U.S. patent application number 10/755092 was filed with the patent office on 2006-01-26 for synthetic dna sequence having enhanced insecticidal activity in maize.
Invention is credited to Cindy Boyce, Nadine Carozzi, John L. Dawson, Nalini Desai, Erik Dunder, Steve Evola, Michael Koziel, Karen Launis, Kelly Lewis, Gary M. Pace, Steven J. Rothstein, Jan Suttie, Gregory Warren, Martha Wright.
Application Number | 20060021095 10/755092 |
Document ID | / |
Family ID | 27118547 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060021095 |
Kind Code |
A1 |
Koziel; Michael ; et
al. |
January 26, 2006 |
Synthetic DNA sequence having enhanced insecticidal activity in
maize
Abstract
DNA sequences optimized for expression in plants are disclosed.
The DNA sequences preferably encode for an insecticidal
polypeptides, particularly insecticidal proteins from Bacillus
thuringiensis. Plant promoters, particular tissue-specific and
tissue-preferred promoters are also provided. Additionally
disclosed are transformation vectors comprising said DNA sequences.
The transformation vectors demonstrate high levels of insecticidal
activity when transformed into maize.
Inventors: |
Koziel; Michael; (Raleigh,
NC) ; Desai; Nalini; (Chapel Hill, NC) ;
Lewis; Kelly; (Cary, NC) ; Warren; Gregory;
(Apex, NC) ; Evola; Steve; (Cary, NC) ;
Wright; Martha; (Overland Park, KS) ; Launis;
Karen; (Franklinton, NC) ; Rothstein; Steven J.;
(Clive, IA) ; Boyce; Cindy; (Raleigh, NC) ;
Dawson; John L.; (Greensboro, NC) ; Dunder; Erik;
(Hillsborough, NC) ; Pace; Gary M.; (Cary, NC)
; Suttie; Jan; (Raleigh, NC) ; Carozzi;
Nadine; (Raleigh, NC) |
Correspondence
Address: |
WHITE & CASE LLP;PATENT DEPARTMENT
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Family ID: |
27118547 |
Appl. No.: |
10/755092 |
Filed: |
January 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09988462 |
Nov 20, 2001 |
6720488 |
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10755092 |
Jan 8, 2004 |
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09547422 |
Apr 11, 2000 |
6320100 |
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09988462 |
Nov 20, 2001 |
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08459504 |
Jun 2, 1995 |
6075185 |
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09547422 |
Apr 11, 2000 |
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07951715 |
Sep 25, 1992 |
5625136 |
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08459504 |
Jun 2, 1995 |
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07772027 |
Oct 4, 1991 |
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07951715 |
Sep 25, 1992 |
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Current U.S.
Class: |
800/302 ;
435/412; 435/468; 536/23.6; 800/320.1 |
Current CPC
Class: |
C12N 9/12 20130101; Y02A
40/146 20180101; C07K 14/325 20130101; C12N 15/8286 20130101; C12N
9/88 20130101 |
Class at
Publication: |
800/302 ;
800/320.1; 536/023.6; 435/412; 435/468 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; C12N 5/04 20060101
C12N005/04; C12N 15/82 20060101 C12N015/82 |
Claims
1. A nucleotide sequence comprising a maize optimized coding
sequence which encodes a protein capable of killing an insect.
2. A nucleotide sequence of claim 1, wherein said coding sequence
encodes a B.t. protein.
3. A nucleotide sequence of claim 2, wherein said coding sequence
encodes a CryIA(b) protein.
4. A nucleotide sequence of claim 3, wherein said coding sequence
comprises Sequence 3, Sequence 4, or the sequence set forth in FIG.
7.
5. A nucleotide sequence of claim 3, wherein coding sequence
encodes a CryIA(b) protein which is heat stable compared to a
native CryIA(b) protein.
6. A nucleotide sequence of claim 5, wherein said coding sequence
comprises a sequence selected from the group of sequences
consisting of FIG. 9, FIG. 11, FIG. 13 and FIG. 15.
7. A nucleotide sequence of claim 2, wherein said coding sequence
encodes a CryIB or a CryIA(c) protein.
8. A nucleotide sequence of claim 7, wherein said coding sequence
comprises the sequence encoding a CryIB protein set forth in FIG.
6.
9. A nucleotide sequence of claim 1, further comprising a first
promoter capable of directing expression of a nucleotide sequence
in a plant cell, operably linked to said coding sequence.
10. A nucleotide sequence of claim 9, wherein said promoter is
capable of directing expression of the associated coding sequence
in a maize cell.
11. A nucleotide sequence of claim 9, wherein said promoter is
selected from the group consisting of inducible promoters,
constitutive promoters, temporal or developmentally-regulated
promoters, tissue-preferred, and tissue-specific promoters.
12. A nucleotide sequence of claim 9, wherein said promoter is
selected from the group consisting of a CaMV 35S promoter, CaMV 19S
promoter, a PEP carboxylase promoter, a pith-preferred promoter,
and a pollen-specific promoter.
13. A nucleotide sequence of claim 9, wherein said promoter is a
pith-preferred promoter comprising the DNA sequence set forth in
FIG. 24.
14. A nucleotide sequence of claim 9, wherein said promoter is a
pollen-specific promoter comprising the DNA sequence set forth in
FIG. 35.
15. A nucleotide sequence of claim 9, further comprising a second
promoter capable of directing expression of an associated coding
sequence in a plant cell, operatively linked to a second coding
sequence.
17. A nucleotide sequence of claim 15, wherein said second promoter
is selected from the group consisting of inducible promoters,
constitutive promoters, temporal or developmentally-regulated
promoters, tissue-preferred, and tissue-specific promoters.
18. A nucleotide sequence of claim 15, wherein said second promoter
is selected from the group consisting of a CaMV 35S promoter, CaMV
19S promoter, a PEP carboxylase promoter, a pith-preferred
promoter, and a pollen-specific promoter.
21. A nucleotide sequence of claim 15, wherein said second coding
sequence is a plant optimized coding sequence which encodes a
protein capable of killing an insect.
22. A nucleotide sequence of claim 21, wherein said second coding
sequence encodes a B.t. protein.
23. A nucleotide sequence of claim 22, wherein said second coding
sequence encodes a CryIA(b) protein.
29. A nucleotide sequence of claim 15, wherein said second coding
sequence is a marker gene.
30. A recombinant vector, comprising a nucleotide sequence of claim
9.
31. A recombinant vector, comprising a nucleotide sequence of claim
9, wherein said coding sequence encodes a B.t. protein.
32. A recombinant vector of claim 31, wherein the B.t. protein is a
CryIA(b) protein.
33. A recombinant vector, comprising a nucleotide sequence of claim
15.
34. A recombinant vector, comprising a nucleotide sequence of claim
18.
35. A recombinant vector, comprising a nucleotide sequence of claim
22.
36. A plant stably transformed with a nucleotide sequence of claim
9.
37. A plant stably transformed with a nucleotide sequence of claim
12.
38. A plant of claim 36, wherein said coding sequence encodes a
B.t. protein.
39. A plant of claim 36, wherein said protein is expressed in said
plant in an amount sufficient to control lepidopteran or
Coleopteran insects.
40. A plant of claim 38, wherein the B.t. protein is a CryIA(b)
protein.
41. A plant of claim 38, which expresses the B.t. insecticidal
protein in an amount sufficient to control Lepidopteran or
Coleopteran pests.
42. A plant of claim 38, wherein the amount is sufficient to
control insects selected from the group consisting of European corn
borer, Sugarcane borer, stalk borers, cutworms, armyworms,
rootworms, wireworms and aphids.
43. A plant stably transformed with a nucleotide sequence of claim
15.
44. A plant stably transformed with a nucleotide sequence of claim
18.
