAAAAAGATTAAATAAGTATTATTAAAGCTTGTG
AATATAAACTTTTTCGAAATTAAAATAATGGCAGAAAAAAAAAAAAAAAA AAAAAAAAAA (SEQ
ID NO:40) >1C05B>msp36>>AY422829
TATTAAAAAAATAACAATTTCTTTTAAAAATAAAATGTATTCCCGTTCAT
CTTTAATTTCTTTTTTTCTTTTAATTAATTTAATTTTGACTCCAATGATT
TTGGCTACTAATAATGATGGTGTTGCTGCTCCGGTTGTTGCTAATAAAGA
TGCTGGGAAAGTTAGAGCGACGGAAATTATAAGAGCACTCCGTGGATTTT
GGAAAGGAGTGGCAGGTGGAGCATTGGTAGGAGGAGGTGCTGTTTTAGCT
GCACGGATATTTCGGAACGCTGGCCGGCGCGGATCGCCCCGACCCATCTG
GATGAGGTCCATCTGGATAATGGAGATGCAGCATGAGCGGTCCTCACAGA
CTTTCCTCGGTCGAACTGGACGCCGCGACGCTGCCGGCGGCGACCGCAGA
GATCGAGCATGAGCGCCGCGTGGCCATCTTCGATCTGGTCGAAAAGAACA
GTTTCGAGCCGGTCGGCGCCGAGGGCGGCCCGTATCAGCTGAAGCTGTCG
CTGCAGGACAACCGGCTGGTGTCCGGCTAAATTCGCATTTAAGGAAATTC
GATGTTTTTAATAATTTAATTTAATAAATTTGTTTTATCTTTAAAAAAAA
AAAAAAAAAAAAAAAAA (SEQ ID NO:41) >1C11B>eng-1>>AF100549
ATTAATTTTAAAAATCTAATTAAAAATGAATTCTCTCTTATTAATAGCAT
TTTTATCCCTCTCATTTTGTGTTCCAATAAAGGCTGCTCCTCCATATGGG
CAATTATCTGTGAAAGGAAGTCAATTAGTGGGCAGTAATGGACAACCAGT
TCAACTTGTTGGAATGTCACTTTTCTGGTCGAGTTGTGGTGAAGGAGAAG
TTTTTTATAATAAAGCAACAGTAAATAGTCTTAAATGCTCTTGGAATTCA
AATGTAGTTAGGGCTGCAATGGGTGTAGAGTATTCAGGGTGCCAACGACC
AGGTTATTTGGATGCCCCAAATGTTGAGCTGGGCAAGGTTGAAGCTGTTG
TTAAGGCCGCAATAGAGTTGGATATGTATGTTATCCTTGACTTTCACGAC
CACAATGCTCAACAACATGTGAAACAAGCTATCGAATTCTTCACATATTT
TGCCCAAAACTACGGATCTAAATACCCTAACATAATCTATGAGACTTTCA
ATGAGCCACTACAAGTAGACTGGAGTGGTGTAAAGTCATATCATGAGCAA
GTTGTTGCAGAAATTAGAAAATATGACACAAAGAATGTCATCGTTCTCGG
TACAACAACATGGTCTCAGGATGTCGATACTGCTGCTAACAATCCTGTAA
GCGGCACAAACCTTTGCTACACTCTACACTTCTACGCAGCAACTCATAAA
CAAAACATAAGAGACAAGGCGCAAGCTGCAATGAATAAAGGAGCTTGTAT
CTTTGTAACTGAATACGGAACTGTTGATGCAAGTGGAGGTGGTGGAGTGG
ATGAAGGTTCGACAAAAGAATGGTATAACTTCATGGATAGTAACAAGATT
TCTAACCTCAACTGGGCTATCTCAAACAAGGCAGAAGGTGCTTCAGCACT
CACATCTGGAACGAGTGCTTCTCAAGTTGGCAATGATGACCGATTGACTG
CCTCCGGTGTTCTAGTGAAGAAGTATATTAAATCAAAGAATACTGGTGTC
AGTTGCAATGGTGCATCACCAGGCAGTGGTTCAGGAAGTAACCCCTCAGG
AAATAAACCGAGCAACTCACAAACCAGCACTGCCAAAACATCAAGCAATT
CAGGAAATAAAGGCGGTAATTCTAACACAGGGAATAATGCAAATAACTCA
GGAAGTAAACCGGGCAACTCCGGAAGTAATACAGGAAATACGGGTAGCAA
TGCCGGAGCCAGTTCAGGAAATACGGGGACCAGTACAAGCGGTAGTTCTG
TTACAGCTTCAGTACAAGTTCCCGATAAATGGGATAATGGCGCAAGATTC
CAATTAGTATTTAAAAACAATGCAAGTACAAAAAAGTGTGCAGTGAAATT
TTCATTGACTTTTGCCTCTGGACAACAAATTACTGGCATTTGGAACGTTC
AAAATGTAACAGGAAATAGTTTTGTTCTTCCAGACTACGTTACAATTGAG
GCAGGGAAACAATATACAGATGCAGGAATGAATATAAATGGGCCAGCAAC
TCCTCCACAAATTAAGGTGCTCGGCGATGGAAAATGCGTTTTTTGAAATT
AAAGACTCCGTCTTAATTGTTGAATTATTTTAATCTTATGATTGTTTAAA
TTGGAAAAAAATATATGTATAATTTGCTTCTGTTAATTTTGTTTATTTTA
AATATACGATAAAAATTA (SEQ ID NO:42) 1D08B>msp37>>AY422830
TAAAAAATGATTTTTATTTCCTTAATTATCCTCGTATTGGCTGCTGAATC
TAATGAAGCAAGCACAAACTGCAAGGATGGTGAAGGCGCGGTAACCTTCT
TGTCCAACCAGCTCGGTAACATACAGGGAATAAAAGGAAATAGTTATTAT
AACAAAACTTGTTCCAACAAAAATACTGCAAAACGTTGCTACCCAAATGA
TGAATCAAATATTAGCGTTTTTAAAATTGTTTGCCCCACAAATATTTGTA
TTTGTGGTAATGTTGATAATCAATGTTACTCTGCAAAAACAGTTAATCCT
GGAGATTTAGACTATATGTTCTATTCTCATAGTGGCAGCATGTTTGTTAA
CCCAAATGTTGGTTCAATTTCATTATCGTCACCTGATAATCATTATTTTG
ATCCAAAGACTAGTGCCCCAAAATTCATGGAATTAACCCCAGGCACAAAA
TCATATCTTAATGGGAATGAGCTTTCTGTTGCTTGTACATCTTGTGCTAA
CTTTAAGCAGCTAACGTGTTGAACAATAAAAAAAAAAAAAAAAAAAA (SEQ D NO:43)
2B02B>peI2>>AY327873
GAAAATAAATTATTCTTTTTAAAATTATCAAAAAATGCCACATTTTTATT
TAAAATTTTTAATTAATTTAATTTTATTAAATTTATTCCCATTACTTATA
AAAAGCGATTTGTGTAAATTTCCAACGGCTAAGGGGAACCAAACTGTTGA
TGAAACAATACCATTAAATAAAGATAAAGATTTTGGGTTTATTCGTCTGA
TAGCTTCTCCAAAGTTGGGAAGTTGTACAATTGACTTTAGTAAGAAAATG
TCGCCAATATTATGGTTATCCGATGGGGTGACTGTTAGTAATTTAATTAT
TGGAACTGAATCTTCTTCAGGCATTTGGTGTAGTGGAAGTTGTACCTTGA
AGAATGTCTATTTTGAACGTGTTTGTACTCACGCCGCAGCTTTTAATGCA
ACAACAGACTTTACAAAAACTGATAGACGTTCATTTACATATACAGTTGA
GGGGGGCGCTGGACTCCATGCTTTAGATAAAATGTTTGTACAATCTGGCC
CCGGAAAGACAATAATTAATAATTTTTGTGGGGATGGATTCCAAAAAGTT
TGGCGATCGTGTGGGACGTGTAATGATGAAGTGAGTCAAAATTCTAAACA
AAGAACTGTTACTATAACAAATTCAAATTTTACTGGCAAAGGACATGTAA
TTGCATCTGGAAATGCCCCTTATAAAGACAAAGTTTCCTTCAATAATGTC
AAAATATTTGGTTATAAAAATCGTTCAACAAGAGTTGTTTATGCCTGTGG
GGAAGTTAAACCAGAAATAAGTGAAGATCATTTAGATACAGGAGCTTCAA
ATTGGTATATACCTGGACGTGCTGGTACTGGAACTGTTTGTAATTATCCC
GCTTCAGCAGTTAAAATTGTTAATTAAACATTAAAAGCTTGATATTTAGA
AAATAGTAATAATAAATGTTATTTATTGTGAATAAAGTTTTATAATTAAA AAAAAAAAAAAAA
(SEQ ID NO:44) 2G06B>cm1>>AY422834
GGTTTAATTACCCAAGTTTAAGACAATAAACTTTTTAATAAAAATATTTA
ATTTTGAATGTCTTTGAATTGGCTTTATTGCAATTTATTTATTGTAATAC
TCCTTTTCAACATTGTAAAGAGTGATACCGATACTAATGCTGATATTGAT
CGATTTGTTGAAATTGCAGACGATCGTTTAACTCTTTCTGATTATGTTGC
TTTATATAAAATTGTTAATAACAAAAGTATTACTGATCCAAAACGAGAAG
AAAAACTTTTGAACGATATGAGAAGTAAGGGAAAGAATCTTTCGTTAAAT
GAGGATTATGTTACTTTAATATTCCAAGACCAAATAAATGCTAGTAAATA
TTTTCAGAATTATTTGGTTAATTTATGGAATCAATCAGGCATTCCACCTA
TTAAAGTTCGAGATTTAAATACAGACTTACGCCCAGCAATTGATCAAATA
AATACAGAAATGCTGCAATTGCTAGTTAAAATACAAAAACTTCCCTCCAA
AGATTGTTTAAAAAAAGTAGATAAGTCTGTAAATAATTTTATTATGAGAG
TTAATCAAATTGATGAACAAAATGATGCTTTGAAAATGGCTGTGAAAGGC
AAAGACCTCTGCCCTGCATGTAAACATAATTAACGTTTAGTTAATTATAA
AGGGAAAAGAAATTATAATTTTGAAAAAATTTTGGGTTTCACCAAAAAAA
AAAAAAAAAAAAAAAAAAAA (SEQ ID NO:45) 4F05B>msp38>>AY422831
AATACACAAAAACTATTTTAAAAAAGGCACTAACTTAAATAATGACTTGT
AGTATTAATATTTTTATTATTTTATTTATTACATTAATTATTGGAATATG
CACGGAGGCAAAAATCCGTAAACAATTTGTTGACTCTCCACAGGAACCAC
AAGCTAAATCGGTTGATTTGAATTTGCAAGTTTTAATCTTTATAAAAAGA
TGCAAATCACAATTATGGGCAGTTGGGTTAAATAATTATAAAACACAATT
TCCAAACTGCTCATTAATTGAGGAAATATATTCTCGTCATTATCCTTTTG
GAATGTTAAAAACTACACAATGGTTATTACAAACACTTCTTTTATTTTCT
GCAATGTATTTTCCATATTTTGAAGTTCATGATATATCTTTGGTTGTTTT
TTTCACCCTGCAATTTTCAGTTTTATTCACTGGCTTTTATATTATTGCGC
AGTTCATGAAAGTCAAAATAATCCAAAACCAATTAATTTGTCTACTCTCT
TCTTTTCTGATATAATCATTTCATATTGCTTCACTATATTATTTATTGTA
CAGTTTATATCATCAGGAAGATATGGGGCATATTTGTTTCTCTTTGGATT
AATTTTGTATGGTGGTTATTCTTTAATTTTAACTTTTGTTTATTTACGTA
ATAATGAAGATGGATCCTTTAAATTCCCAATTTCAATAAAAATAAATGTT
GAAATTATTCAAAAATCGGATAAAGAATTAAAACAGGAAAAAAAAAAAAA AAAAAAAAAAAAA
(SEQ ID NO:46) 5A12B>eng3>>AY422836
GACAACACAAATCAAATTAATTTTAAAAATCTAATTAAAAATGAATTCTC
TCTTATTAATAGCATTTTTATCCCTCTCATTTTGTGTTCCAATAAAGGCT
GCTCCTCCATATGGGCAATTATCTGTGAAAGGAAGTCAATTAGTGGGCAG
TAATGGACAACCAGTTCAACTTGTTGGAATGTCACTTTTCTGGTCGAGTT
GTGGTGAAGGAGAAGTTTTTTATAATAAAGCAACAGTAAATAGTCTTAAA
TGCTCTTGGAATTCAAATGTAGTTAGGGCTGCAATGGGTGTAGAGTATTC
AGGGTGCCAACGACCAGGTTATTTGGATGCCCCAAATGTTGAGCTGGGCA
AGGTTGAAGCTGTTGTTAAGGCCGCAATAGAGTTGGATATGTATGTTATC
CTTGACTTTCACGACCACAATGCTCAACAACATGTGAAACAAGCTATCGA
ATTCTTCACATATTTTGCCCAAAACTACGGATCTAAATACCCTAACATAA
TTTATGAGACTTTCAATGAGCCACTACAAGTAGACTGGAGTGGTGTAAAG
TCATATCATGAGCAAGTTGTTGCAGAAATTAGAAAATATGACACAAAGAA
TGTCATCGTTCTCGGTACAACAACATGGTCTCAAGATGTCGATACTGCTG
CTAACAATCCTGTAAGCGGCACAAACCTTTGCTACACTCTACACTTCTAC
GCAGCAACTCATAAACAAAACTTAAGAGACAAGGCTCAGGCTGCAATGAA
TAAGGGAGCTTGTATCTTTGTAACTGAATACGGAACTGTTGATGCAAGTG
GAGGTGGTGGAGTGGATGAAGGTTCGACAAAAGAATGGTATAACTTCATG
GATAGTAACAAGATTTCTAACCTCAACTGGGCTATCTCAAACAAGGCAGA
AGGTGCTTCAGCACTCACATCTGGAACGAGTGCTTCTCAAATTGGCAATG
ATGACCGATTGACTGCCTCCGGTCTTATAGTGAAGAAGTATATTAAATCA
AAGAATACTGGTGTCAGTTGCAATGGTGCATCATCAGGCAGTGGTTCCGG
AAATAACCCCTCAGGAAATGAACCGAGCAACTCACAAACCAGCACTGCCA
AAACATCAAGCAATTCAGGAAATAAAGGCGGTAATTCTAACACAGGGAAT
AATGCAAATAACTCAGGAAGTAAACCGGGCAACTCCGGAAGTAATACAGG
AAATACGGGCAGCAATGCTGGGGCAAATTCAGGAAATACGGGGACCAGTA
CAGGCAGTAGTTCTGTTACAGCTTCTGTGCAAGTTCCCGATAAATGGGAT
AATGGCGCAAGATTCCAATTAGTATTTAAAAACAATGCGAGTACAAAAAA
GTGTGCAGTGAAATTTTCATTGACTTTTGCCTCTGGACAACAAATTACTG
GCATTTGGAATGCCCAAAATGTAACAGGAAATAATTTTGTTCTTCCAGAC
TACGTTACAATTGGAGCAGGGAAACAATATACAGATGCAGGAATGAATAT
AAATGGGCCAGCAACTCCTCCACAAATTAAGGTGCTCGGCGATGGAAAAT
GCGTTTTTTGAAATTAAAGACTCCGTCTAAATTGTTGAATTATTTAATCT
TATGATTGTTTAAATTGGAAAATAAATATATGTATAATTTGCTTCTGTTA
ATTTTGTTTATTTAAATATACGATAAAAATTAAAAAAAAAAAAAAAAAAA AAAAAA (SEQ ID
NO:47) 5C03B>msp39>>AY422832
TAAATTTCTTCCCTAAAATTTATTTAAAATTTTATAACAAAAAAATGTTT
TCAATTCAAGGATTATCTTCTTTTCACTTCATTTTCCTCTCATTATTGAT
ATTATTGCAAAACTCTTCTACTGTATTTTCTCAACTTGGTTGTGATTATG
GATCAATGTATGGCGGGGGAATGAGTGGTTATGGCCAAGCAGGTTATGGA
AATGAAAGTACACACATCACTTCTGCCCACATTATATTGGCCAAAGTGAA
TCACATGGTTTCTCCTGACTTCAACAAGCAGGGCATGAATAATCTAACCT
CCCACAAAGAACACGACTAGGAAAGAAAATAGAATAATTGGCAAACACTA
ATGCAATCTACTACAGAAGTCAATGGAGAATTTACCTCCTAAACAGGAAA
ATGATTTGTGCCTAAAAGGAAGGAAGAAGAACCTCCTCTTTGTTGAGGGG
AAAAGTCCATAACACAGGAGTGCTTGGACCCAAGTACACAAATATAAGAA
CCCTTCTAGGAAAACACGAGCTGGGGAAGCAGTTTCTCTTTGCTATTTTG
TGAGAAAATAAATGCCAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:48)
6D09B>cm2>>AY422835
GGTTTAATTACCCAAGTTTAAGAAAATAAACTTTTTAATAAAAATATTTA
ATTTTGAATGTCTTTTAATTGGCTTTATTGCAATTTATTTATTGCAATAC
TCTTTTTCAACATTGTAAAGAGTGATACCGATACTAATCCTGATATTGAT
CGATTTGTTGAAATTGCAGACGATCGTTTAACTCTTTCTGATTATGTTGC
TTTATATAAAGTTGTTAATAATCAAAGTATTACTGATCCAAAACGAGAAG
AAAAACTTTTGAACGATATGAGAAGTAAGGGAAAGAATTTTTCGTTAAAT
GAGGATTATGTTACTTTAATATTCCAAGACCAAATAAATGCTAGTAAATA
TTTTCAGAATTATTTAGTTAATTTATGGAATCAATCAGGCATACCACTTA