45. A plant of claim 43, wherein the first and second coding
sequences each encode a B.t. protein.
46. A plant of claim 38, which expresses the B.t. insecticidal
proteins in an amount sufficient to control lepidopteran or
Coleopteran pests.
47. A plant of claim 46, wherein the amount is sufficient to
control stalk borers.
48. An isolated and purified promoter capable of directing
pith-preferred expression of an associated structural gene in a
plant.
49. A promoter of claim 48, isolated from a monocot.
50. A promoter of claim 48, isolated from a maize plant.
51. A promoter of claim 48, isolated from a plant tryptophan
synthase-alpha (TrpA) subunit gene.
52. A promoter of claim 51, isolated from a maize tryptophan
synthase-alpha (TrpA) subunit gene.
53. A promoter of claim 48, comprising the sequence set forth in
FIG. 24.
54. A recombinant DNA molecule comprising a promoter of claim 48,
operably associated with a structural gene encoding a protein of
interest.
55. A recombinant DNA molecule of claim 54, wherein said structural
gene encodes an insecticidal protein.
56. A recombinant DNA molecule of claim 55, wherein said structural
gene encodes a Bacillus thuringiensis protein.
57. A vector, comprising a recombinant DNA molecule of claim
54.
58. A vector of claim 57, wherein said structural gene encodes an
insecticidal protein.
59. A vector of claim 57, wherein said structural gene encodes a
Bacillus thuringiensis protein.
60. A vector, comprising at least two recombinant DNA molecules of
claim 54, wherein at least one of the two structural genes encodes
an insecticidal protein.
61. A plant stably transformed with recombinant DNA molecule of
claim 54.
62. A plant of claim 61, which is a maize plant.
63. A purified promoter capable of directing pollen-specific
expression of an associated structural gene in a plant, wherein
said promoter is isolated from a plant calcium-dependent phosphate
kinase (CDPK) gene.
64. A promoter of claim 63, isolated from a monocot CDPK gene.
65. A promoter of claim 63, isolated from a maize CDPK gene.
66. A promoter of claim 65, comprising the sequence set forth in
FIG. 35.
67. A recombinant DNA molecule, comprising a promoter of claim 63,
operably associated with a structural gene encoding a protein of
interest.
68. A recombinant DNA molecule, of claim 67, wherein said
structural gene encodes an insecticidal protein.
69. A recombinant DNA molecule of claim 68, wherein said structural
gene encodes a Bacillus thuringiensis protein.
70. A vector, comprising at least one recombinant DNA molecule of
claim 67.
71. A vector of claim 70, wherein said structural gene encodes an
insecticidal protein.
72. A vector of claim 71, wherein said structural gene encodes a
Bacillus thuringiensis protein.
73. A vector of claim 70, comprising two recombinant DNA molecules,
wherein at least one of the two structural genes encodes an
insecticidal protein.
74. A plant stably transformed with a recombinant DNA molecule of
claim 67.
75. A plant of claim 74, which is maize plant.
76. A maize plant stably transformed with at least one recombinant
DNA molecule: wherein said DNA molecule comprises a promoter
operably linked to a nucleotide sequence encoding an insecticidal
protein. wherein said promoter is capable of directing
tissue-preferred or tissue-specific expression of said gene in said
maize plant.
77. A maize plant of claim 76, wherein said gene encodes a Bacillus
thuringiensis protein.
78. A plant of claim 76, wherein said promoter is obtained from a
monocot.
79. A plant of claim 78, wherein said monocot is maize.
80. A maize plant of claim 76, wherein said promoter is capable of
directing pith-preferred expression of said gene in said maize
plant.
81. A plant of claim 76, wherein said promoter is obtained from a
plant tryptophan synthase-alpha subunit gene.
82. A plant of claim 76, wherein said promoter is obtained from a
maize tryptophan synthase-alpha subunit gene.
83. A plant of claim 82, wherein said promoter comprises the
sequence set forth in FIG. 24.
84. A plant of claim 76, wherein said promoter is capable of
directing green tissue-specific expression of said gene in said
maize plant.
85. A plant of claim 84, wherein said promoter is a PEP carboxylase
promoter.
86. A maize plant of claim 76, wherein said promoter is capable of
directing pollen-specific expression of said gene in said maize
plant.
87. A maize plant of claim 76, wherein said promoter is obtained
from a plant calcium-dependent phosphate kinase gene.
88. A maize plant of claim 76, wherein said promoter is obtained
from a maize calcium-dependent phosphate kinase gene.
89. A maize plant of claim 76, wherein said promoter comprises the
sequence set forth in FIG. 35.
90. A method of producing a maize optimized coding sequence for an
insecticidal B.t. protein, comprising: determining the amino acid
sequence of a predetermined insecticidal B.t. protein, and altering
the coding sequence of the protein by substituting codons which are
most preferred in maize for corresponding native codons.
91. A method of protecting a maize plant against at least one
insect pest, comprising: stably transforming a maize plant with at
least one nucleotide sequence of claim 9, wherein the coding
sequence encodes an insecticidal protein; whereby the transformed
maize plant expresses the insecticidal protein in an amount
sufficient to protect the plant against the pest.
92. The method of claim 91, wherein said insecticidal protein is a
B.t. protein.
93. The method of claim 91, wherein said promoter is a
tissue-specific or a tissue-preferred promoter.
94. A plant which has been stably transformed with a nucleotide
sequence wherein said nucleotide sequence comprises a maize
optimized coding sequence for an insecticidal protein, wherein said
protein is expressed in said transformed plant at least 100 fold
greater than expression of the protein using a native coding
sequence.
95. A transgenic maize seed comprising a chimeric gene comprising a
heterologous promoter sequence operatively linked to a synthetic
DNA coding sequence that encodes a Bacillus thuringiensis (Bt)
insecticidal protein selected for optimized expression in a plant,
wherein said synthetic DNA coding sequence is produced by a method
comprising: (a) obtaining the amino acid sequence of said Bt
insecticidal protein; (b) reverse-translating said amino acid
sequence into a synthetic DNA coding sequence comprising a
sufficient number of the single codons that most frequently encode
each amino acid in maize, wherein said synthetic DNA coding
sequence has at least about 60% G+C content, and wherein the single
codons that most frequently encode each amino acid in maize are
determinable by (i) pooling a plurality of gene sequences from
maize, (ii) calculating a codon usage profile from said pooled
maize gene sequences, and (iii) determining which single codon most
frequently encodes each amino acid in maize; and (c) synthesizing
said DNA coding sequence.
96. A transgenic maize seed according to claim 95, wherein said
Bacillus thuringiensis insecticidal protein is CryIA(b).
97. A transgenic maize seed according to claim 95, wherein said
Bacillus thuringiensis insecticidal protein is CryIB.
98. A transgenic maize plant grown from the transgenic maize seed
of claim 95.
99. A method of controlling insect pests, comprising contacting the
insect pests with the transgenic plant according to claim 98.
100. The method of claim 99, wherein the insect pests are
lepidopteran insect pests.
101. The method of claim 100, wherein the insect pests are European
corn borers.
102. A method of producing an insect-resistant maize plant,
comprising growing the transgenic maize seed of claim 95, wherein
said synthetic DNA coding sequence is expressed in said plant in an
effective amount to control insect pests.
103. The method of claim 102, wherein the insect pests are
lepidopteran insect pests.
104. The method of claim 103, wherein the insect pests are European
corn borers.