TTAAAGTTCGAAATTTAACAACAGACTTACGCCCAGCAATTGATCAAATA
AATACAGAAATGCTGCAATTGCTAGTTAAAATACAAAAACTTCCCTCCAA
AGATTGTTTAAAAAAAGTAGATAAGTCTGTAAATAATTTTATTATGATAG
TTAATCAAATTGATGAACAAAATGATGCTTTGAAAATGGCTGTGAAAGGC
AAAGACCTCTGCCCAGCATGTAAACATAATTAACGAAAAAAAAAAAAAAA AAAAAAAAAA (SEQ
ID NO:49) 8E08B>eng4AY422837
ACGCGGGGAACACAAATCGAAATATTTTTAAAAATTTAATTAAATGTTTT
CCCTCTCATTAGTAGCATTTTTATCCCTCACATTTTGTATTCAAATTAAT
GCTGCTCCTCCGTATGGGCAATTATCTGTGAAAGGAAGTCAATTAGTGGG
CAGTAATGGACAACCAGTTCAACTTGTTGGAATGTCACTTTTCTGGTCGA
GTTGTGGTGAAGGGGAAGGTTTCTATAACAGAGAAACTGTAAATAGTCTT
AAATGCTCTTGGAATTCAAATGTTGTTAGAGCTGCAATGGGTGTAGAATA
TTCTGGATGCCAACGACCAGGTTACCTTGATGCCCCAAATGTTGAGCTGG
CAAAGGTTGAAGCTGTAGTGAAGGCGGCGATTGAGTTGGATATGTATGTT
ATTCTTGATTTTCACGACCACAATGCTCAGGGTCATGTGAAACAAGCTAA
ACAATTCTTCGCATATTTTGCCCAAAACTACGGATCTAAATACCCAAATA
TCATTTATGAGACTTTCAATGAGCCACTACAAGTAGACTGGAATGGTGTA
AAATCATATCATGAGCAAGTTGTTGCAGAAATTAGAAAATATGACAATAA
GAATGTCATCGTTCTTGGTTCAACAACTTGGTCTCAAGATGTTGATACTG
CCGCTAATAATCCTGTACGAGGTTCAAACCTTTGCTATTCTTTACACTAC
TACGCAGCAACTCATAAACAAAACTTAAGAGACAAGGCACAGGCTGCAAT
TAATAAAGGAGCCTGTATCTTCGTAACTGAGTACGGAACCGTTGATGCAA
GTGGAGGTGGTGGAGTGGATGAAGGCTCGACAAAAGAGTGGTATAACTTC
TTGGATAGCAAGAAAATTTCTAACCTCAACTGGGCTATCTCGAACAAGGC
AGAAGGGGCTGCAGCACTCACCCCTGGAACGACTTCTTCTCAAGTTGGCA
ATGATGACCGATTGACTGCCTCCGGTCGTCTAGTGAAAAGTTATATTAAA
TCAAAGAATACTGGTGTCAGATGCAATGGAGGGGGTGCTGCAAAAAAAGG
CTCTTCATCATCTAATACTGGTTCAAAAAAAGACAAACAAAAATTCAAAG
AACAAAAATTCAAAGAAAAAATCTAACAACGCCAAACTGCCGAAAAAAAG
GTCCCAAAAAGAACACTTAGACAAATATCAAGGAATTTAATGTTAAATGG
AATATAATTGTTTTAAATTAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:50)
8E10B>msp40>>AY422833
GGTCATTCTTATAACTAAAAACCTTCAAACTTCAAAAAATATTCCTTAAA
CTTCTTCAGAAAAATAATTGAAAAATGTTATTAAAATTCTTTTTCCCATT
ATTGCTTTTGCTTACCCTTATCTATTTGGGTTGTTCTGAGGAGGATAAGG
GAGACATTGCAAATGGTCCTCAGGAATCTGAGAATCAGGTTGATCAAGAA
TTGGTTAGATTGAAAAGAGATGATGAAGAAGAGGAGGGAGAGAAGGCCGA
AGATGAAGAGAAGGCTGAAGAGGATGGAGATAAAGCTGAAGATGCTGAGA
GTGCAGAGGAGGGAGATAAGGCTGAAGATGCTGATGAGGGAGAAAAAAAG
AGTGAAGATGAAGAGAAAAAGAGTGAAGGTGACGAAGAAAAAGCGGAAGG
TGAAGAGGAAGAAAAAAAGGATGGAACTGAGGAAGAAAAGGAGGATGAAG
ATGAAGAAGAGAAAAAAGATGATGATGAAGAAAAAAATGAGGAAGAAGAA
AAAAAGGATGACGAAGAAGAGAATGGAGATAAAGAAGAAAAGAAGGATGA
TACGGAAGAGAAAGAGGATAAACACACAAAGGATAAAAGTAAGAAGAAGG
ATAGTAAGTCCGTTCAAAAGGATAAAAAGGAGGAAGATGACAAGGAGAAA
AAGGAAAAAAGTTCAAGTGGTGATAATTCTAAAACAGATAAATCACAAAA
TCAAAAACAAAGCAAAGAATCATGTAATGGGGATACTGCTTACAACTGTC
CTAAACTATCAGGTCTTTGTGAATCAAAAATTCAAGTACAACAAGACTTC
ATGGGTGAAAAATGTTGTGCTACGTGCAAAAATTCGGCTCCTGCTGCGAA
GAAAGATATACCCCTATGCACTGATTTGGCTGATAATTGTGATCAAATAG
CATCCACCTGTGGGGAAGAGGCGTGGCAACCGACTATGATTTCTGATTGT
GCTGAGACCTGCGATAAGTGTGAATTACATTTTCAAATGTTGGAGAAGAG
ACTTGCAGCAGCTGCTGCTTAAAATTTTGAAAGGAAAAGAATTTTATCAA
AAATATATGTGTATCATATTCACTAAGCAAGAAATTTTCTTTGATTTTCA
CACCTTTAATACGTAAAATTTCAATCTATTCATCCGTGTTTCTCGTAATT
ATGTTTTATTAATTTTTTCGAAATTTAGTAAAAATGCCTCCAAAAAAAAA
AAAAAAAAAAAAAAAAA (SEQ ID NO:51)
Sequence CWU 1
1
791817DNAMeloidogyne incognita 1gagaaaataa aatataaatt attcctcaaa
aataccataa aggttagcca atattaattc 60ttttgaaatt ttctttgctt ccataaatta
aaaaaaattg tttttaagtg agggaatgtg 120gattaagcat ctttcttatt
tttaaaattt ttgatagagt gtagcgacag tcaatcaaaa 180tattttgatt
tttttaaagt taaaaattaa ggatgataaa gaagtttaaa atgtaggtgg
240aaatataagt ataccgaaaa acatctttta tttttaagtt taaacaagca
gtaaaacttt 300gtctggtttt atcaccgggc aactgtaagg gaagctttaa
taaaaatttt gtaagatacg 360aaaatcattg tccccagtag cttgagtgat
cgaagcgcct ggttgccatt aagttttttg 420cttgagactt atataacaag
tatatatcaa accggattat aaagttaaag aacagaaaaa 480atttcacgga
ataaatattg gctaaccact caatttattt aattattctt caatcaaaaa
540atgtttacta attcaattaa aaatttaatt atttatttaa tgcctttaat
ggttacttta 600atgcttttgt ctgtctcatt tgtggatgca ggcaaaaagc
ctagtgggcc aaatcctgga 660ggaaataatt gaagaaaaat gattgaagaa
aaacgtttaa attaaacgat aaatgggaaa 720taatggaatt taaattaagc
taattttgat ggtttccttt gttaatttca acataaaatt 780aattgaattt
actgaataaa attatatctg aaaaaaa 8172364DNAMeloidogyne incognita
2gagaaaataa aatataaatt attcctcaaa aataccataa agttaattat tcttcaatca
60aaaaaatgtt tactaattca attaaaaatt taattattta tttaatgcct ttaatggtta
120ctttaatgct tttgtctgtc tcatttgtgg atgcaggcaa aaagcctagt
gggccaaatc 180ctggaggaaa taattgaaga aaaatgattg aagaaaaacg
tttaaattaa acgataaatg 240ggaaataatg gaatttaaat taagctaatt
ttgatggttt cctttgttaa tttcaacata 300aaattaattg aatttactga
ataaaattat atctgaaaaa aaaaaaaaaa aaaaaaaaaa 360aaaa
3643108DNAartificial sequence16D10 plasmid 3ctcgagggca aaaagcctag
tgggccaaat cctggaggaa ataattgagg taccatcgat 60tcaattattt cctccaggat
ttggcccact aggctttttg cctctaga 1084566DNAartificial sequence16D10
plasmid 4ctcgagcctc aaaaatacca taaagttaat tattcttcaa tcaaaaaaat
gtttactaat 60tcaattaaaa atttaattat ttatttaatg cctttaatgg ttactttaat
gcttttgtct 120gtctcatttg tggatgcagg caaaaagcct agtgggccaa
atcctggagg aaataattga 180agaaaaatga ttgaagaaaa acgtttaaat
taaacgataa atgggaaata atggaattta 240aattaagcta attttgatgg
tttcctttgt taatttcggt accatcgatg aaattaacaa 300aggaaaccat
caaaattagc ttaatttaaa ttccattatt tcccatttat cgtttaattt
360aaacgttttt cttcaatcat ttttcttcaa ttatttcctc caggatttgg
cccactaggc 420tttttgcctg catccacaaa tgagacagac aaaagcatta
aagtaaccat taaaggcatt 480aaataaataa ttaaattttt aattgaatta
gtaaacattt ttttgattga agaataatta 540actttatggt atttttgagg tctaga
5665694DNAMeloidogyne incognita 5gatcaaacaa tctcctcaac aactaaaaaa
actcaaaaaa caccccaaaa ccaaactaaa 60aaatcaaaaa tgtccatctt cctcacttct
gctcttctaa tcatttcatt aatcgctatg 120accgagggag caggcgatcg
aagcgcttca acctctactg gttgtacaac ctattttgga 180atgctagatc
atgcggatac caaggaaaat aacaaaagaa aaactttcaa acccaacgat
240aaaaccatat ccaacacttt gcaagtgatt ggtgggacaa agttcagcaa
tacctcggtg 300gcgttggttg tcggtgatga ggtgttatgt atggctaaga
caggaggttc aggcgattgc 360ggaatgcgct acgatgcgtt gactggatca
atgaaattta tcatttctga taatattatt 420gttgaggttc catttgaagg
cgtttttttc ttcaccgaca acaagtgtgt catccagctt 480gtaggctacg
atattaaaac taatataact cttctcaaaa ttaatgatgt cgacttcaaa
540attgtcccta ctgataagaa aatttccccg aaggcttgta ctatgaaaat
gtgagggaaa 600aaagtaaaga aaatgtgtaa atatggaagg ataaaaacta
aacaaaaaag aatgtgaagt 660aagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
6946776DNAMeloidogyne incognita 6ggatttaaaa aattaattta aaaaaagtga
aaaattcaat taaaattaaa aaatattttt 60caatgaattt attttctatt tttttatttt
tatttccaat cgggtttatt tgggctgaat 120gtagcggaga ttgttctata
gagaaccaat ataattataa atgtgaggat agaagtgaat 180tttgtgaaga
atggggaaaa tactgcgaaa atgtctttct tcacaaatgt gtaagaaagg
240cttgtccaaa gaaatgtaaa gtttgtcata gttctggtga agaacctaaa
ccaaatccta 300caactataac aacggcatca acaataacaa caccattagc
aacaacacct caaaactcag 360cagttacttc ggcaacctca aaaagtgcta
ctccatcaaa aacttattca accgagacaa 420ccgaatgtgc taacacaact
actgaggaat atgaagcaac tattgaggaa tatcaaacaa 480ctacagaaga
atatgaagag gtaacaaccc ctataattac aaccaccaat ccaacaactt
540atttattaat gactacaata gttgaagaaa ttagtgacga cgaattcaaa
gacgcaaaga 600agatgaaatg taaatcatgt aatgcaaaaa ggaagaaatt
ggctgaaatt tatgacaaat 660attatccgaa agttaagatt catgctaaat
tgtaaattat gatggaaaat gtttttgaat 720tgtgaaaata aaaatttaat
taacccaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 7767999DNAMeloidogyne
incognita 7gatagcacag atctattttt agattttttt agctttttag aaaattttaa
tttaaaaatt 60atgtttattc ttccaaaacc tttttatctt ttaattttac taattatttc
aacaattttc 120cttttatttt taattcttcg tttgccttca atttcttacc
aaacaaatca atgtcaacat 180ttatgggact cttccgagtg caaaaattta
aataattctt taaattggca tccaataact 240tgttttattg acggaaagca
aaaaagagtt ccctgtcttc aacaaaatga tttaaaagaa 300gtttatcttc
cattcaattc atttttgaaa aaacaatttg atttgtatgg agagactgac
360aaaagcattt ttttaatttt aaaatttatt tttaacaaaa tttgcggaca
gataaaattt 420gtccgcaaaa tatttgcgga caaataattt tttttgttgt
taggcttttt tagggtattt 480ttttctacaa ttttttggag tttttttcta
caattttttg gagttaattc taaattataa 540ttttattata tattttataa
ttttaaaatt tttttctttt taagaaaaca attcttttga 600ttattttact
tcaaacattc ctccacgtct ttttaaaaat aaaaataaaa tggtggctgc
660aaatccaatt gaacaattta gcaatgtggc tattcgtcaa agaataaaat
gtttaaaacc 720tgaaaatgga ttaccaatga gcgttcaatg gagtccaatt
ccctacttct atcctgttca 780aatactccaa tttggctttg attattttat
gagaaatcga acagaacaga ggaaattaat 840tgaaagaagg ttatcaaaca
aagatgattt cttggtacta aaaagtggag agaaagttag 900cgaattttca
acttttttct gatttgccat tttacctttt ctgcaaaatt gaatcaatgg
960atgcttcctt gtaatatttt tgagaagatt gggggaatt 9998785DNAMeloidogyne
incognita 8gacaataaac gatccaattt cctaaaattt tttaaaaatt tttaaaattt
attttatgcc 60cctttttgtt tatttaaaca aatttgcttg attattaatg ccaaaattaa
ttttattatt 120ttatttaatt atttatggaa ttttattgtt aataagttta
agtgaagcat ttgggtttgg 180tggaggatgt ggatgccctt gtatgccgca
accatgtatt ccacaaccac ctccaattgc 240tttaccttct ctatgtttcc
ctcaaatcca attgccctgt ccccctccat cttgtggatg 300ttgtggtaga
agaaaaagag aaagtggagc ttcagcatta ttaacagcag tttcaacaaa
360gtcgggaatt aaaagaattg gagaagaaaa aaatcattgt aataatccac