105. A transgenic plant seed comprising a chimeric gene comprising
a heterologous promoter sequence operatively linked to a synthetic
DNA coding sequence that encodes a Bacillus thuringiensis (Bt)
insecticidal protein selected for optimized expression in a plant,
wherein said synthetic DNA coding sequence is produced by a method
comprising: (a) obtaining the amino acid sequence of said Bt
insecticidal protein; (b) reverse-translating said amino acid
sequence into a synthetic DNA coding sequence comprising a
sufficient number of the following codons: Ala, GCC; Arg, CGC; Asn,
AAC; Asp, GAC; Cys, TGC; Gln, CAG; Glu, GAG; Gly, GGC; His, CAC;
Ile, ATC; Leu, CTG; Lys, AAG; Met, ATG; Phe, TTC; Pro, CCC; Ser,
AGC; Thr, ACC; Trp, TGG; Tyr, TAC; and Val, GTG; such that said
synthetic DNA coding sequence has at least about 60% G+C content;
and (c) synthesizing said DNA coding sequence.
106. A transgenic plant seed according to claim 105, wherein said
Bacillus thuringiensis insecticidal protein is CryIA(b).
107. A transgenic plant seed according to claim 105, wherein said
Bacillus thuringiensis insecticidal protein is CryIB.
108. A transgenic plant grown from the transgenic plant seed of
claim 105.
109. A transgenic plant according to claim 108, which is maize.
110. A method of controlling insect pests, comprising contacting
the insect pests with the transgenic plant according to claim
108.
111. The method of claim 110, wherein the insect pests are
lepidopteran insect pests.
112. The method of claim 110, wherein said transgenic plant is
maize.
113. The method of claim 112, wherein the insect pests are European
corn borers.
114. A method of producing an insect-resistant plant, comprising
growing the transgenic seed of claim 105, wherein said synthetic
DNA coding sequence is expressible in said plant in an effective
amount to control insect pests.
115. The method of claim 114, wherein the insect pests are
lepidopteran insect pests.
116. The method of claim 114, wherein said plant is maize.
117. The method of claim 116, wherein the insect pests are European
corn borers.
Description
[0001] This is a continuation of U.S. application Ser. No.
09/547,422, filed Apr. 11, 2000, which is a continuation of U.S.
application Ser. No. 08/459,504, filed Jun. 2, 1995, now U.S. Pat.
No. 6,075,185, which is a division of U.S. application Ser. No.
07/951,715, filed Sep. 25, 1992, now U.S. Pat. No. 5,625,136, which
is a continuation-in-part of U.S. application Ser. No. 07/772,027,
filed Oct. 4, 1991, now abandoned, which disclosures are herein
incorporated in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to DNA sequences encoding
insecticidal proteins, and expression of these sequences in
plants.
BACKGROUND OF THE INVENTION
[0003] Expression of the insecticidal protein (IP) genes derived
from Bacillus thuringiensis (Bt) in plants has proven extremely
difficult. Attempts have been made to express chimeric promoter/Bt
IP gene combinations in plants. Typically, only low levels of
protein have been obtained in transgenic plants. See, for example,
Vaeck et al., Nature 328:33-37, 1987; Barton et al., Plant Physiol.
85:1103-1109, 1987; Fischoff et al., Bio/Technology 5:807-813,
1987.
[0004] One postulated explanation for the cause of low expression
is that fortuitious transcription processing sites produce aberrant
forms of Bt IP mRNA transcript. These aberrantly processed
transcripts are non-functional in a plant, in terms of producing an
insecticidal protein. Possible processing sites include
polyadenylation sites, intron splicing sites, transcriptional
termination signals and transport signals. Most genes do not
contain sites that will deleteriously affect gene expression in
that gene's normal host organism. However, the fortuitous
occurrence of such processing sites in a coding region might
complicate the expression of that gene in transgenic hosts. For
example, the coding region for the Bt insecticidal crystal protein
gene derived from Bacillus thuringiensis strain kurstaki (GENBANK
BTHKURHD, accession M15271, B. thuringiensis var. kurstaki, HD-1;
Geiser et al. Gene 48:109-118 (1986)) as derived directly from
Bacillus thuringiensis, might contain sites which prevent this gene
from being properly processed in plants.
[0005] Further difficulties exist when attempting to express
Bacillus thuringiensis protein in an organism such as a plant. It
has been discovered that the codon usage of a native Bt IP gene is
significantly different from that which is typical of a plant gene.
In particular, the codon usage of a native Bt IP gene is very
different from that of a maize gene. As a result, the mRNA from
this gene may not be efficiently utilized. Codon usage might
influence the expression of genes at the level of translation or
transcription or mRNA processing. To optimize an insecticidal gene
for expression in plants, attempts have been made to alter the gene
to resemble, as much as possible, genes naturally contained within
the host plant to be transformed.
[0006] Adang et al., EP 0359472 (1990), relates to a synthetic
Bacillus thuringiensis tenebrionis (Btt) gene which is 85%
homologous to the native Btt gene and which is designed to have an
A+T content approximating that found in plants in general. Table 1
of Adang et al. show the codon sequence of a synthetic Btt gene
which was made to resemble more closely the normal codon
distribution of dicot genes. Adang et al. state that a synthetic
gene coding for IP can be optimized for enhanced expression in
monocot plants through similar methods, presenting the frequency of
codon usage of highly expressed monocot proteins in Table 1. At
page 9, Adang et al. state that the synthetic Btt gene is designed
to have an A+T content of 55% (and, by implication, a G+C content
of 45%). At page 20, Adang et al. disclose that the synthetic gene
is designed by altering individual amino acid codons in the native
Bt gene to reflect the overall distribution of codons preferred by
dicot genes for each amino acid within the coding region of the
gene. Adang et al. further state that only some of the native Btt
gene codons will be replaced by the most preferred plant codon for
each amino acid, such that the overall distribution of codons used
in dicot proteins is preserved.
[0007] Fischhoff et al., EP 0 385 962 (1990), relates to plant
genes encoding the crystal protein toxin of Bacillus thuringiensis.
At table V, Fischhoff et al. disclose percent usages for codons for
each amino acid. At page 8, Fischoff et al. suggest modifying a
native Bt gene by removal of putative polyadenylation signals and
ATTTA sequences. Fischoff et al. further suggest scanning the
native Bt gene sequence for regions with greater than four
consecutive adenine or thymine nucleotides to identify putative
plant polyadenylation signals. Fischoff et al. state that the
nucleotide sequence should be altered if more than one putative
polyadenylation signal is identified within ten nucleotides of each
other. At page 9, Fischoff et al. state that efforts should be made
to select codons to preferably adjust the G+C content to about
50%.
[0008] Perlak et al., PNAS USA, 88:3324-3328 (1991), relates to
modified coding sequences of the Bacillus thuringiensis cryIA(b)
gene, similar to those shown in Fischoff et al. As shown in table 1
at page 3325, the partially modified cryIA(b) gene of Perlak et al.
is approximately 96% homologous to the native cryIA(b) gene (1681
of 1743 nucleotides), with a G+C content of 41%, number of plant
polyadenylation signal sequences (PPSS) reduced from 18 to 7 and
number of ATTTA sequences reduced from 13 to 7. The fully modified
cryIA(b) gene of Perlak et al. is disclosed to be fully synthetic
(page 3325, column 1). This gene is approximately 79% homologous to
the native cryIA(b) gene (1455 of 1845 nucleotides), with a G+C
content of 49%, number of plant polyadenylation signal sequences
(PPSS) reduced to 1 and all ATTTA sequences removed.
[0009] Barton et al., EP 0431 829 (1991), relates to the expression
of insecticidal toxins in plants. At column 10, Barton et al.
describe the construction of a synthetic AaIT insect toxin gene
encoding a scorpion toxin using the most preferred codon for each
amino acid according to the chart shown in FIG. 1 of the
document.
SUMMARY OF THE INVENTION
[0010] The present invention is drawn to methods for enhancing
expression of heterologous genes in plant cells. Generally, a gene
or coding region of interest is constructed to provide a plant
specific preferred codon sequence. In this manner, codon usage for
a particular protein is altered to increase expression in a
particular plant. Such plant optimized coding sequences can be
operably linked to promoters capable of directing expression of the
coding sequence in a plant cell.
[0011] Specifically, it is one of the objects of the present
invention to provide synthetic insecticidal protein genes which
have been optimized for expression in plants.
[0012] It is another object of the present invention to provide
synthetic Bt insecticidal protein genes to maximize the expression
of Bt proteins in a plant, preferably in a maize plant. It is one
feature of the present invention that a synthetic Bt IP gene is
constructed using the most preferred maize codons, except for
alterations necessary to provide ligation sites for construction of
the full synthetic gene.
[0013] According to the above objects, we have synthesized Bt
insecticidal crystal protein genes in which the codon usage has
been altered in order to increase expression in plants,
particularly maize. However, rather than alter the codon usage to
resemble a maize gene in terms of overall codon distribution, we
have optimized the codon usage by using the codons which are most
preferred in maize (maize preferred codons) in the synthesis of the
synthetic gene. The optimized maize preferred codon usage is
effective for expression of high levels of the Bt insecticidal
protein. This might be the result of maximizing the amount of Bt
insecticidal protein translated from a given population of
messenger RNAs. The synthesis of a Bt IP gene using maize preferred
codons also tends to eliminate fortuitous processing sites that
might occur in the native coding sequence. The expression of this
synthetic gene is significantly higher in maize cells than that of
the native IP Bt gene.
[0014] Preferred synthetic, maize optimized DNA sequences of the
present invention derive from the protein encoded by the cryIA(b)
gene in Bacillus thuringiensis var. kurstaki, HD-1; Geiser et al.,
Gene, 48:109-118 (1986) or the cryIB gene (AKA Crya4 gene)
described by Brizzard and Whiteley, Nuc. Acids. Res., 16:2723
(1988). The DNA sequence of the native kurstaki HD-1 cryIA(b) gene
is shown as SEQ ID NO:1. These proteins are active against various
lepidopteran insects, including Ostrinia nubilalis, the European
Corn Borer.
[0015] While the present invention has been exemplified by the
synthesis of maize optimized Bt protein genes, it is recognized
that the method can be utilized to optimize expression of any
protein in plants.
[0016] The instant optimized genes can be fused with a variety of
promoters, including constitutive, inducible, temporally regulated,
developmentally regulated, tissue-preferred and tissue-specific
promoters to prepare recombinant DNA molecules, i.e., chimeric
genes. The maize optimized gene (coding sequence) provides
substantially higher levels of expression in a transformed plant,
when compared with a non-maize optimized gene. Accordingly, plants
resistant to Coleopteran or Lepidopteran pests, such as European
corn borer and sugarcane borer, can be produced.
[0017] It is another object of the present invention to provide
tissue-preferred and tissue-specific promoters which drive the
expression of an operatively associated structural gene of interest
in a specific part or parts of a plant to the substantial exclusion
of other parts.
[0018] It is another object of the present invention to provide
pith-preferred promoters. By "pith-preferred," it is intended that
the promoter is capable of directing the expression of an
operatively associated structural gene in greater abundance in the
pith of a plant than in the roots, outer sheath, and brace roots,
and with substantially no expression in seed.
[0019] It is yet another object of this invention to provide
pollen-specific promoters. By "pollen-specific," it is intended
that the promoter is capable of directing the expression of an
operatively associated structural gene of interest substantially
exclusively in the pollen of a plant, with negligible expression in
any other plant part. By "negligible," it is meant functionally
insignificant.
[0020] It is yet another object of the present invention to provide
recombinant DNA molecules comprising a tissue-preferred promoter or
tissue-specific promoter operably associated or linked to a
structural gene of interest, particularly a structural gene
encoding an insecticidal protein, and expression of the recombinant
molecule in a plant.
[0021] It is a further object of the present invention to provide
transgenic plants which express at least one structural gene of
interest operatively in a tissue-preferred or tissue-specific
expression pattern.
[0022] In one specific embodiment of the invention disclosed and
claimed herein, the tissue-preferred or tissue-specific promoter is
operably linked to a structural gene encoding an insecticidal
protein, and a plant is stably transformed with at least one such
recombinant molecule. The resultant plant will be resistant to
particular insects which feed on those parts of the plant in which
the gene(s) is(are) expressed. Preferred structural genes encode
B.t. insecticidal proteins. More preferred are maize optimized B.t.
IP genes.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIG. 1 is a comparison of the full-length native Bt cryIA(b)
gene (BTHKURHD; SEQ ID NO:1), a full-length synthetic maize
optimized Bt cryIA(b) gene (flsynbt.fin; SEQ ID NO:4) and a
truncated synthetic maize optimized Bt cryIA(b) gene (bssyn; SEQ ID
NO:3). This figure shows that the full-length synthetic maize
optimized cryIA(b) gene sequence matches that of the native
cryIA(b) gene at about 2354 out of 3468 nucleotides (approximately
68% homology).
[0024] FIG. 2 is a comparison of the truncated native Bt cryIA(b)
gene (nucleotides 1 to 1947 of BTHKURHD; SEQ ID NO:1) and a
truncated synthetic maize optimized Bt gene (bssyn; SEQ ID NO:3).
This figure shows that the truncated synthetic maize optimized
cryIA(b) gene sequence matches that of the native cryIA(b) gene at
about 1278 out of 1947 nucleotides (approximately 66%
homology).
[0025] FIG. 3 is a comparison of the pure maize optimized Bt gene
sequence (syn1T.mze; SEQ ID NO:2) with a truncated synthetic maize
optimized Bt gene (bssyn; SEQ ID NO:3) and a full-length synthetic
maize optimized Bt gene modified to include restriction sites for
facilitating construction of the gene (synful.mod; SEQ ID NO:4).
This figure shows that the truncated synthetic maize optimized
cryIA(b) gene sequence matches that of the pure maize optimized
cryIA(b) gene at 1913 out of 1947 nucleotides (approximately 98%
homology).
[0026] FIG. 4 is a comparison of a native truncated Bt cryIA(b)
gene (nucleotides 1 to 1845 of BTHKURHD SEQ ID NO:1) with a
truncated synthetic cryIA(b) gene described in Perlak et al., PNAS
USA, 88:3324-3328 (1991) (PMONBT SEQ ID NO:5) and a truncated
synthetic maize optimized Bt gene (bssyn SEQ ID NO:3). This figure
shows that the PMONBT gene sequence matches that of the native
cryIA(b) gene at about 1453 out of 1845 nucleotides (approximately
79% homology), while the truncated synthetic maize optimized Bt
cryIA(b) gene matches the native cryIA(b) gene at about 1209 out of
1845 nucleotides (approximately 66% homology).
[0027] FIG. 5 is a comparison of a truncated synthetic cryIA(b)
gene described in Perlak et al., PNAS USA, 88:3324-3328 (1991)
(PMONBT SEQ ID NO:5) and a truncated synthetic maize optimized Bt
cryIA(b) gene (bssyn SEQ ID NO:3). This figure shows that the
PMONBT gene sequence matches that of the truncated synthetic maize
optimized Bt cryIA(b) gene at about 1410 out of 1845 nucleotides
(approximately 77% homology).
[0028] FIG. 6 is a full-length, maize optimized CryIB gene (SEQ ID
NO:6) encoding the CryIB protein (SEQ ID NO:7).
[0029] FIG. 7 is a full-length, hybrid, partially maize optimized
DNA sequence of a CryIA(b) gene (SEQ ID NO:8) which is contained in
pCIB4434. The synthetic region is from nucleotides 1-1938 (amino
acids 1-646 SEQ ID NO:9), and the native region is from nucleotides
1939-3468 (amino acids 647-1155 SEQ ID NO:9). The fusion point
between the synthetic and native coding sequences is indicated by a
slash (/) in the sequence.