acattaaaag 420aattatttta aagaatttaa ttattggaga ttgggttggt
acaagaaatg caatatattc 480agaattaaga gctaaattag gggggaatta
tataattaat tgtgctcatg ccccctcatt 540tgcgtattct ggtgattctg
tgattgatta ttgtgtggat ggacatcagg caataacttg 600tgcagtcttc
aaaattcaat gagaataaaa tcagaatgaa ttctattttt ttaataaata
660taaaaatttt tataatatat tttgagcatt ataaatattt ataaattagt
tttttttgat 720aaattaattt gaaaatgtat aaattagttt ttactcaaaa
aaaaaaaaaa aaaaaaaaaa 780aaaaa 7859864DNAMeloidogyne incognita
9gacgcaattc cattttgcgt tcaatcaatt tagaaaaagg ctggaaataa tgattcatca
60acaactttat tattagcgct tagtgttccg ggctttcatt tcaacagaga aatttcaaat
120tcacctattt ggagcttgga tcagttcttc tatcgacttg tagtcctaac
ctatactaaa 180aattttttaa attaaccaca atggaacttg ctattaacag
tcgattgtta tcatttttgt 240ctttattcct attcatattt cctttaaatg
ttgttgctca acggcatcgt tacccacaca 300atcaaggaaa ttatttcagc
agacaaaagc tgcaagaaat acagaaggag gaaaatgagg 360ctgaaaattc
tttaccaaaa atcttttgcg cgcatggagc ttcagtagcc ggccgttgcg
420tatgtgatca tggttgggcc ggtactaatt gccagcggga aatgcattgt
gctacttttg 480agcgaaatgc taatggaagc tgcccagtct gtcagcccaa
ttttcaaggg gataagtgcg 540aatatattga atgccaaaat ggaggccaag
aatcattgga aactcagaat tgtaactgcc 600caaagcctta ttctggccgt
ttttgtgatg aattactcac aggaaatgtc tactactact 660ataactctaa
agtagcaacc cttggtcctc ttggacttat ttctgttata ccaatgattt
720gtctttatgt tttatgtgaa aagatttgca aggaaaagac aagtgagacg
gattgagaaa 780acttggaatt tacagagcag taaaactgtg aatcctgctc
atattgaatt tctattaagg 840gaaaaaaaaa aaaaaaaaaa aaaa
86410977DNAMeloidogyne incognita 10gaccttaatc aataaaaaat attttttaca
taaaaatgtt ttatttattt tattttaaat 60tatttttatt ttccttaatt tctttaaata
aagttaatgg attttgtatg aagactattt 120gttctgcgga caccgattct
cgacaccctg taaatcgagt aatcggtatt ggttctgatg 180gaattagtgg
ggaatataaa gctttgagac gaaatgatca aattgttgaa gctgtagatt
240taagttgtag agaaggaagt tttgtttatt ctccctttga aggggaaatt
tctgcttgga 300gaccttttga tggaaatggg caagatgaaa ttagaacgga
tgaaaataag aagaatacag 360atggatgcag acctgaccga ggagttagaa
ttgatggaaa aggacaatgg cagggatatc 420acgtccttat tggctctgtt
cgtttattcc gttacagtgg acatgttaat gctggacaaa 480aaattggtgt
atctttggat attgaatgtg aattgaaatt aaataaacaa aagatgaata
540aacgtcctcg aaaagaagaa aattttgtca gagtttattt acacaaggaa
ggacgtccaa 600ttgatccaac acatcattta attgattgta tgtgtataaa
ccaagtctgt gagacaaaca 660gaattaatgc tttggaagga ccgttattta
aatttgacag tcgttttaac ggtgttagag 720gatgggaaat taaatgtcca
gatattcaac aaattgaaga agaaaattct tcagaagaag 780aggaagaaaa
gaaaaaagaa gaaaataatt taaatgaaga atggggaact ccaaaattta
840ttcacctata gaaggggaat tggttggaag aattagagtt aatagtgaac
ctggggcaca 900gacttatact ggatgtacta atgaaggaat atttatggtt
ggggctggaa agtggaatga 960ttatgaagtt cgaattt 977111339DNAMeloidogyne
incognita 11gttcatttaa aaattttttc ctaaaaaact tcaaaaaagc aacttttatt
gcgtaaatga 60aagaaaatct gtttaaaaag agccttatag gcctattttt gttgttagca
ttcaatttta 120ctgaagctaa ggactctgga gagaatacta gtcttgaagc
tagtttgaaa ccaactaaaa 180gtattgaaaa tgcttcccta gaagaaaaga
atcaaaaaga agaaaatgga gtaacattcc 240cggcagaagg tcatgaaatt
gtcgaaacaa aaaaagaaat caactcacca gaagaggtga 300cagattcaac
taaaggacag gaaaattccg aggatcgtaa agtgacaatg aatggtgatg
360agtctgaggc cgataaatta aacaatgaaa atgttgaggg tgaagaaaag
aaagcaactg 420aaaacaagaa tgaagttgag gaaaaagaag ttttagagga
tgagaagaca aaagaagagg 480aagataaaat tagcgatgag cctgtgaaga
caaaggaaat gaaatcaaca aacaatgata 540aggaagttga agatttgaaa
gaagaggaag agaaagtcga ggtaaaaggt aacaaggatg 600aagaagaaaa
taaggaagag aagaaggaag ataagaagac aaaggatgaa aaaaaggttc
660cagaggttat tgagggagag aagaaaacac ccaaggaaaa ggaacacaaa
agccattggt 720ttatggacaa atttaaacat gctttctgtt tcataactca
ttacttcttt tgtccatcta 780actctgcaga aaaaggcaaa gaatcccatc
atgaaggaaa agaatcacac cgtggaaagc 840gtcttaactc tgattttagt
tctttaagca gtgatgagga aatgattgag aattttgaaa 900atgcccacga
atttagtgaa gaaattgaag aaaatgggga atttaaagct aaaatgaatg
960ttggtgcaac atacttcaaa gctgagacag ataattctgg aaagatgcgc
ggcaaaattg 1020aaaaatttaa tgctgaaatg cataattgaa aagattgtaa
ggatggtggg tgtgctgatg 1080agtaaaacaa aaaaaagcaa tccgatttta
ttctaaattt tattttttaa agtgattcca 1140acaagtgatt ccattaaccc
ctcaaattta tttaaaaaaa cgaaatttta aaagttctgg 1200atttatgtcc
caaaaattgt acaaattatt caaacaaact caatggtttt ggacattata
1260tttttttatt attttctaac aatttttatt aatgttgaag taaaagatta
attcaaaaaa 1320aaaaaaaaaa aaaaaaaaa 133912762DNAMeloidogyne
incognita 12attcttaatt tatttaaaga atttattctg catgatgaaa ttaattaata
ttttattttt 60attttttgtt attttactga attctatggc tttcggaagg ttttctttat
ttttggaaaa 120atcaaaattt caattgaata ttttgttttc attccaagat
tttctcacac agatcccgct 180tagtgtgaga tatcggataa agcttcataa
cctctacaat tttaagatat ccacattact 240ccgtccaaat tcccttatat
ctcctttgat ccctacaaat ctaccgatat cccctccacc 300gatattttcc
ttttccgaac cttaacttcc gatcaatccg ctatctggac aaatcgttat
360tcctctaaac aagaatttat gcttttaaat gtataaaacc aatctttaat
attcttcaaa 420aaaattttca gtccttctct caattcagtg cgtgctaaac
gtcaaggctg gggaggatgg 480ggttggaacc ctcaagttca aacagatatt
gatcgtcttc gtattgataa agacaaactg 540cgattagata tggaccgttt
aagactagat caggatagct cttggggatg gggaaaatga 600gagaatcaaa
cgactaattt aagtgtaacg atttttaatt aacgatttat aaattaataa
660atacttgatt gatacacaat ttagataatt taaaataaat tttattaaat
gataaaatta 720aattgccgtt ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa
76213778DNAMeloidogyne incognita 13gaccatcaaa tcatctcctc atcaactaaa
aatccctaaa aacaccccaa aacatccata 60aaaacaacca cgaaaatggc cacctttttc
acttttaccc ttctaatcat ttcaattatt 120gccacaactg agggaatgaa
tactaatcga agtgcttcaa cctccgattc tctcaaagcc 180caaaaggatt
gtaaagtgat atatggcatg tttgtgcctg tagcagggtc aaaaatgcat
240ggagacgcca aaagcgcaat gaagccaaac aatccaagtc tctccaataa
attaattgta 300tcaggtggca actcaaaata ttcagtgact ttacaggttg
aaaaccagcc gaagtgtgtt 360gcccaaaatg acggaaaccc tgtagaatgc
caaattcaag gagacaaact ttcaggaaaa 420ttgatttatg atattgaaaa
cggcccttct gtcaacgttc ccttcaaaga caccccaatc 480tttgttggaa
ataaatgcga aattgttttt gtagactacg ataaggacca caaattaact
540cttcttatga ataaagtaaa gctgatgatt gagccgactg aaaagcaaat
tgtaaaggct 600tgtggagtga aaaattagat ggaaaaatga tatatgaatg
aatgaatgtg agagggaggg 660aaagaaaaat atttttaaaa ttgaagaaag
cattcaaaaa aattaaaaaa aaacaattct 720tcaaataata taaccttaaa
atttctgata aattatgttt ttacaaaaaa aaaaaaaa 77814757DNAMeloidogyne
incognita 14gccatcaaat aatctcctca acaactaaaa aactcaaaaa aacaccccaa
aacaactcta 60aaatggcggc tctcctcttc acttctaccc ttctaatcat ttcattggct
tttattgcca 120tagctgaggg agcaggcgat cgaaatgcat cagcttcaag
ccctggttgt atgcaggttg 180caacccttat tcatataggg gaaattcgcc
cagcaaaagc aaacaaacca ggtgtacaaa 240atactctaaa aatgtctgga
aatgttcaaa cattcaaaac tactcaagtg acattacaag 300tagctgggca
agagccttgt accgttaaaa ttaataatgg cgaaaccaaa tgtaaaataa
360ccggagatga attaaatgga aaattaattt tcaaaactga aaaaggaact
gaaatttctg 420cttatttcga actggttcca ttattttctg aaaataagtg
tgttattgaa cttgacactt 480ataacaagga aacccatgaa actaaactta
aaattaatgg aaataatttt atgattaaaa 540agaaggaagg taatgtgtca
attaagtgtg gtggaagagc taatactgtt taaattttaa 600aagtgtgaat
tgaaagagga agagaataaa caaatgtgaa gatgagaaaa aaatattttg
660aagaaagcat tataaaaata ttaaaaaaaa ttaattcttc aaatttttat
ttgatttttg 720aataaattat tttattaaaa aaaaaaaaaa aaaaaaa
757151091DNAMeloidogyne incognita 15gctcattaat tagttaaaaa
atttaaaaaa taatttaaaa aatgaaaatt tattttaatt 60taattgtttt tctatttatt
ttaaattttt attttgtcga attggcaaaa aggaaggcaa 120cggatactga
gattcctgag caaaataaaa agcaaaatac aagcaaccat gcccatcaac
180aattaactcc ttcttcttca aatgctgata atgagaagca aggaaatctt
tcctctgaag 240cttcaaatat tcgaggaaaa aatattctgc atgatcagtc
tgctattaaa aacaattcgt 300taactaatca acaattagga gcctcctctt
ctaatgctgg gcaacagaga aataataatt 360cggatctttt aaaattaaca
attataaatc atttgttatc ccatcgccaa tttaatgcct 420cttcttcaaa
tgctggtcaa cacaaaaata ttccctccga aaatctaaat tttcatcaaa
480aaactattcc aattgctact aaaaataatt tgttccccaa tcagcaattt
attgcatctt 540cttcaaatga tcttgatttt caacaaaaaa atattccata
tggaactaaa aagaaggtgt 600tacatcaatt tatgccatct tcttccaatg
ctaataaacg caaaaatagt tccacggaat 660atttaaaata tgcaattaaa
aatagatttt tatctaatca gccatttgat gacgacattt 720atggtaaaaa
gaaaaatgtt tccccggaat atcaaaatat tcaacaaaaa aatcttccat
780atgtccaata tgctattgat aataatttga aattgccaat tccaaaaaat
cctaaagcac 840ttccatatga tttgtctaaa tacgcattta acttccccaa
tatgaacaag aaaaatattt 900atgaaggagc atatgatcct tattatatta
attttcaaca ataacagatt tggctaataa 960aacgttggaa aacgactaag
aagttataca tttgacataa attaaataaa taaaattaaa 1020ttactattat
aaaattgtta attatcgtaa taaaattttt taactcaaaa aaaaaaaaaa
1080aaaaaaaaaa a 1091161282DNAMeloidogyne incognita 16ctagtcagtc
atttaaaata atttaatatt cctctaaaaa tccctaaatt aatttaaata 60tttctttaat
caatttttct tcaaaaaatt taaagaagga aatgttttta caaaaacaat
120tattgttttt ggttgttctt ctattagcct tttctcttgt aaagggagta
accgagaata 180agaataaaag cgaaataaaa aatgaaacaa ccacaaaagt
aattcaaaca tcaactggag 240gttatgatga taacgaaaaa gcagactatg