[0030] FIG. 8 is a map of pCIB4434.
[0031] FIG. 9 is a full-length, hybrid, maize optimized DNA
sequence (SEQ ID NO:10) encoding a heat stable CryIA(b) protein
(SEQ ID NO:11), contained in pCIB5511.
[0032] FIG. 10 is a map of pCIB5511.
[0033] FIG. 11 is a full-length, hybrid, maize optimized DNA
sequence (SEQ ID NO:12) encoding a heat stable CryIA(b) protein
(SEQ ID NO:13), contained in pCIB5512.
[0034] FIG. 12 is a map of pCIB5512.
[0035] FIG. 13 is a full-length, maize optimized DNA sequence (SEQ
ID NO:14) encoding a heat stable CryIA(b) protein (SEQ ID NO:15),
contained in pCIB5513.
[0036] FIG. 14 is a map of pCIB5513.
[0037] FIG. 15 is a full-length, maize optimized DNA sequence (SEQ
ID NO:16) encoding a heat-stable CryIA(b) protein (SEQ ID NO:17),
contained in pCIB5514.
[0038] FIG. 16 is a map of pCIB5514.
[0039] FIG. 17 is a map of pCIB4418.
[0040] FIG. 18 is a map of pCIB4420.
[0041] FIG. 19 is a map of pCIB4429.
[0042] FIG. 20 is a map of pCIB4431.
[0043] FIG. 21 is a map of pCIB4428.
[0044] FIG. 22 is a map of pCIB4430.
[0045] FIG. 23A is a table containing data of cryIA(b) protein
levels in transgenic maize.
[0046] FIG. 23B is a table which summarizes results of bioassays of
Ostrinia and Diatraea on leaf material from maize progeny
containing a maize optimized CryIA(b) gene.
[0047] FIG. 23C is a table containing data of cryIA(b) protein
levels in transgenic maize.
[0048] FIG. 23D is a table which summarizes the results of
bioassays of Ostrinia and Diatraea on leaf material from maize
progeny containing a synthetic Bt. maize gene operably linked to a
pith promoter.
[0049] FIG. 23E is a table containing data on expression of the
cryIA(b) gene in transgenic maize using the pith-preferred
promoter. Leaf samples from small plantlets transformed with
pCIB4433 using procedures described elsewhere were analyzed for the
presence of the cryIA(b) protein using ELISA. All plants expressing
cryIA(b) were found to be insecticidal in the standard European
corn borer bioassay. Note that the pith-preferred promoter has a
low, but detectable level of expression in leaf tissue of maize.
Detection of CryIA(b) protein is consistent with this pattern of
expression.
[0050] FIG. 24 is a complete genomic DNA sequence (SEQ ID NO:18)
encoding a maize tryptophan synthase-alpha subunit (TrpA) protein
(SEQ ID NO:19). Introns, exons, transcription and translation
starts, start and stop of cDNA are shown. $=start and end of cDNA;
+1=transcription start; 73*******=primer extension primer; +1=start
of translation; +++=stop codon; bp 1495-99=CCAAT Box; bp
1593-1598=TATAA Box; bp 3720-3725=poly A addition site; # above
underlined sequences are PCR primers.
[0051] FIGS. 25A, 25B, 25C and 25D are Northern blot analyses which
show differential expression of the maize TrpA subunit gene in
maize tissue at 2 hour, 4 hour, 18 hour, and 48 hour intervals,
respectively, at -80.degree. C. with DuPont Cronex intensifying
screens. P=pith; C=cob; BR=brace roots; ES=ear shank; LP=lower
pith; MP=middle pith; UP=upper pith; S=seed; L=leaf; R=root;
SH=sheath; and P(upper left)=total pith.
[0052] FIG. 26 is a Northern blot analysis, the two left lanes of
which show the maize TrpA gene expression in the leaf (L) and pith
(P) of Funk inbred lines 211D and 5N984. The five right lanes
indicate the absence of expression in Funk 211D seed total RNA.
S(1, 2, 3, 4 and 5)=seed at 1, 2, 3, 4 and 5 weeks post
pollenation. L=leaf; P=pith; S#=seed # weeks post pollenation.
[0053] FIG. 27 is a Southern blot analysis of genomic DNA Funk line
211D, probed with maize TrpA cDNA 8-2 (pCIB5600), wherein B denotes
BamHI, E denotes EcoRI, EV denotes EcoRV, H denotes HINDIII, and S
denotes SacI. 1.times., 5.times. and 10.times. denote reconstructed
gene copy equivalents.
[0054] FIG. 28A is a primer extension analysis which shows the
transcriptional start of the maize TrpA subunit gene and sequencing
ladder at a 1 hour exposure against film at -80 C with Dupont
Cronex intensifying screens. Lane +1 and +2 are 1.times.+0.5.times.
samples of primer extension reaction.
[0055] FIG. 28B is an analysis of RNase protection from +2 bp to
+387 bp at annealing temperatures of 42.degree. C., 48.degree. C.
and 54.degree. C., at a 16 hour exposure against film at
-80.degree. C. with DuPont Cronex intensifying screens.
[0056] FIG. 29 is A map of the original Type II pollen-specific
cDNA clone. The subcloning of the three EcoRI fragments into
pBluescript vectors to create pCIB3168, pCIB3169 and II-.6 is
illustrated.
[0057] FIG. 30 shows the DNA sequence of the maize pollen-specific
calcium dependent protein kinase gene cDNA (SEQ ID NO:20), as
contained in the 1.0 kb and 0.5 kb fragments of the original Type
II cDNA clone. The EcoRI site that divides the 1.0 kb and 0.5 kb
fragments is indicated. This cDNA is not full length, as the mRNA
start site maps 490 bp upstream of the end of the cDNA clone. The
translated protein is disclosed as SEQ ID NO:21.
[0058] FIG. 31 illustrates the tissue-specific expression of the
pollen CDPK mRNA. RNA from the indicated maize 211D tissues was
denatured, electrophoresed on an agarose gel, transferred to
nitrocellulose, and probed with the pollen CDPK cDNA 0.5 kb
fragment. The mRNA is detectable only in the pollen, where a strong
signal is seen.
[0059] FIG. 32 is an amino acid sequence (sequence line 1, amino
acids 13 to 307 of SEQ ID NO:22) comparison of the pollen CDPK
derived protein sequence and the rat calmodulin-dependent protein
kinase 2 protein sequence (sequence line 3; SEQ ID NO:23) disclosed
in Tobimatsu et al., J. Biol. Chem. 263:16082-16086 (1988). The
Align program of the DNAstar software package was used to evaluate
the sequences. The homology to protein kinases occurs in the 5' two
thirds of the gene, i.e. in the 1.0 kb fragment.
[0060] FIG. 33 is an amino acid sequence comparison of the pollen
CDPK derived protein sequence (sequence line 1; amino acids 311 to
450 of SEQ ID NO:22) and the human calmodulin protein sequence
(sequence line 3; SEQ ID NO:24) disclosed in Fischer et al., J.
Biol. Chem. 263:17055-17062 (1988). The homology to calmodulin
occurs in the 3' one third of the gene, i.e. in the 0.5 kb
fragment.
[0061] FIG. 34 is an amino acid sequence comparison of the pollen
CDPK derived protein sequence (sequence line 1; SEQ ID NO:22) and
soybean CDPK (SEQ ID NO:25). The homology occurs over the entire
gene.
[0062] FIG. 35 illustrates the sequence of the maize
pollen-specific CDPK gene (SEQ ID NO:26). 1.4 kb of sequence prior
to the mRNA start site is shown. The positions of the seven exons
and six introns are depicted under the corresponding DNA sequence.
The site of polyadenylation in the cDNA clone is indicated.
[0063] FIG. 36 is a map of pCIB4433.
[0064] FIG. 37 is a full-length, hybrid, maize-optimized DNA
sequence (SEQ ID NO:77) encoding a heat stable cryIA(b) protein
(SEQ ID NO:28).
[0065] FIG. 38 is a map of pCIB5515.
DESCRIPTION OF THE SEQUENCES
[0066] SEQ ID NO:1 is the DNA sequence of a full-length native Bt
cryIA(b) gene.
[0067] SEQ ID NO:2 is the DNA sequence of a full-length pure maize
optimized synthetic Bt cryIA(b) gene.
[0068] SEQ ID NO:3 is the DNA sequence of an approximately 2 Kb
truncated synthetic maize optimized Bt cryIA(b) gene.
[0069] SEQ ID NO:4 is the DNA sequence of a full-length synthetic
maize optimized Bt cryIA(b) gene.
[0070] SEQ ID NO:5 is the DNA sequence of an approximately 2 Kb
synthetic Bt gene according to Perlak et al.
DETAILED DESCRIPTION OF THE INVENTION
[0071] The following definitions are provided in order to provide
clarity with respect to the terms as they are used in the
specification and claims to describe the present invention.
[0072] Maize preferred codon: Preferred codon refers to the
preference exhibited by a specific host cell in the usage of
nucleotide codons to specify a given amino acid. The preferred
codon for an amino acid for a particular host is the single codon
which most frequently encodes that amino acid in that host. The
maize preferred codon for a particular amino acid may be derived
from known gene sequences from maize. For example, maize codon
usage for 28 genes from maize plants are listed in Table 4 of
Murray et al., Nucleic Acids Research, 17:477-498 (1989), the
disclosure of which is incorporated herein by reference. For
instance, the maize preferred codon for alanine is GCC, since,
according to pooled sequences of 26 maize genes in Murray et al.,
supra, that codon encodes alanine 36% of the time, compared to GCG
(24%), GCA (13%), and GCT (27%). Table 4 of Murray et al. is
reproduced below TABLE-US-00001 Soybean Maize CAB RuBP SSU n = 29 n
= 26 n = 17 n = 20 AmAcid Codon No. % No. % No. % No. % Gly GGG 90
16 95 16 42 8 16 9 Gly GGA 189 33 78 13 167 32 95 51 Gly GGT 193 33
129 21 196 37 32 17 Gly GGC 102 18 302 50 118 23 43 23 Glu GAG 310
51 368 81 178 71 139 74 Glu GAA 301 49 84 19 73 29 49 26 Asp GAT
244 62 87 24 53 29 39 33 Asp GAC 150 38 277 76 128 71 79 67 Val GTG
219 37 227 40 62 21 93 36 Val GTA 77 13 36 6 24 8 7 3 Val GTT 227
38 99 17 118 39 87 33 Val GTC 75 12 209 37 96 32 73 28 Ala GCG 42 8
211 24 26 5 16 5 Ala GCA 170 30 115 13 61 12 42 14 Ala GCT 208 37
237 27 225 45 110 38 Ala GCC 139 25 324 36 192 38 125 43 Arg AGG 88
22 109 26 21 15 17 12 Arg AGA 119 30 28 7 33 24 31 21 Ser AGT 117
18 29 5 15 5 21 8 Ser AGC 129 20 150 28 84 27 56 22 Lys AAG 278 58
367 90 186 85 176 85 Lys AAA 204 42 43 10 34 15 30 15 Asn AAT 168
40 56 19 52 30 35 26 Asn AAC 248 60 246 81 119 70 102 74 Met ATG
184 100 210 100 111 100 115 100 Ile ATA 109 24 35 8 10 6 1 1 Ile
ATT 219 49 100 24 61 40 63 43 Ile ATC 118 27 284 68 83 54 83 56 Thr
ACG 29 7 114 26 10 6 5 3 Thr ACA 128 29 48 11 35 22 21 13 Thr ACT
151 35 72 16 61 38 59 36 Thr ACC 124 29 212 47 54 34 79 48 Trp TGG
82 100 84 100 99 100 86 100 End TGA 5 18 7 26 15 88 2 11 Cys TGT 63
40 29 21 16 39 7 9 Cys TGC 95 60 110 79 25 61 72 91 End TAG 9 32 14
52 0 0 1 5 End TAA 14 50 6 22 2 12 16 84 Tyr TAT 135 49 38 14 23 19
17 10 Tyr TAC 139 51 240 86 99 81 151 90 Leu TTG 175 24 116 13 118
30 79 36 Leu TTA 79 11 28 3 15 4 6 3 Phe TTT 166 46 69 20 106 40 32
20 Phe TTC 193 54 278 80 160 60 125 80 Ser TCG 39 6 89 16 17 5 10 4
Ser TCA 125 19 56 10 46 15 48 19 Ser TCT 140 22 75 14 83 26 33 13
Ser TCC 94 15 145 27 69 22 89 34 Soybean Maize CAB RuBP SSU AmAcid
Codon No. % No. % No. % No. % Arg CGG 17 4 54 13 7 5 1 1 Arg CGA 41
10 13 3 6 4 3 2 Arg CGT 70 18 45 11 50 36 48 33 Arg CGC 64 16 165
40 20 15 44 31 GIn CAG 181 41 311 59 36 37 75 51 GIn CAA 261 59 219
41 60 62 73 49 His CAT 124 63 49 29 16 32 4 18 His CAC 73 37 122 71
34 68 18 82 Leu CTG 75 10 289 31 29 7 27 12 Leu CTA 60 8 78 9 6 2 9
4 Leu CTT 184 26 147 16 134 34 56 25 Leu CTC 148 21 261 28 88 23 43
20 Pro CCG 55 8 149 27 29 10 13 6 Pro CCA 346 47 126 23 137 47 72
34 Pro CCT 236 32 109 20 73 25 60 29 Pro CCC 95 13 164 30 54 18 66
31 n = the number of DNA sequences in the sample. No. is the number
occurrences of a given codon in the sample. % is the percent
occurrence for each codon within a given amino acid in the sample.
(See text of Murray et al., Nucleic Acids Research, 17: 477-498
(1989) for a description of the samples).
[0073] Pure maize optimized sequence: An optimized gene or DNA
sequence refers to a gene in which the nucleotide sequence of a
native gene has been modified in order to utilize preferred codons
for maize. For example, a synthetic maize optimized Bt cryIA(b)
gene is one wherein the nucleotide sequence of the native Bt
cryIA(b) gene has been modified such that the codons used are the
maize preferred codons, as described above. A pure maize optimized
gene is one in which the nucleotide sequence comprises 100 percent
of the maize preferred codon sequences for a particular
polypeptide. For example, the pure maize optimized Bt cryIA(b) gene
is one in which the nucleotide sequence comprises 100 percent maize
preferred codon sequences and encodes a polypeptide with the same
amino acid sequence as that produced by the native Bt cryIA(b)
gene. The pure nucleotide sequence of the optimized gene may be
varied to permit manipulation of the gene, such as by altering a
nucleotide to create or eliminate restriction sites. The pure
nucleotide sequence of the optimized gene may also be varied to
eliminate potentially deleterious processing sites, such as
potential polyadenylation sites or intron recognition sites.
[0074] It is recognized that "partially maize optimized," sequences
may also be utilized. By partially maize optimized, it is meant
that the coding region of the gene is a chimeric (hybrid), being
comprised of sequences derived from a native insecticidal gene and
sequences which have been optimized for expression in maize. A
partially optimized gene expresses the insecticidal protein at a
level sufficient to control insect pests, and such expression is at
a higher level than achieved using native sequences only. Partially
maize optimized sequences include those which contain at least
about 5% optimized sequences.