gcgatttggc tgcagaattg gctaaacttg 300ttgaggagga agatgaatta
aataaaaaga agaatgcttt gagttcggag aatggaaata 360aaaatagcac
aggaaagcct tatattcaaa aagataaaag taaaaaatat ttggaagaag
420ataaaggaaa atatgaggaa agaaattcta gaaataaata tgaaaactcg
gatgaaaccc 480atgaaagtga atcaggttca agttcggatg aggatttaga
tgaagataat ttagaaagat 540tgccagggcc ttcgccacac aatgaaggaa
tttctaggcg aagagttgaa aaggaaaaag 600gtggagaaga tgaggaggag
gaagaaaaag agcaagaaaa ttctaatgat aaagaagaaa 660gaaagaagaa
aaggaacacc aaatataatc caaaagatga gagtgaggaa gatatttctt
720ttgatggtca aatacctaaa agtgtacgta aattacttaa acaattagca
gctggtggaa 780agaatcctgt aattatacct ttaattataa ataacaacaa
tataccgaat cgaagagaag 840atgagtctga ggaatggaat aaaaaaagac
atgggagacc tcatagatta aatgattgga 900ataatccgtt tcctccattc
tttcaatctt caatgtttca accaccaatg tttcaaccac 960ctatgtttcc
accacaacag ccaccttttg gtggccctcc aacatttgct cagcacttaa
1020tcttcctgga gggcctctcg gaggaggtct tgctggcagt cttcccaaca
caaatccatt 1080tttatcacaa ctaaatcgtg gtgtaagtcc taatcaattt
cccaatcctc cctctaatca 1140cgttccacct tttgggcaac aaaatcaatt
ctatcctcct caacaacaac aacaaaatca 1200agtcaaccca cagggagcag
atggcaatga tgtgaaaaaa gtgaattaaa caaaaaaaaa 1260aaaaaaaaaa
aaaaaaaaaa aa 1282171228DNAMeloidogyne incognita 17gatcgtcatt
cttgtaaact aaaaatcttc aaacttcaaa aaatattcct taaacttctt 60cacaaaaaga
attgaaaaat gttattaaaa ttctttctcc cattattgct tttggttacc
120cttatctatt tggggtgttc tgaggaggat aaggaagaca ttgcaaatgg
tcctcaggaa 180tctgagaatc aggttgatca agaattggtt agattgaaaa
gagatgatga agaagaggag 240ggagagaagg ctgaagatga agagaagcct
gaagaggagg gagaaaaggc cgaagatgct 300gagaatgcag aaggagatgc
tgataaagga gatgctgatg aggaagaaaa aaaagaaagt 360gaagatgaag
agaaaaagag tgaaggtgaa gaagaaaaag cggaaggtga agaggaagaa
420aaaaaggatg gaattgagga agaaaagaag gatgaagatg aagaagagaa
gaaagatgat 480gatgaagaaa aaaacgagga agaaggaaaa aaggatgatg
aagaagaaaa cgtagacaaa 540gaagaaaaga aagatgatac ggaagagaaa
gaggataaac attcaaagga taaaagtaag 600aaggatagta agtccgttca
aaaggataaa aaggaggaga aggagaaaaa ggataaaagt 660tcaagtggtg
ataattctaa aacagataaa tcagataaat cacatagtaa tcaaaaacaa
720gacagcaaag aaccatgtaa tggggatact gcttacaact gtcctaaact
atcaggtctt 780tgtgaatcaa aaattcaagt acaacaagac ttcatgggtg
aaaaatgttg tgctacatgc 840aaaaattcgg ttcctgtcgc gaagaaagat
atacccttat gcactgattt ggctgataat 900tgtgatcaaa tagcatccac
ctgtggggaa gaggcgtggc aaccgactat gatttctgat 960tgtgctcaga
catgcgataa gtgtgaatta cattttcaaa tgttggaaaa gaaacttgca
1020gcagctgctg cttaaaaatt ttgaaaggaa aagaattttt atcaaaaata
tatgtatcaa
1080aaatatattt tctttgattt tcacaccctt aatactaaaa tttcaattta
ttcatcagtg 1140tttctcgtaa ttatatttta ttaatttgtt tcgagattta
gtaaagatgc tttaaaccaa 1200aaaaaaaaaa aaaaaaaaaa aaaaaaaa
122818737DNAMeloidogyne incognita 18ggaatttttc aaaaaaagta
ggctggagaa taaatttatt gaaaaaccag aattcttaaa 60gtttcaacca tttaaaaaat
gtcaaacaat tttaaaactt gcccagcttt attatattta 120ttgcttctgt
tgggaaaagc aagttgcaat tattttgaat cagaattaag cttagctaat
180gacaaaactt ctatagttcg caaatgttgt cctaaggaga agattagaca
ccatcggaga 240ccgttgcatt gctgccagga tgggttattc cgtgatgaag
ttgatggtta tttattaaaa 300gaatgtgcag atcaaggtga ttccatagtc
aaaacaatta gatgtgctca acaagaaata 360catggtgaaa atgcagtgga
gatttgcaaa gcctattgct gcgaattatt cagagataat 420aattgttcca
aaatatgcct aacaaacatt accaaagtaa acatgtctat tgaaatatta
480tttgagctgt taaaaaaatg caggaatcat gagaattatg gggaagtcca
tgactgtatc 540cattcaaaaa gaccaaaaaa catggatgcc gcagagttgg
aaatttattg taaaagggct 600attaatatgg tttaaatctg gaatttattt
tttaatttat tctactcgat ctccttttat 660ctatttaatt attaatttat
ttttggcaat aaaatttaat aaaaaatgta aaaaaaaaaa 720aaaaaaaaaa aaaaaaa
73719656DNAMeloidogyne incognita 19ggtcgtcatt cttgtaaact aaaaatcttc
aaacttcaca aaaatattcc ttaaacttct 60tcacaaaaag aattgaaaaa tgttattaaa
attctttctc ccattattgc ttttggttac 120ccttatctat ttggggtgtt
ctgaggagga taaggaagac attgcaaatg gtcctcagga 180atctgagaat
caggttgatc aagaattggt tagattgaaa agagatgatg aagaagagga
240gggagagaag gctgaagatg aagagaagcc tgaagaggag ggagaaaagg
ccgaagatgc 300tgagaatgca gaaggagatg ctgataaagg agatgctgat
gaggaagaaa aaaaagaaag 360tgaagatgaa gagaaaaaga gtgaaggtga
agaagaaaaa gcggaaggtg aagaggaaga 420aaaaaaggat ggaattgagg
aagaaaagaa ggatgaagat gaagaagaga agaaaggtga 480tgatgaagaa
aaaaacgagg aagaaggaaa aaaggatgat gaagaagaaa acgtagacaa
540agaagaaaag aaagatgata cggaagagaa agaggaataa acattcaaag
gataaaagta 600agaaggatag taagtccgtt caaaaggaca aaaaaaaaaa
aaaaaaaaaa aaaaaa 656201499DNAMeloidogyne incognita 20atatttattt
tttaatttaa caaaaatatt tttaattaaa attatttatt taatgtttaa 60attgttgttt
ttcattttgt ttgccttatt aaattctgtt gattgtcttt taaaattacg
120aacactggat aaagaacatc ttctggttga ggagagatat gccaaggaag
atacgcttta 180tctttttgtt tttcctagaa catcaaatgc cccatatttt
ggagcaatgt gtctttatgt 240tgaagctgtt ttaacttgga aaggaattcc
ttttcataga ataagtaacc aattctttct 300tggttcaaaa actgatggag
caattccttt tgctatttat aacgggaaat atttggatgg 360agcagaaaaa
ataattgaag aagttagaaa aaagggaaat aaaaaattga gtgatgaaca
420tgatgataat attagaaaat ttgcaactag aaccttgcta aagactctaa
ttgctgatag 480aacatttcgg agagatcttc cccatgcaac aattccaaaa
aataattccg aaacacaaat 540agcctcttct tcattatcaa atagtgcacc
agcaactccc aagggtggaa tccctacaag 600aaagagattt agtccaattg
atattaaaat ccctcatact aaaaatgaag aaataataat 660ggcaaaatct
gaggggcatt ctcctggaag ttctttcttt tctagaacta ttgctcattt
720aaaattacat aataataatt ctccaaagaa aggtccgggt ggtcttgatt
ggatgttaaa 780agatgaagga gttcgtgaac aattaattcc agttattcca
gaggcttttt tagaagaaag 840tatgagtgat gaatattttg attccccggt
aaaagataaa aatgaaaaga aatcaaaaag 900agaggaggaa gatgaaagtg
atgaaacaaa aatatctaaa attaaatatt ccattaaatt 960gacgttaagt
ccagaattgt ggaaagatta ttttaatatt ttaaataaaa taaaaataaa
1020tggaagggaa aatagagaag aaattaattt attgaaaata aattttcttc
aagaatattt 1080cggattctta gcaagaattg atgatgattg ggaacgtgta
aattctattc tgaaaaatac 1140aattaacgat attttaaaga aattaattgt
tgatagccaa ataccttttt gttgggaaaa 1200aaggttgaga gagattaatg
ggaaaaatat taatgaagtt gaagtattta atgaatttaa 1260agataaaata
aaatcgttgg gtataataaa aagttgactg aggcagagac taaaaataat
1320tttttgcatg gaaataatcc aactttggct gattttgccc tttttgcttt
tctcaatcaa 1380ttttttgaat ttcctttaaa tattccagaa tttaaagaat
tatttacccc agaaaagctc 1440agtaatgagg aaaaagaatt aattgcgaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1499211266DNAMeloidogyne incognita
21ggcatcaaac aatctcctca acaactaata aactcaaaaa acaccccaaa acaaccctaa
60aaacaaccca aaaatgtcca tcttccctac ttctgctctt ctagtcattt caataatcgc
120tatgaccgag ggtgcaggcg atcgaagcgc ttcaacctct actggttgta
caacctattt 180tggcatgctt gatcatgcgg ataccaagga aaataacaaa
agaaaaactt tcaaacccaa 240cgataaaacc aaatccaaca ccttgcaagt
gactggtggg gcaatgttca gcaatacctc 300ggtggcgttg gttgtcggtg
ataaggcgtt atgtatggct aagacaggaa gttcagacga 360ttgcggaatg
cgctacgatg ctttgactgg aacaatgaaa tttatcattt ctgataatat
420tactgttgaa gtgggtgtgg gtttatataa ttggtgccag acaaatggaa
aggcccctgt 480cactaacata ccatccggag cgttcatgtt gccccggaag
taactggtgg cccacaaaag 540ggcaatcact attgattaca acccaaatat
ctataggatt ttgacatttt ctggcataat 600ttaggtattt tctgacattt
ttctgacatt tttaactaga attaattcaa ttgaaaacaa 660aataatagga
ttgacctaaa tgagcgtttc ttggatatcc ttttaacagg agcagtctct
720aattttgtaa gagctcctaa tgtttaccct cctccatctc cctccccctc
tatgctccta 780ccaatgactg attaagttaa aaatcgtaca taaaatggag
agtgtataaa tctgggtgta 840tatacaatca ggattcgact ttataacatt
tgaaggttcc atttgaagac gtttttttct 900tcaccgacaa caagtgtgtc
atccagcttg taagctacga taataaaact aataaaactc 960ttctcaaaat
taatgatgtc gacttcaaaa ttatccctac tgataagaaa atttccccga
1020aggcttgtac tatgaaaatg tgagcttgta ctatgaaaat gtgagggaaa
aaagtaaaga 1080aaagaataac aaaaagtgta aatatggaag gataaaaacg
aaacaaaaat gaatgtgaag 1140taaaaaataa aaagaaattc aagtagattt
aaaaaaaatg ttaagcttca caatatctgt 1200ctccttttgt ttatgttttt
cgaataaatc gcattaccaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaa
126622723DNAMeloidogyne incognita 22gaatcacaaa aatggccacc
tttttcactt ttacccttct aatcatttca attattgcca 60caactgaggg aatgaatact
aatcgaagtg cttcaacctc cgattctctc aaagaccaaa 120aggattgtaa
agtgatatat ggcatgtttg tgcctgtagc agggtcaaaa atgcatggag
180acgccaaaag cgcaatgaag ccaaacaatc caagtctccc caataaatta
attgtatcag 240gtggcaactc aaaatattca gtgactttac aggttgaaaa
ccagccgaag tgtgttgccc 300aaaatgacgg aaaccctgta gaatgccaaa
ttcaaggaga caaactttca ggaaaattga 360tttatgatat tgaaaacggc
ccttctgtca acgttccctt caaagacacc ccaatctttg 420ttggaaataa
atgcgaaatt gtttttgtag cctacgataa ggaccacaaa ttaactcttc
480ttatgaataa agtaaagctg atgattgagc cgacaaataa gcaaattgta
aaggcttgtg 540gagcgaaaaa ttatatggaa aaatgatgaa tgaatgaatg
tgggagggaa ggaaatgaaa 600aatattttta aaattgaaga aagcattcaa
aatttaaaaa aaaaacaatt cttcaaataa 660tatataactt taatattttt
gataaatttt atttcataaa aaaaaaaaaa aaaaaaaaaa 720aaa
72323824DNAMeloidogyne incognita 23ggcatcaaac aatctcctca acaactaata
aactcaaaaa acaccccaaa acaaccctaa 60aaacaaccca aaaatgtcca tcttccttac
ttctgctctt ctaatcattt caataatcgc 120tatgaccgag ggtgcaggcg
atcgaagcgc ttcaacctct actggttgta caacctattt 180tggcatgctt
gatcatgcgg ataccaagga aaataacaaa agaaaaactt tcaaacccaa
240cgataaaacc aaatccaaca ccttgcaagt gactggtggg gcaatgttca
gcaatacctc 300ggtggcgttg gttgtcggtg ataaggcgtt atgtatggct
aagacaggaa gtccagacga 360ttgcggaatg cgctacgatg ctttgactgg
aacaatgaaa tttatcattt ctgataatat 420tactgttgaa gttccatttg
aagacgtttt tttcttcacc gacaacaagt gtgtcatcca 480gcttgtaagc
tacgataata aaactaataa aactcttctc aaaattaatg atgtcgactt
540caaaattatc cctactgata agaaaatttc cccgaaggct tgtactatga
aaatgtgagc 600ttgtactatg aaaatgtgag ggaaaaaagt aaagaaaaga
ataacaaaaa gtgtaaatat 660ggaaggataa aaacgaaaca aaaatgaatg
tgaagtaaaa aataaaaaga aattcaagta 720gatttaaaaa aaatgttaag
cttcacaata tctgtctcct tttgtttatg tttttcgaat 780aaatcgcatt
agcagcaaaa aaaaaaaaaa aaaaaaaaaa aaaa 82424696DNAMeloidogyne
incognita 24gaaataatct cctcaacaac taaaaaaact caaaaaaaca ctccaaaaca
actctaaatg 60gctttcctct tcacttctac ccttctaatc atttcattgg cttttattgc
catagctgag 120ggagcaggcg atcgaaatgc atcagcttca agccctggtt
gtatgcaggt tgcaaccctt 180attcatatag gggaaattcg cccagcaaaa
gcaaacaaac caggtgtaca aaatactcta 240aaaatgtctg gaaatgttca
aacattcaaa actactcaag tgacattaca agtagctggg 300caagagcctt
gtaccgttaa aattaataat ggcgaaacca aatgtaaaat aaccggagat
360gaattaaatg gaaaattaat tttcaaaact gaaaaaggaa ctgaaatttc
tgcttatttc 420gaactggttc cattattttc tgaaaataag tgtgttattg
aacttgacac ttataacaag 480gaaacccatg aaactaaact tataattaat
ggaaataatt ttatgattaa aaagaaggaa 540ggtagtgttt caactaagtg
tggtggaaga gctaatactg tttaaatttt aaaagtgtga 600attgaaagag
gaagagaata taaacaaatg tgaggatgag aaaaaaatat ttttgaagaa
660agcattacaa aaaaaaaaaa aaaaaaaaaa aaaaaa 696251687DNAMeloidogyne
incognita 25gattcaaaaa atattattta aaaattcttt accatttaat taacaaattg
taataaaaga 60aagacaatta aaaaatgagt ccttcctcat tcaccttaac ggcagtactt
cttgaggcga 120ttgtttttct ttacaaccgt caagtagcgg caatgctttc
catgcatccg agctgttctg 180gccgttcatc aaccattgag aataaattga
aaatgagcgg gggtggtaac ggcatcaata 240aatttacacc gggaaatgtt
tcattcccgg tagcatgcca ataccattca aagaatctca 300aagcaacaaa
taaaaaggaa tataaaatct cagaagattt gcctatgaat caagaaaagc
360ttacaaacag taaggaagat gatctcattc ataaggtaaa aaagatagat
aagggcaatg 420gagctgctgt tccttataaa acaaacaaga acaatgaaat
tggagatgga gccgagaatg 480gaaaagctgt caaaattaga gaaattattt
ttactgaaga gcaaaagaaa atgactagcg 540aagaatttga gcattatttg
tatagtgttc catatgacaa aaacaagaaa aacaaaattg 600gaaaaaacga
aaatggtgaa aaagttgata aaccaagcaa agaaggagga gatacaatgt
660tttattcaaa agctgggata attgctaaaa agataaaaga atatgtcccc
actaatggcg 720aattcaagat ccagactgga cttgtatatc gtaacaatag
ttttaatgct tcccaagatg 780atagtaaaaa tttactaaat atttcgcata
ttttaatggc tttaaatgaa aatgagaggg 840attctcaaga aaatttgaga
aatgctgctg atttgtttgt ggcacttcat gagtgttacc 900aactcttttc
ggcaattcct ctagtttttg aagtagaaat ggttttgaaa aaacttgagg
960aagagggaaa caaagacgat ccaataaaat tactcgaata tttccgtttg
ccaacaatta 1020aatatccatt attggatttg attaaaattg agaactcaac
tgtgtctcca gatgagttga 1080ttgaaaacgt cactaaaact attcacaaag
cagacaattt tattgctaaa aacatccatg 1140cattcttcat aaatgacaac
gaaacatttt ttaatgaaat aatttctcgt cttgaaacag 1200ctgatatggt
tttggccagt atcaaaaaaa ttcttaaaat gttcaataac tttaatgaga
1260aaattcccga aaacttttcg atgctaaaac gtttaaaacc aattgaaatg
cacgatttat 1320tcgaaaattc taaactgctt caaaagcttc atgcagcaat
tttgcctgga gatgaaatga 1380aattttgaaa tgagagttaa atatttttaa
aaaaattttg cgaacacaaa aacaacaaca 1440aattagaaga atattaaaat
tattaatgaa acaagagttg ccgcggctga tcggaaatat 1500taattaaatc
caatttagct gacgttgcct gctcaatcac caaataaatc aatttatgat
1560tttgcccatt ctctatcatt accttatttc ctatttgtac attttttttt
cttttttaaa 1620aattattttt agttttgttc ttgaatgttc gcttaaataa
attctaaaaa aaaaaaaaaa 1680aaaaaaa 168726783DNAMeloidogyne incognita
26gatcaaacaa tctccttaac cactaaaaaa ctcaaaaaac cccctaaaag cagcccaaaa
60aatacccaaa aatggccatc ctctttactt ctacccttct aatcatttcc cttttgggaa
120ttaccgaggg agtgaataca ggcattccga gcggatcttc tccaccctct
tctgcttgtg 180agacttacaa gggcaaaatt gagcacatgc cagaaaccgc
cagaaaaatt gaatggaagg 240aaaatactcc cggaggaaag catttaatcc
ttaaaaagtc tattcaaggt ctagacaaag 300taaccctcaa aattgaaggc
aaagaatgta gtgcttccct caacaaccct ggaacatgtc 360aagtcgatgg
acagtcccat gccggtcaat tagtctttgt aacttcaaag gctaaaattg
420aggttgactt tggggaagct caaatcttct ctgggaacaa gtgcgagatt
gaaattgaga 480agtatgaccg tgctacctac gtaactctaa tcaaaattaa
tgggggtgac ttcaaaatta 540cgcctgattc gccacctatg ccgatgccat
gcaaaaatat gatgaactaa aaagtgagga 600ggagggaagg aaaagtgaag
gaagagacca tgtaaaattg aaatattgaa gaaagcattc 660aaaaatttaa
tttttaaaaa atctgtttgt ttagtaacta aatatagctt tctattgttc
720ttatattttg tttatcttta tcaaattaaa atgaaaaact caaaaaaaaa
aaaaaaaaaa 780aaa 78327798DNAMeloidogyne incognita 27gacattcatt
taaacattca ttaattacct aaaattgttt ttcaaattgt ttgcctctga 60gttttgctca
actgagaaga aaaatgcttc cttactcaat tctatttcaa ttgggaatag
120tttcgttgct tctacctcat gcaaatggaa tgcagtctgg cagtagcaaa
attatgaaca 180aagcatctga aaagaaatat gctttggttg ttgctccaaa
ctttcttaaa gttcatttta 240aaatgaacag tgtctttgcc aatgcgttga
ccaaaaagtt ttttgtgcac tttctaattc 300tgaacaccaa aaatgaagaa
attggagata atttcgacta tggaattgat ctcgaaaaat 360ttgaagaagg
aacgggaaat acatatcaag ttgtaaattt tccagatgat tatcccgaaa
420aattgaacga aggcgtgaag aatttagaga acaaattcat taagagaggt
tacgaacaga 480gtagtcaaat tctgaaaaat gaagctttca ccgtttataa
agatttattt gaaaacaatg 540gagctattgt tcattacttg aaggaggcaa
agtttgattt aggggttttt gacacttggg 600acactggagc tctcttcatt
ctccatgcag caggaattaa aaatgttttt ggcattaaca 660acattcaact
taatgcttat caatttaaat atgctgggaa agaatttcca aaaaatattc
720cagaaattta attcggcaca aacaggcgat aatgaattat caccaacaaa
ggaaaaaaaa 780aaaaaaaaaa aaaaaaaa 79828704DNAMeloidogyne incognita
28ggcatcaaac aatctcctct cctcaacaac taaaaaactc aaaaaacacc ccaaaacaac
60tctaaaatgt cggctctcct cttcacttct acccttctaa tcatatcatt ggcttttatt
120gccatagctg agggaacagg cgatcgaaat gcatcagctt caagccctgg
ttgtatgcag 180gttgcaaccc ttattcatat aggggaaatt cgcccagcaa
aagcaaacaa accaggtgta 240caaaatactc ttaaaatgtc tggaaatgct
caaatattca aaactactca agtgacatta 300caagtagctg ggcaagagcc
ttgtaccgtt aaaattaata atggtgaaac caaatgtaaa 360ataaccggag
atgaattaaa tggaaaatta attttcaaaa ctgaaaaagg aactgaaatt
420tctgcttctt tcgaacaggc taaattgttt tctgaaaata agtgtgttat
tgaacttgac 480acttataaca aggaaaccca tgaaactaaa cttaaaatta
atggaaataa ttttatgatt 540aaaaagaagg aaggtagtgt gtcaataaag
tgtggtggaa gagctaatac tgtttaaatt 600ttaaaaatgt gaattgaaag
aggaagagaa tataaacaaa tgtgaagatg tgaaaaaata 660ttttgaagaa
agcattccaa aaaaaaaaaa aaaaaaaaaa aaaa 70429414DNAMeloidogyne
incognita 29gacattcatt taaacattca ttaattacct aaaattgttt ttcaaattgt
gattttattt 60atttctattt aatatcttta aatgcggagt gctttaaaaa ctttaattgt
tttgtggcct 120cctttgcttg gacattttat tttgttaatt cctagtggag
tagcttttgt agttaaagag 180aatgttcaag aagtatcgcc tgttattcct
gataaacccg gagtaattgg aggtgatgtt 240attgataaaa gcgcaaaaac
tagtcaacta aaaaagggaa gtgaaagtct gatttctgga 300attgaacgta
gccatgttga ggaattaaag gaggaaatta aaggagaagg taagaaagta
360cccaaaatga atggacagga taatgaaagc cttgaaacta aaattgttga aaag
41430748DNAMeloidogyne incognita 30gatcaaccaa tcccctcaac aactaaaaga
ctcaaaaaca ccccaaaaca actctaatat 60ggctctcctc ttcagttcta cccttctaat
catttcattt attgccatag ctgagggagc 120aggcgatcga aatgcatcag
cttcaagccc tggttgtatg caggttgcaa cccttattca 180tataggggaa
attcgcccag caaaagcaaa caaaccaggt gtacaaaata ctctaaaaat
240gtctggaaat gctcaaatat tcaaaactac tcaagtgaca ttacaagtag
ctgggcaaga 300gccttgtacc gttaaaatta ataatggcga aaccaaatgt
aaaataaccg gagatgaatt 360aaatggaaaa ttaattttca aaactgaaaa
gggaactgaa atttctgctt ctttcgaaca 420ggctaaattg ttttctgaaa
ataagtgtgt tattgaactt gacacttata acaaggaaac 480ccatgaaact
aaacttaaaa ttaatggaaa taattttatg attaaaaaga aggaaggtaa
540tgtgtcaatt aagtgtggtg gaagagctaa tactgtttaa attttaaaag
agtgaattga 600aagaggaaga aaatataaac aaatgtgaag atgagaaaaa
aatattttga agaaagcatt 660ccaaaaaata aaaaaaaatt aattcttcaa
atccatttat tttttgaata aaacatttta 720ctaaaaaaaa aaaaaaaaaa aaaaaaaa
748311511DNAMeloidogyne incognita 31gatttttatt aattttaaaa
attattaact ctccaaaatg aagtgtttgc tccccttctt 60ttggatttta ttaacaattt
ttgtttcttg cactaatggc acttcaaatg agtatagtga 120acttgttttg
gttcaagctt tgtggagaca cggtgatcgt tcacccacaa aaaccttcaa
180aacggataaa tatcaagaaa aggattggcc tcaaggatgg gggcaattaa
cacctacagg 240aatggctcaa catgtagagt tgggaagacg actaagacag
cgatatatag aggaattgaa 300atttgttggt cctcggtata atagccatga
aatttatgtt agaagtactg actggaatag 360aacattaact agtgctatat
cgaattttat tggcttctac ggccccggaa atgatgatga 420atacccaaag
gatttgggcg caaacaaatg gccaggatgg tttttcccaa tagcgataca
480ttcactccct ggaaacgaag attttatggc tcctggagaa tcggaatgta
aacgatttga 540acaaataaaa gaacggataa ctttaacaga agaatacaac
tcgactttga ttaaatacaa 600atggctactc gattttttga gtgaaaagac
gggacagaat gtcgaccctt tcgatatgtg 660gatgattaac gatgcttttt