[0075] Full-length Bt Genes: Refers to DNA sequences comprising the
full nucleotide sequence necessary to encode the polypeptide
produced by a native Bt gene. For example, the native Bt cryIA(b)
gene is approximately 3.5 Kb in length and encodes a polypeptide
which is approximately 1150 amino acids in length. A full-length
synthetic cryIA(b) Bt gene would be at least approximately 3.5 Kb
in length.
[0076] Truncated Bt Genes: Refers to DNA sequences comprising less
than the full nucleotide sequence necessary to encode the
polypeptide produced by a native Bt gene, but which encodes the
active toxin portion of the polypeptide. For example, a truncated
synthetic Bt gene of approximately 1.9 Kb encodes the active toxin
portion of the polypeptide such that the protein product exhibits
insecticidal activity.
[0077] Tissue-preferred promoter: The term "tissue-preferred
promoter" is used to indicate that a given regulatory DNA sequence
will promote a higher level of transcription of an associated
structural gene or DNA coding sequence, or of expression of the
product of the associated gene as indicated by any conventional RNA
or protein assay, or that a given DNA sequence will demonstrate
some differential effect; i.e., that the transcription of the
associated DNA sequences or the expression of a gene product is
greater in some tissue than in all other tissues of the plant.
[0078] "Tissue-specific promoter" is used to indicate that a given
regulatory DNA sequence will promote transcription of an associated
coding DNA sequence essentially entirely in one or more tissues of
a plant, or in one type of tissue, e.g. green tissue, while
essentially no transcription of that associated coding DNA sequence
will occur in all other tissues or types of tissues of the
plant.
[0079] The present invention provides DNA sequences optimized for
expression in plants, especially in maize plants. In a preferred
embodiment of the present invention, the DNA sequences encode the
production of an insecticidal toxin, preferably a polypeptide
sharing substantially the amino acid sequence of an insecticidal
crystal protein toxin normally produced by Bacillus thuringiensis.
The synthetic gene may encode a truncated or full-length
insecticidal protein. Especially preferred are synthetic DNA
sequences which encode a polypeptide effective against insects of
the order Lepidoptera and Coleoptera, and synthetic DNA sequences
which encode a polypeptide having an amino acid sequence
essentially the same as one of the crystal protein toxins of
Bacillus thuringiensis variety kurstaki, HD-1.
[0080] The present invention provides synthetic DNA sequences
effective to yield high expression of active insecticidal proteins
in plants, preferably maize protoplasts, plant cells and plants.
The synthetic DNA sequences of the present invention have been
modified to resemble a maize gene in terms of codon usage and G+C
content. As a result of these modifications, the synthetic DNA
sequences of the present invention do not contain the potential
processing sites which are present in the native gene. The
resulting synthetic DNA sequences (synthetic Bt IP coding
sequences) and plant transformation vectors containing this
synthetic DNA sequence (synthetic Bt IP genes) result in
surprisingly increased expression of the synthetic Bt IP gene,
compared to the native Bt IP gene, in terms of insecticidal protein
production in plants, particularly maize. The high level of
expression results in maize cells and plants that exhibit
resistance to lepidopteran insects, preferably European Corn Borer
and Diatrea saccharalis, the Sugarcane Borer.
[0081] The synthetic DNA sequences of the present invention are
designed to encode insecticidal proteins from Bacillus
thuringiensis, but are optimized for expression in maize in terms
of G+C content and codon usage. For example, the maize codon usage
table described in Murray et al., supra, is used to reverse
translate the amino acid sequence of the toxin produced by the
Bacillus thuringiensis subsp. kurstaki HD-1 cryIA(b) gene, using
only the most preferred maize codons. The reverse translated DNA
sequence is referred to as the pure maize optimized sequence and is
shown as Sequence 4. This sequence is subsequently modified to
eliminate unwanted restriction endonuclease sites, and to create
desired restriction endonuclease sites. These modifications are
designed to facilitate cloning of the gene without appreciably
altering the codon usage or the maize optimized sequence. During
the cloning procedure, in order to facilitate cloning of the gene,
other modifications are made in a region that appears especially
susceptible to errors induced during cloning by the polymerase
chain reaction (PCR). The final sequence of the maize optimized
synthetic Bt IP gene is shown in Sequence 2. A comparison of the
maize optimized synthetic Bt IP gene with the native kurstaki
cryIA(b) Bt gene is shown in FIG. 1.
[0082] In a preferred embodiment of the present invention, the
protein produced by the synthetic DNA sequence is effective against
insects of the order Lepidoptera or Coleoptera. In a more preferred
embodiment, the polypeptide encoded by the synthetic DNA sequence
consists essentially of the full-length or a truncated amino acid
sequence of an insecticidal protein normally produced by Bacillus
thuringiensis var. kurstaki HD-1. In a particular embodiment, the
synthetic DNA sequence encodes a polypeptide consisting essentially
of a truncated amino acid sequence of the Bt CryIA(b) protein.
[0083] The insecticidal proteins of the invention are expressed in
a plant in an amount sufficient to control insect pests, i.e.
insect controlling amounts. It is recognized that the amount of
expression of insecticidal protein in a plant necessary to control
insects may vary depending upon species of plant, type of insect,
environmental factors and the like. Generally, the insect
population will be kept below the economic threshold which varies
from plant to plant. For example, to control European corn borer in
maize, the economic threshold is 0.5 eggmass/plant which translates
to about 10 larvae/plant.
[0084] The methods of the invention are useful for controlling a
wide variety of insects including but not limited to rootworms,
cutworms, armyworms, particularly fall and beet armyworms,
wireworms, aphids, corn borers, particularly European corn borers,
sugarcane borer, lesser corn stalk borer, Southwestern corn borer,
etc.
[0085] In a preferred embodiment of the present invention, the
synthetic coding DNA sequence optimized for expression in maize
comprises a G+C percentage greater than that of the native cryIA(b)
gene. It is preferred that the G+C percentage be at least about 50
percent, and more preferably at least about 60 percent. It is
especially preferred that the G+C percent be about 64 percent.
[0086] In another preferred embodiment of the present invention,
the synthetic coding DNA sequence optimized for expression in maize
comprises a nucleotide sequence having at least about 90 percent
homology with the "pure" maize optimized nucleotide sequence of the
native Bacillus thuringiensis cryIA(b) protein, more preferably at
least about 95 percent homology, and most preferably at least about
98 percent.
[0087] Other preferred embodiments of the present invention include
synthetic DNA sequences having essentially the DNA sequence of SEQ
ID NO:4, as well as mutants or variants thereof; transformation
vectors comprising essentially the DNA sequence of SEQ ID NO:4; and
isolated DNA sequences derived from the plasmids pCIB4406,
pCIB4407, pCIB4413, pCIB4414, pCIB4416, pCIB4417, pCIB4418,
pCIB4419, pCIB4420, pCIB4421, pCIB4423, pCIB4434, pCIB4429,
pCIB4431, pCIB4433. Most preferred are isolated DNA sequences
derived from the plasmids pCIB4418 and pCIB4420, pCIB4434,
pCIB4429, pCIB4431, and pCIB4433.
[0088] In order to construct one of the maize optimized DNA
sequences of the present invention, synthetic DNA oligonucleotides
are made with an average length of about 80 nucleotides. These
oligonucleotides are designed to hybridize to produce fragments
comprising the various quarters of the truncated toxin gene. The
oligonucleotides for a given quarter are hybridized and amplified
using PCR. The quarters are then cloned and the cloned quarters are
sequenced to find those containing the desired sequences. In one
instance, the fourth quarter, the hybridized oligonucleotides are
cloned directly without PCR amplification. Once all clones of four
quarters are found which contain open reading frames, an intact
gene encoding the active insecticidal protein is assembled. The
assembled gene may then be tested for insecticidal activity against
any insect of interest including the European Corn Borer (ECB) and
the sugarcane borer. (Examples 5A and 5B, respectively). When a
fully functional gene is obtained, it is again sequenced to confirm
its primary structure. The fully functional gene is found to give
100% mortality when bioassayed against ECB. The fully functional
gene is also modified for expression in maize.