atattgagaa attaaaaggc aaaaaattgg tagactgggc 720agaggggaac
caaacacttt tggatacgat tgctgaactt gacaatttac aagaaagatg
780gatggttggg ttagatttaa aacctctggg tgatgccaac tttcgcgaag
aacttccaaa 840aattttgggc gggccaatct tatggaaatt tataacaaat
atgcaggaga agttggcttg 900ttcaaagcga atgaattctg taaaagaaat
tgacagggaa atagagggaa gaaaatcgcc 960aatggggacg cccttgtgta
aatggatgaa caaaatgcgc tattttgcgt actctgcgca 1020cgacagcaca
attattgcaa tttttgcagc tttgggttta aacaaaacga attatgacga
1080ggatggttac ccgaagtatt ctacttgtgt aacttttgaa ttgtggaggg
agaagaatac 1140tggtcaattt gatgttaagg tatttttatg gagacctaac
accaacgaga cttcccctaa 1200agaaataacg acagatattg aaggctgtca
aagcaattca actctagaac aatttgttga 1260aagatcaaaa aattatcaaa
tgctgccttc acccaaagac tattgttcac aacttctaca 1320acccctaaat
aatgctgcac gtatgttaat tcagtggaaa ttggaaatgc ttattctaat
1380gggaattcct tcaattgttg ctaatgttgt atagagaatt tttttgtttt
tggaaattat 1440ttagttgcac ctattcatca aaagaagggc aaaataaatt
tttatcccct aaaaaaaaaa 1500aaaaaaaaaa a 151132994DNAMeloidogyne
incognita 32gatgcttcaa ttgacattta aaattaaaaa gacccaggtt tcttaattaa
agaatgtttt 60taaaaatttt aactttcctt ttaattataa acaaaattat tgcggatgat
tctaatagcg 120gagatagtgg caatgaaaat tctaatagta agcccagtga
tgagcttgcc gactctgttg 180atgttcgaga gcatgataat gagcaacatc
catccaattc gatcgacaag caaaatcttc 240aagacccaca atttattaaa
gaagatgtta caaatgtatt gccactatta aataacgatg 300agaataatct
cattgatgaa tacacaacag aaaaaattaa agaagatgag gaagaccaac
360taaataatga aggatctggt atagacaatg aatttcctga ggaagataat
gatgtaaatg 420gattggatat taatacaact gcaaaatatg ctaatgatgt
agatgataat aataacaatg 480aaggtgatgg tcaatcatgc gtttatgagg
atggtgtaat taacgataat ggtgacgaac 540gcacacctac atatgaagaa
caacaacaaa ttgaagaata tcttcaagaa atgcgtgaat 600ttgaagagca
aatggtcaaa gacagcgcta attttatgag aaatttggca caatttgtga
660tgagtcaatt cgaaaacatt tttggttctt ctacctcgtc tctatcaggt
aacaataata 720atttgttgga gaaaaaacct ttagaagcac caacacctcc
ttgtttatgc aaaaaatgtg 780acagtatgac atttatacaa aataagcaaa
ccaaatatct taaaaatttt gctaattaat 840taacaaaaat ttgaagaata
atttaaataa tgtttatctc tttcttgaga ttttcaaatt 900atttaatcca
tttatatata aatttaaatt
cattttcttt tacaaaaagc tgaagagatt 960aaattttaat gtttgaaaaa
aaaaaaaaaa aaaa 99433362DNAMeloidogyne incognita 33ggaataaaaa
gcggcgaaat tttactttat tcatcaaagt actttaaaat attctataat 60ctaaaatgaa
attcagccaa ttatttgttc tcttaataat tgctttacaa tttgtggttg
120ctcaagggtt gatttacgat gcgaaagcaa tagccaaagg aaaaggaaaa
ccgttcaggg 180cgctgaatat ttggtatttg tcttgggcaa ctatgtaatg
aagaaacgat atctttatgt 240aattgataaa taaaaataac ctaagtatca
aaaaatgttt atgggaaata aaagatttat 300cttcatttaa aatctaataa
atttgtcaat cccaaaaaaa aaaaaaaaaa aaaaaaaaaa 360aa
362341124DNAMeloidogyne incognita 34ggtttaatta cccaagttta
aggggtaaaa aatgttttca agcaaaacta gcttcaattt 60ccttcttcta atttcttcat
ttgctttatg taaggccgac ttttggccta aagcaagaaa 120taatattacg
gtatccgaaa caatacaaat tactaaccgt gactgtaatt ttgatcgtta
180tattcccgat ccgagtaaac ttggaaacgg aggtcagaac gagcatcaag
gctacgtttt 240tgaaataaaa aatggtggtt ctttatctaa ttgtataatt
ggtgctaggc ctgggactaa 300aggctctgct catggagttc tttgtgatgg
agattgcgat ataaacaatg tttggtttga 360ggatgttggg gaagatgcta
ttaattttaa tggagattct gatggttgtg tttataatgt 420taatggtggt
ggtgctaaga atggagaaga caaagttatg caatttgacg gaaaggggac
480actgaatgtt aacaactatt atgtagacaa ttatgtccgt ttttgtcgct
cctgtggcga 540ctgcggtgac caacatcaac gccatatcgt gattactaat
ctgacagcgg ttcatggcca 600agctggtcaa ttcgtttgtg gagtaaatag
caattatcag gatacgtgta ccttgcatga 660tataaaaatg gagaagggta
ttcacccctg caaggttttt gatggcaatt ctgatggatc 720tgagccaact
tcgaataacg acgaagagga ccacggagac gggaaatttt gtatttataa
780gaagggcgat attaaatata ttggatccaa accaaagccg aaaagcaaaa
agagcgcaaa 840gaattaagtg ccggaagtta aaaagccttg aagttaaaaa
cgtttaaagg gataaattgt 900agggttgtcg gttctgaacc gaaccgagcc
gaagaaccga tgatttttcg gttcggttcc 960ggatatccaa agattttcca
agagccgaca accctagtag tatgagtaga atctattatt 1020atttggaata
ctaatttaat tttgtgaaat ttctttttac tatattaatc ctgtccaata
1080aaattatgaa atcgaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
112435782DNAMeloidogyne incognita 35ggcatcaaac aatctactca
acaaataaaa atccctaaaa acaccccaaa acaaccctaa 60aagcatacca aaaatggcca
cctttttcac atttaccctt ctaatcattt caattattgc 120cacaactgag
ggaatgcata ctaatcgaag tgcttcaacc tccgattctc tcaaagccca
180aaaggattgt aaagtgatat atggaatgtt tgtgcctgta gcagggtcag
aaatgcatgg 240agacgccaaa agcgcaatga agccaaacaa tccaagtgtc
cccaataaat taactgtatc 300aggtggcaac tcaaaatatt cagtgacttt
acaggttgaa aaccagccga agtgtgttgc 360ccaaaatgac ggaaaccctg
tagaatgcca aattcaagga gacaaacttt caggaaaatt 420gatttatgat
attgaaaacg gcccttctgt caacgttccc ttcaaagaca ctccaatctt
480tgttggaaat aaatgcgaaa ttgtttttgt agactacgat aaggaccaca
aattaactct 540tttcatgaat aaagtaaagc ttatgatttc cccgactgat
aagcaaattg taaaggcttg 600tggggtgaaa aattagaaag aaaaatgatg
aatgaatgaa ggtgagaggg aaggaaagaa 660aaaatatttt taaaattgaa
gaaagcattc aaaaattaaa aaaaacaatt cttcaagata 720atatataacg
tttaactctt tttgataaat tttatttcaa aaaaaaaaaa aaaaaaaaaa 780aa
78236801DNAMeloidogyne incognita 36attgaagaag atgatacatt caatgttcct
gtcatgggag aagaaaatca tagagatatt 60cctgtcgaag aagctaatta tcaagttcct
ccttctgctg atttcacttt tacaagctct 120gagcatggaa gacgtacatc
atttacagtt ggaacaccgc atcatcgata catgcaacca 180ggcactcgag
aagcatattt attgccccat ccaggagggg aaggtgcaac gcttatacgc
240aatgaagttc gtcgagatgg aacgcaaatt tcccaacaag acacacttca
aaacattgaa 300ggagggagag gttatgttta ttcttcatcg tcccacactc
aaaacgaatc aagtagtagt 360tcaagaataa cttcgagaat tcgttttggg
aataatgaaa gacatgggaa aaaatgagga 420ataaagggaa gatttaggaa
gacatggaaa aaagtgagga atggaggaag agatttatac 480taattataaa
atgatagaaa aattgaagag atattctctt attctttcct atatctttac
540tttcacatac aaaattctat aatggcaatt tatgatttaa cattaaaatt
gaatttagaa 600atatttttta aaattatttt agttttcatt tttatcaatt
ttttgatatt taaatacgtc 660ttgtatttat cttcatataa ttgttgatta
aacttttctt tatcattctt ttgtaggtat 720tctaaaatta aataattata
tgtaatattt tttaattttc aatttgaata aaattttctg 780caaaaaaaaa
aaaaaaaaaa a 801372210DNAMeloidogyne incognita 37gacattcctc
agcttcatta cccatccatt tttcatagac aacatccccc ttgccaaaca 60ttaaaaattg
agtaacgctg aatgaagctt tttgtcctgt taattggagt tttagccttc
120acggttctaa atgtccatgg aggagtcagc cattcgacat tgactcacag
aaacccgcga 180agcaacgaaa tcgaacaatt aactgatgtg tcgttggacg
ataccccatc ctcgcctcct 240caagctgtgt tggacattgg aatgtcagga
cagcgaaatt tgcaacgtcg agaagctcca 300atgtcgattg ggaaaaaagt
ggtggctgta atttttttat ttcttctgtc ttttacatcg 360ttatatctat
tggccgtgcc aaaacaacaa attcaacaag ttgaatacaa acaatttcca
420caaccctata aatttgttcc gattagtaac attgtcaagt gcgacagaaa
aacacggcaa 480tgttcaataa agttagagaa tttggatccg acaaacaact
atagcctcta tactgcaaag 540aatgacaagg ggagaggaga taaagtaaag
ttaactaaag ttgcagatat agatttggac 600aaatgtcaac tcgacaagaa
tgtaaaacca gaagtaaatg gggaagaaat ttgtaatcag 660attgtcaaag
gaattgatga taacgcaaaa gccgaaacta ttgaggttaa cagtggagaa
720atagaatttg gttcggaatt agaaggaacg gaggattatg cgatagttga
aaaagcaatg 780aatgagaaga atgaacataa aaatcaacaa gcggttgagc
atgttcatat ccctgggcca 840ggggaacaac cagttgaaca caatcagccg
acaatagaat atccaacaaa ttccaaacaa 900gttcatccag ctgacaaata
tcaacataaa ctagaagagc gcgccaaaaa atttgggctt 960agcgacttca
aacatggaga tttatatgag gattatcgcc aacaaaaaac ggtccaagaa
1020gatgaaaagg ataaacgata tcaaaaggtt ctaggaacac taggagacca
taaacatcca 1080tcgctagttg atcaatataa cgaagataaa ggaaaattca
atcaacgtgt taaaagtgac 1140cccacaggca ataaagttga aaaggcaaaa
aattctgatt ctaatggact tgaacaaaaa 1200ttagaaaaac tggcactgag
tgacttcaaa catggagatt tatataaaga ttatcagcaa 1260caaatcacgg
tcagagatga tgaaaaggat aaacgatatc aaaaggttct aggaacacta
1320ggagaccata aacatccatc gctagttgaa caatataaca gagataaagg
aaaattcaat 1380caacgcgtta aaagtgaccc cactagcaat tggcatgaag
atttattcgg aaaggattac 1440cgacgtgcta tgagcgattt cgatcattta
aaggctaaac aacgtgaaaa gatccttgga 1500acactagaag atcataagca
tccatcgcta attgatcaat ataacaaagg aagcttaaat 1560caacgcgcta
aaagtgaccc cacaggcaat aatattggaa aggcaaaaaa ttctaatttt
1620aatgggtctg aacaaaaatt agaaaaactg gcactgagtg acttcaaaca
tggagattta 1680ttaggtcgaa aaggaggaat taaacaacgc actataaatg
ttctcgctgg caaaaaaata 1740gaacatgaaa aaggaagtga ttttaatgca
aacgttgaag aaatgatagg ggcagaaaac 1800ggcaaggcta atcaagtgaa
tcccaaatta actggacgca aactagctga atttaatcat 1860attccagctg
ttgacagaat tcttggtttt aaacgtggag gtcatgcgct agaggagcct
1920cataaaaatt gagatatttt gcctgaagag ttggattgaa cgatgtatat
aagatttttt 1980aaccatgtaa atatttttaa aaaagatttt attagagcca
ggaaattacg atactgaatc 2040ccgaaaaata tcgtaatggc tcttaatttt
ttatttttta acttttccat tgcaaagatt 2100tttttaaaat ttttcccgat
tgtctggtaa acttgtgatg agataaactg attttgattg 2160ataataatcg
tccattttcc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 221038836DNAMeloidogyne
incognita 38tacctaaaat tgtttttaaa ttgtttgcct ctgagttttg ctcaactgag
aagaaaaatg 60cttccttact caattctatt tcaattggga atagtttcgt tgcttctacc
tcatgcaaat 120ggaatgcagt ctggcagtag caaaattatg aacaaagcat
ctgaaaagaa atatgctttg 180gttgttgctc caaactttct taaagttcat
tttaaaatga acagtgtctt tgccaatgcg 240ttgaccaaaa agttttttgt
gcactttcta attctgaaca ccaaaaatga agaaattgga 300gataatttcg
actatggaat tgatctcgaa aaatttgaag aaggaacggg aaatacatat
360caagttgtaa attttccaga tgattatccc gaaaaattga acgaaggcgt
gaagaattta 420gagaacaaat tcattaagag aggttacgaa cagagtagtc
aaattctgaa aaatgaagct 480ttcaccgttt ataaaggtta aaatccaaaa
tattttgcct tctaaaattg ttatttgatt 540aataatatat aaaatattta
agatttattt gaaaacaatg gagctattgt tcattacttg 600aaggaggcaa
agtttgattt aggggttttt gacacttggg acactggagc tctcttcatt
660ctccatgcag caggaattaa aaatgttttt ggcattaaca acattcaact
taatgcttat 720caatttaaat atgctgggaa agaatttcca aaaaatattc
cagaaattta ttcggcacaa 780acaggcgata atgaattatc accaccaagg
gaaaaaaaaa aaaaaaaaaa aaaaaa 83639398DNAMeloidogyne incognita
39gacatttaat tttttaaatt tcttaacatt aaataaattc aaaaagaaaa ttgagaaaaa
60aaatctttta atttaaaaaa aaagaaaaaa gaaaaatgta tccttggaca atttttcttt
120tattaattat tttgttggct atggccattg aaataattgg aggaaaaggt
cgaaagttaa 180ggaagagaga caaagaggaa aaaggtcatg cctcaatttt
ctgttgggca ttcatctagg 240gaaggtttcg aggaaaagct tgatgaaatg
gttgaatcaa cttcaaatat gttaataaat 300cttggtaaaa aagtaaagaa
aggagggaag aaagttgtaa aaggagttgt agaaactgcg 360cagctgatca
aaaaaaaaaa aaaaaaaaaa aaaaaaaa 39840860DNAMeloidogyne incognita
40caagtttgag cgtcagcaat tttaaattaa aaaaagacaa actataaaat ctctcttatt
60taaaataagc agtataccct tcaatctatc cacaatccaa taaaactttc taataaaaat
120cctccactaa aatggcatcc tttttttatt tcttatttat ttctgttagt
cttttgattc 180tagctaatgc tgatgatgct ggtagatatc cttcaggaga
tgatttagtt gaaggtacta 240ctgctgctcg gcttcattcg tcttctgacc
taccagacga tgatgaagaa gaatgcgagt 300gcgaagatga cgacgagaca
acagtcgcaa ctcacatttc tacacgcagc aatggttacc 360cttctaataa
cggagccccc actagcacta aacgcccttc aaacaacgga agctcaaaca
420atggaggctc aagctctgtc acaggatctg ttatattgag agataaatgg
gtaaatggcg 480ccaattgtat tttagctttc aagaataatg gaaacgctag
agcatgtggt gtcaagttcg 540agctgactct cggtgataat caaagaatcc
aaagcatttg gaacgttgag aaagtcggag 600ataaagttta cagaattccg
gactacatcc aacttggtcc aggagtcgaa aacagagata 660ttggagttgt
ttacaatgat gtgccagaac tcttccacaa tcaaggtctt ggacaagaag
720aaggatgcaa cattattgaa taaaaaatat ggatataaaa atatttaaaa
aagattaaat 780aagtattatt aaagcttgtg aatataaact ttttcgaaaa
ttaaaataat ggcagaaaaa 840aaaaaaaaaa aaaaaaaaaa
86041617DNAMeloidogyne incognita 41tattaaaaaa ataacaattt cttttaaaaa
taaaatgtat tcccgttcat ctttaatttc 60tttttttctt ttaattaatt taattttgac
tccaatgatt ttggctacta ataatgatgg 120tgttgctgct ccggttgttg
ctaataaaga tgctgggaaa gttagagcga cggaaattat 180aagagcactc
cgtggatttt ggaaaggagt ggcaggtgga gcattggtag gaggaggtgc
240tgttttagct gcacggatat ttcggaacgc tggccggcgc ggatcgcccc
gacccatctg 300gatgaggtcc atctggataa tggagatgca gcatgagcgg
tcctcacaga ctttcctcgg 360tcgaactgga cgccgcgacg ctgccggcgg
cgaccgcaga gatcgagcat gagcgccgcg 420tggccatctt cgatctggtc
gaaaagaaca gtttcgagcc ggtcggcgcc gagggcggcc 480cgtatcagct
gaagctgtcg ctgcaggaca accggctggt gtccggctaa attcgcattt
540aaggaaattc gatgttttta ataatttaat ttaataaatt tgttttatct
ttaaaaaaaa 600aaaaaaaaaa aaaaaaa 617421668DNAMeloidogyne incognita
42attaatttta aaaatctaat taaaaatgaa ttctctctta ttaatagcat ttttatccct
60ctcattttgt gttccaataa aggctgctcc tccatatggg caattatctg tgaaaggaag
120tcaattagtg ggcagtaatg gacaaccagt tcaacttgtt ggaatgtcac
ttttctggtc 180gagttgtggt gaaggagaag ttttttataa taaagcaaca
gtaaatagtc ttaaatgctc 240ttggaattca aatgtagtta gggctgcaat
gggtgtagag tattcagggt gccaacgacc 300aggttatttg gatgccccaa
atgttgagct gggcaaggtt gaagctgttg ttaaggccgc 360aatagagttg
gatatgtatg ttatccttga ctttcacgac cacaatgctc aacaacatgt
420gaaacaagct atcgaattct tcacatattt tgcccaaaac tacggatcta
aataccctaa 480cataatctat gagactttca atgagccact acaagtagac
tggagtggtg taaagtcata 540tcatgagcaa gttgttgcag aaattagaaa
atatgacaca aagaatgtca tcgttctcgg 600tacaacaaca tggtctcagg
atgtcgatac tgctgctaac aatcctgtaa gcggcacaaa 660cctttgctac
actctacact tctacgcagc aactcataaa caaaacataa gagacaaggc
720gcaagctgca atgaataaag gagcttgtat ctttgtaact gaatacggaa
ctgttgatgc 780aagtggaggt ggtggagtgg atgaaggttc gacaaaagaa
tggtataact tcatggatag 840taacaagatt tctaacctca actgggctat
ctcaaacaag gcagaaggtg cttcagcact 900cacatctgga acgagtgctt
ctcaagttgg caatgatgac cgattgactg cctccggtgt 960tctagtgaag
aagtatatta aatcaaagaa tactggtgtc agttgcaatg gtgcatcacc
1020aggcagtggt tcaggaagta acccctcagg aaataaaccg agcaactcac
aaaccagcac 1080tgccaaaaca tcaagcaatt caggaaataa aggcggtaat
tctaacacag ggaataatgc 1140aaataactca ggaagtaaac cgggcaactc
cggaagtaat acaggaaata cgggtagcaa 1200tgccggagcc agttcaggaa
atacggggac cagtacaagc ggtagttctg ttacagcttc 1260agtacaagtt
cccgataaat gggataatgg cgcaagattc caattagtat ttaaaaacaa
1320tgcaagtaca aaaaagtgtg cagtgaaatt ttcattgact tttgcctctg
gacaacaaat 1380tactggcatt tggaacgttc aaaatgtaac aggaaatagt
tttgttcttc cagactacgt 1440tacaattgag gcagggaaac aatatacaga
tgcaggaatg aatataaatg ggccagcaac 1500tcctccacaa attaaggtgc
tcggcgatgg aaaatgcgtt ttttgaaatt aaagactccg 1560tcttaattgt
tgaattattt taatcttatg attgtttaaa ttggaaaaaa atatatgtat
1620aatttgcttc tgttaatttt gtttatttta aatatacgat aaaaatta
166843547DNAMeloidogyne incognita 43taaaaaatga tttttatttc
cttaattatc ctcgtattgg ctgctgaatc taatgaagca 60agcacaaact gcaaggatgg
tgaaggcgcg gtaaccttct tgtccaacca gctcggtaac 120atacagggaa
taaaaggaaa tagttattat aacaaaactt gttccaacaa aaatactgca
180aaacgttgct acccaaatga tgaatcaaat attagcgttt ttaaaattgt
ttgccccaca 240aatatttgta tttgtggtaa tgttgataat caatgttact
ctgcaaaaac agttaatcct 300ggagatttag actatatgtt ctattctcat
agtggcagca tgtttgttaa cccaaatgtt 360ggttcaattt cattatcgtc
acctgataat cattattttg atccaaagac tagtgcccca 420aaattcatgg
aattaacccc aggcacaaaa tcatatctta atgggaatga gctttctgtt
480gcttgtacat cttgtgctaa ctttaagcag ctaacgtgtt gaacaataaa
aaaaaaaaaa 540aaaaaaa 54744963DNAMeloidogyne incognita 44gaaaataaat
tattcttttt aaaattatca aaaaatgcca catttttatt taaaattttt 60aattaattta
attttattaa atttattccc attacttata aaaagcgatt tgtgtaaatt
120tccaacggct aaggggaacc aaactgttga tgaaacaata ccattaaata
aagataaaga 180ttttgggttt attcgtctga tagcttctcc aaagttggga
agttgtacaa ttgactttag 240taagaaaatg tcgccaatat tatggttatc
cgatggggtg actgttagta atttaattat 300tggaactgaa tcttcttcag
gcatttggtg tagtggaagt tgtaccttga agaatgtcta 360ttttgaacgt
gtttgtactc acgccgcagc ttttaatgca acaacagact ttacaaaaac
420tgatagacgt tcatttacat atacagttga ggggggcgct ggactccatg
ctttagataa 480aatgtttgta caatctggcc ccggaaagac aataattaat
aatttttgtg gggatggatt 540ccaaaaagtt tggcgatcgt gtgggacgtg
taatgatgaa gtgagtcaaa attctaaaca 600aagaactgtt actataacaa
attcaaattt tactggcaaa ggacatgtaa ttgcatctgg 660aaatgcccct
tataaagaca aagtttcctt caataatgtc aaaatatttg gttataaaaa
720tcgttcaaca agagttgttt atgcctgtgg ggaagttaaa ccagaaataa
gtgaagatca 780tttagataca ggagcttcaa attggtatat acctggacgt
gctggtactg gaactgtttg 840taattatccc gcttcagcag ttaaaattgt
taattaaaca ttaaaagctt gatatttaga 900aaatagtaat aataaatgtt
atttattgtg aataaagttt tataattaaa aaaaaaaaaa 960aaa
96345720DNAMeloidogyne incognita 45ggtttaatta cccaagttta agacaataaa
ctttttaata aaaatattta attttgaatg 60tctttgaatt ggctttattg caatttattt
attgtaatac tccttttcaa cattgtaaag 120agtgataccg atactaatgc
tgatattgat cgatttgttg aaattgcaga cgatcgttta 180actctttctg
attatgttgc tttatataaa attgttaata acaaaagtat tactgatcca
240aaacgagaag aaaaactttt gaacgatatg agaagtaagg gaaagaatct
ttcgttaaat 300gaggattatg ttactttaat attccaagac caaataaatg
ctagtaaata ttttcagaat 360tatttggtta atttatggaa tcaatcaggc
attccaccta ttaaagttcg agatttaaat 420acagacttac gcccagcaat
tgatcaaata aatacagaaa tgctgcaatt gctagttaaa 480atacaaaaac
ttccctccaa agattgttta aaaaaagtag ataagtctgt aaataatttt
540attatgagag ttaatcaaat tgatgaacaa aatgatgctt tgaaaatggc
tgtgaaaggc 600aaagacctct gccctgcatg taaacataat taacgtttag
ttaattataa agggaaaaga 660aattataatt ttgaaaaaat tttgggtttc
accaaaaaaa aaaaaaaaaa aaaaaaaaaa 72046763DNAMeloidogyne incognita
46aatacacaaa aactatttta aaaaaggcac taacttaaat aatgacttgt agtattaata
60tttttattat tttatttatt acattaatta ttggaatatg cacggaggca aaaatccgta
120aacaatttgt tgactctcca caggaaccac aagctaaatc ggttgatttg
aatttgcaag 180ttttaatctt tataaaaaga tgcaaatcac aattatgggc
agttgggtta aataattata 240aaacacaatt tccaaactgc tcattaattg
aggaaatata ttctcgtcat tatccttttg 300gaatgttaaa aactacacaa
tggttattac aaacacttct tttattttct gcaatgtatt 360ttccatattt
tgaagttcat gatatatctt tggttgtttt tttcaccctg caattttcag
420ttttattcac tggcttttat attattgcgc agttcatgaa agtcaaaata
atccaaaacc 480aattaatttg tctactctct tcttttctga tataatcatt
tcatattgct tcactatatt 540atttattgta cagtttatat catcaggaag
atatggggca tatttgtttc tctttggatt 600aattttgtat ggtggttatt
ctttaatttt aacttttgtt tatttacgta ataatgaaga 660tggatccttt
aaattcccaa tttcaataaa aataaatgtt gaaattattc aaaaatcgga
720taaagaatta aaacaggaaa aaaaaaaaaa aaaaaaaaaa aaa
763471706DNAMeloidogyne incognita 47gacaacacaa atcaaattaa
ttttaaaaat ctaattaaaa atgaattctc tcttattaat 60agcattttta tccctctcat
tttgtgttcc aataaaggct gctcctccat atgggcaatt 120atctgtgaaa
ggaagtcaat tagtgggcag taatggacaa ccagttcaac ttgttggaat
180gtcacttttc tggtcgagtt gtggtgaagg agaagttttt tataataaag
caacagtaaa 240tagtcttaaa tgctcttgga attcaaatgt agttagggct
gcaatgggtg tagagtattc 300agggtgccaa cgaccaggtt atttggatgc
cccaaatgtt gagctgggca aggttgaagc 360tgttgttaag gccgcaatag
agttggatat gtatgttatc cttgactttc acgaccacaa 420tgctcaacaa
catgtgaaac aagctatcga attcttcaca tattttgccc aaaactacgg
480atctaaatac cctaacataa tttatgagac tttcaatgag ccactacaag
tagactggag 540tggtgtaaag tcatatcatg agcaagttgt tgcagaaatt
agaaaatatg acacaaagaa 600tgtcatcgtt ctcggtacaa caacatggtc
tcaagatgtc gatactgctg ctaacaatcc 660tgtaagcggc acaaaccttt
gctacactct acacttctac gcagcaactc ataaacaaaa 720cttaagagac
aaggctcagg ctgcaatgaa taagggagct tgtatctttg taactgaata
780cggaactgtt gatgcaagtg gaggtggtgg agtggatgaa ggttcgacaa
aagaatggta 840taacttcatg gatagtaaca agatttctaa cctcaactgg
gctatctcaa acaaggcaga 900aggtgcttca gcactcacat ctggaacgag
tgcttctcaa attggcaatg atgaccgatt 960gactgcctcc ggtcttatag
tgaagaagta tattaaatca aagaatactg gtgtcagttg 1020caatggtgca
tcatcaggca gtggttccgg aaataacccc tcaggaaatg aaccgagcaa
1080ctcacaaacc agcactgcca aaacatcaag caattcagga aataaaggcg
gtaattctaa 1140cacagggaat aatgcaaata actcaggaag taaaccgggc
aactccggaa gtaatacagg 1200aaatacgggc agcaatgctg gggcaaattc
aggaaatacg gggaccagta caggcagtag 1260ttctgttaca gcttctgtgc
aagttcccga taaatgggat aatggcgcaa gattccaatt 1320agtatttaaa
aacaatgcga gtacaaaaaa gtgtgcagtg aaattttcat tgacttttgc
1380ctctggacaa caaattactg gcatttggaa tgcccaaaat gtaacaggaa
ataattttgt
1440tcttccagac tacgttacaa ttggagcagg gaaacaatat acagatgcag
gaatgaatat 1500aaatgggcca gcaactcctc cacaaattaa ggtgctcggc
gatggaaaat gcgttttttg 1560aaattaaaga ctccgtctaa attgttgaat
tatttaatct tatgattgtt taaattggaa 1620aataaatata tgtataattt
gcttctgtta attttgttta tttaaatata cgataaaaat 1680taaaaaaaaa
aaaaaaaaaa aaaaaa 170648589DNAMeloidogyne incognita 48taaatttctt
ccctaaaatt tatttaaaat tttataacaa aaaaatgttt tcaattcaag 60gattatcttc
ttttcacttc attttcctct cattattgat attattgcaa aactcttcta
120ctgtattttc tcaacttggt tgtgattatg gatcaatgta tggcggggga
atgagtggtt 180atggccaagc aggttatgga aatgaaagta cacacatcac
ttctgcccac attatattgg 240ccaaagtgaa tcacatggtt tctcctgact
tcaacaagca gggcatgaat aatctaacct 300cccacaaaga acacgactag
gaaagaaaat agaataattg gcaaacacta atgcaatcta 360ctacagaagt
caatggagaa tttacctcct aaacaggaaa atgatttgtg cctaaaagga
420aggaagaaga acctcctctt tgttgagggg aaaagtccat aacacaggag
tgcttggacc 480caagtacaca aatataagaa cccttctagg aaaacacgag
ctggggaagc agtttctctt 540tgctattttg tgagaaaata aatgccaaaa
aaaaaaaaaa aaaaaaaaa 58949660DNAMeloidogyne incognita 49ggtttaatta
cccaagttta agaaaataaa ctttttaata aaaatattta attttgaatg 60tcttttaatt
ggctttattg caatttattt attgcaatac tctttttcaa cattgtaaag
120agtgataccg atactaatcc tgatattgat cgatttgttg aaattgcaga
cgatcgttta 180actctttctg attatgttgc tttatataaa gttgttaata
atcaaagtat tactgatcca 240aaacgagaag aaaaactttt gaacgatatg
agaagtaagg gaaagaattt ttcgttaaat 300gaggattatg ttactttaat
attccaagac caaataaatg ctagtaaata ttttcagaat 360tatttagtta
atttatggaa tcaatcaggc ataccactta ttaaagttcg aaatttaaca
420acagacttac gcccagcaat tgatcaaata aatacagaaa tgctgcaatt
gctagttaaa 480atacaaaaac ttccctccaa agattgttta aaaaaagtag
ataagtctgt aaataatttt 540attatgatag ttaatcaaat tgatgaacaa
aatgatgctt tgaaaatggc tgtgaaaggc 600aaagacctct gcccagcatg
taaacataat taacgaaaaa aaaaaaaaaa aaaaaaaaaa 660501242DNAMeloidogyne
incognita 50acgcggggaa cacaaatcga aatattttta aaaatttaat taaatgtttt
ccctctcatt 60agtagcattt ttatccctca cattttgtat tcaaattaat gctgctcctc
cgtatgggca 120attatctgtg aaaggaagtc aattagtggg cagtaatgga
caaccagttc aacttgttgg 180aatgtcactt ttctggtcga gttgtggtga
aggggaaggt ttctataaca gagaaactgt 240aaatagtctt aaatgctctt
ggaattcaaa tgttgttaga gctgcaatgg gtgtagaata 300ttctggatgc
caacgaccag gttaccttga tgccccaaat gttgagctgg caaaggttga
360agctgtagtg aaggcggcga ttgagttgga tatgtatgtt attcttgatt
ttcacgacca 420caatgctcag ggtcatgtga aacaagctaa acaattcttc
gcatattttg cccaaaacta 480cggatctaaa tacccaaata tcatttatga
gactttcaat gagccactac aagtagactg 540gaatggtgta aaatcatatc
atgagcaagt tgttgcagaa attagaaaat atgacaataa 600gaatgtcatc
gttcttggtt caacaacttg gtctcaagat gttgatactg ccgctaataa
660tcctgtacga ggttcaaacc tttgctattc tttacactac tacgcagcaa
ctcataaaca 720aaacttaaga gacaaggcac aggctgcaat taataaagga
gcctgtatct tcgtaactga 780gtacggaacc gttgatgcaa gtggaggtgg
tggagtggat gaaggctcga caaaagagtg 840gtataacttc ttggatagca
agaaaatttc taacctcaac tgggctatct cgaacaaggc 900agaaggggct
gcagcactca cccctggaac gacttcttct caagttggca atgatgaccg
960attgactgcc tccggtcgtc tagtgaaaag ttatattaaa tcaaagaata
ctggtgtcag 1020atgcaatgga gggggtgctg caaaaaaagg ctcttcatca
tctaatactg gttcaaaaaa 1080agacaaacaa aaattcaaag aacaaaaatt
caaagaaaaa atctaacaac gccaaactgc 1140cgaaaaaaag gtcccaaaaa
gaacacttag acaaatatca aggaatttaa tgttaaatgg 1200aatataattg
ttttaaatta aaaaaaaaaa aaaaaaaaaa aa 1242511217DNAMeloidogyne
incognita 51ggtcattctt ataactaaaa accttcaaac ttcaaaaaat attccttaaa
cttcttcaga 60aaaataattg aaaaatgtta ttaaaattct ttttcccatt attgcttttg
cttaccctta 120tctatttggg ttgttctgag gaggataagg gagacattgc
aaatggtcct caggaatctg 180agaatcaggt tgatcaagaa ttggttagat
tgaaaagaga tgatgaagaa gaggagggag 240agaaggccga agatgaagag
aaggctgaag aggatggaga taaagctgaa gatgctgaga 300gtgcagagga
gggagataag gctgaagatg ctgatgaggg agaaaaaaag agtgaagatg
360aagagaaaaa gagtgaaggt gacgaagaaa aagcggaagg tgaagaggaa
gaaaaaaagg 420atggaactga ggaagaaaag gaggatgaag atgaagaaga
gaaaaaagat gatgatgaag 480aaaaaaatga ggaagaagaa aaaaaggatg
acgaagaaga gaatggagat aaagaagaaa 540agaaggatga tacggaagag
aaagaggata aacacacaaa ggataaaagt aagaagaagg 600atagtaagtc
cgttcaaaag gataaaaagg aggaagatga caaggagaaa aaggaaaaaa
660gttcaagtgg tgataattct aaaacagata aatcacaaaa tcaaaaacaa
agcaaagaat 720catgtaatgg ggatactgct tacaactgtc ctaaactatc
aggtctttgt gaatcaaaaa 780ttcaagtaca acaagacttc atgggtgaaa
aatgttgtgc tacgtgcaaa aattcggctc 840ctgctgcgaa gaaagatata
cccctatgca ctgatttggc tgataattgt gatcaaatag 900catccacctg
tggggaagag gcgtggcaac cgactatgat ttctgattgt gctgagacct
960gcgataagtg tgaattacat tttcaaatgt tggagaagag acttgcagca
gctgctgctt 1020aaaattttga aaggaaaaga attttatcaa aaatatatgt
gtatcatatt cactaagcaa 1080gaaattttct ttgattttca cacctttaat
acgtaaaatt tcaatctatt catccgtgtt 1140tctcgtaatt atgttttatt
aattttttcg aaatttagta aaaatgcctc caaaaaaaaa 1200aaaaaaaaaa aaaaaaa
12175213PRTMeloidogyne incognita 52Gly Lys Lys Pro Ser Gly Pro Asn
Pro Gly Gly Asn Asn1 5 105313PRTartificial sequenceshared amino
acids of the functional domain of Arabidopsis CLV3-like proteins
53Lys Xaa Xaa Xaa Pro Ser Gly Pro Asn Pro Xaa Xaa Asn1 5
105413PRTArabidopsis sp. 54Lys Arg Leu Val Pro Ser Gly Pro Asn Pro
Leu His Asn1 5 105527DNAartificial sequenceprimer 55gagaaaataa
aatataaatt attcctc 275619DNAartificial sequenceprimer 56cagatataat
tttattcag 195732DNAartificial sequenceprimer 57cggggtacct
agatgtttac taattcaatt aa 325831DNAartificial sequenceprimer
58cggggtacct agatgggcaa aaagcctagt g 315926DNAartificial
sequenceprimer 59gctctagatc aattatttcc tccagg 266027DNAartificial
sequenceprimer 60ggggtaccat ggattctaaa agctttg 276133DNAartificial
sequenceprimer 61ccactaggct ttttgccaag gaacaagaag cag
336236DNAartificial sequenceprimer 62cttctgcttc ttgttccttg
gcaaaaagcc tagtgg 366326DNAartificial sequenceprimer 63gctctagatc
aattatttcc tccagg 266431DNAartificial sequenceprimer 64gatctttgcc
ggaaaacaat tggaggatgg t 316532DNAartificial sequenceprimer
65cgacttgtca ttagaaagaa agagataaca gg 326620DNAartificial
sequenceprimer 66ccggtcgtgg tcttactgat 206720DNAartificial
sequenceprimer 67gcaccgattg tgatgacttg 206839DNAartificial
sequenceprimer 68taatacgact cactataggg cctcaaaaat accataaag
396939DNAartificial sequenceprimer 69taatacgact cactataggg
gaaattaaca aaggaaacc 397039DNAartificial sequenceprimer
70taatacgact cactataggg ggcaaaaagc ctagtgggc 397138DNAartificial
sequenceprimer 71taatacgact cactataggg tcaattattt cctccagg
387228DNAartificial sequenceprimer 72ccgctcgagg gcaaaaagcc tagtgggc
287327DNAartificial sequenceprimer 73cggggtacct caattatttc ctccagg
277426DNAartificial sequenceprimer 74ccatcgattc aattatttcc tccagg
267527DNAartificial sequenceprimer 75gctctagagg caaaaagcct agtgggc
277628DNAartificial sequenceprimer 76ccgctcgagc ctcaaaaata ccataaag
287728DNAartificial sequenceprimer 77cggggtaccg aaattaacaa aggaaacc
287827DNAartificial sequenceprimer 78ccatcgatga aattaacaaa ggaaacc
277927DNAartificial sequenceprimer 79gctctagacc tcaaaaatac cataaag
27
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