[0089] The maize optimized gene is tested in a transient expression
assay, e.g. a maize transient expression assay. The native Bt
cryIA(b) coding sequence for the active insecticidal toxin is not
expressed at a detectable level in a maize transient expression
system. Thus, the level of expression of the synthesized gene can
be determined. By the present methods, expression of a protein in a
transformed plant can be increased at least about 100 fold to about
50,000 fold, more specifically at least about 1,000 fold to at
least about 20,000 fold.
[0090] Increasing expression of an insecticial gene to an effective
level does not require manipulation of a native gene along the
entire sequence. Effective expression can be achieved by
manipulating only a portion of the sequences necessary to obtain
increased expression. A full-length, maize optimized CryIA(b) gene
may be prepared which contains a protein of the native CryIA(b)
sequence. For example, FIG. 7 illustrates a full-length, maize
optimized CryIA(b) gene which is a synthetic-native hybrid. That
is, about 2 kb of the gene (nucleotides 1-1938 SEQ ID NO:8) is
maize optimized, i.e. synthetic. The remainder, C-terminal
nucleotides 647-1155 SEQ ID NO:8, are identical to the
corresponding sequence native of the CryIA(b) gene. Construction of
the illustrated gene is described in Example 6, below.
[0091] It is recognized that by using the methods described herein,
a variety of synthetic/native hybrids may be constructed and tested
for expression. The important aspect of hybrid construction is that
the protein is produced in sufficient amounts to control insect
pests. In this manner, critical regions of the gene may be
identified and such regions synthesized using preferred codons. The
synthetic sequences can be linked with native sequences as
demonstrated in the Examples below. Generally, N-terminal portions
or processing sites can be synthesized and substituted in the
native coding sequence for enhanced expression in plants.
[0092] In another embodiment of the present invention, the maize
optimized genes encoding cryIA(b) protein may be manipulated to
render the encoded protein more heat stable or temperature stable
compared to the native cryIA(b) protein. It has been shown that the
cryIA(b) gene found in Bacillus thuringiensis kurstaki HD-1
contains a 26 amino acid deletion, when compared with the cryIA(a)
and cryIA(c) proteins, in the --COOH half of the protein. This
deletion leads to a temperature-sensitive cryIA(b) protein. See M.
Geiser, EP 0 440 581, entitled "Temperaturstabiles Bacillus
thuringiensis-Toxin". Repair of this deletion with the
corresponding region from the cryIA(a) or cryIA(c) protein improves
the temperature stability of the repaired protein. Constructs of
the full-length modified cryIA(b) synthetic gene are designed to
insert sequences coding for the missing amino acids at the
appropriate place in the sequence without altering the reading
frame and without changing the rest of the protein sequence. The
full-length synthetic version of the gene is assembled by
synthesizing a series of double-stranded DNA cassettes, each
approximately 300 bp in size, using standard techniques of DNA
synthesis and enzymatic reactions. The repaired gene is said to
encode a "heat stable" or "temperature-stable" cryIA(b) protein,
since it retains more biological activity than its native
counterpart when exposed to high temperatures. Specific sequences
of maize optimized, heat stable cryIA(b) genes encoding temperature
stable proteins are set forth in FIGS. 9 (SEQ ID NO:10), 11 (SEQ ID
NO:12), 13 (SEQ ID NO:14), and 15 (SEQ ID NO:16), and are also
described in Example 8A, below.
[0093] The present invention encompasses maize optimized coding
sequences encoding other polypeptides, including those of other
Bacillus thuringiensis insecticidal polypeptides or insecticidal
proteins from other sources. For example, cryIB genes can be maize
optimized, and then stably introduced into plants, particularly
maize. The sequence of a maize optimized cryIB gene constructed in
accordance with the present invention is set forth in FIG. 6 (SEQ
ID NO:6).
[0094] Optimizing a Bt IP gene for expression in maize using the
maize preferred codon usage according to the present invention
results in a significant increase in the expression of the
insecticidal gene. It is anticipated that other genes can be
synthesized using plant codon preferences to improve their
expression in maize or other plants. Use of maize codon preference
is a likely method of optimizing and maximizing expression of
foreign genes in maize. Such genes include genes used as selectable
or scoreable markers in maize transformation, genes which confer
herbicide resistance, genes which confer disease resistance, and
other genes which confer insect resistance.
[0095] The synthetic cryIA(b) gene is also inserted into
Agrobacterium vectors which are useful for transformation of a
large variety of dicotyledenous plant species. (Example 44). Plants
stably transformed with the synthetic cryIA(b) Agrobacterium
vectors exhibit insecticidal activity.
[0096] The native Bt cryIA(b) gene is quite A+T rich. The G+C
content of the full-length native Bt cryIA(b) gene is approximately
39%. The G+C content of a truncated native Bt cryIA(b) gene of
about 2 Kb in length is approximately 37%. In general, maize coding
regions tend to be predominantly G+C rich. The modifications made
to the Bt cryIA(b) gene result in a synthetic IP coding region
which has greater than 50% G+C content, and has about 65% homology
at the DNA level with the native cryIA(b) gene. The protein encoded
by this synthetic CryIA(b) gene is 100% homologous with the native
protein, and thus retains full function in terms of insect
activity. The truncated synthetic CryIA(b) IP gene is about 2 Kb in
length and the gene encodes the active toxin region of the native
Bt kurstaki CryIA(b) insecticidal protein. The length of the
protein encoded by the truncated synthetic CryIA(b) gene is 648
amino acids.
[0097] The synthetic genes of the present invention are useful for
enhanced expression in transgenic plants, most preferably in
transformed maize. The transgenic plants of the present invention
may be used to express the insecticidal CryIA(b) protein at a high
level, resulting in resistance to insect pests, preferably
coleopteran or lepidopteran insects, and most preferably European
Corn Borer (ECB) and Sugarcane Borer.
[0098] In the present invention, the DNA coding sequence of the
synthetic maize optimized gene may be under the control of
regulatory elements such as promoters which direct expression of
the coding sequence. Such regulatory elements, for example, include
monocot or maize and other monocot functional promoters to provide
expression of the gene in various parts of the maize plant. The
regulatory element may be constitutive. That is, it may promote
continuous and stable expression of the gene. Such promoters
include but are not limited to the CaMV 35S promoter; the CaMV 19S
promoter; A. tumefaciens promoters such as octopine synthase
promoters, mannopine synthase promoters, nopaline synthase
promoters, or other opine synthase promoters; ubiquitin promoters,
actin promoters, histone promoters and tubulin promoters. The
regulatory element may be a tissue-preferential promoter, that is,
it may promote higher expression in some tissues of a plant than in
others. Preferably, the tissue-preferential promoter may direct
higher expression of the synthetic gene in leaves, stems, roots
and/or pollen than in seed. The regulatory element may also be
inducible, such as by heat stress, water stress, insect feeding or
chemical induction, or may be developmentally regulated. Numerous
promoters whose expression are known to vary in a tissue specific
manner are known in the art. One such example is the maize
phosphoenol pyruvate carboxylase (PEPC), which is green
tissue-specific. See, for example, Hudspeth, R. L. and Grula, J.
W., Plant Molecular Biology 12:579-589, 1989). Other green
tissue-specific promoters include chlorophyll a/b binding protein
promoters and RubisCO small subunit promoters.
[0099] The present invention also provides isolated and purified
pith-preferred promoters. Preferred pith-preferred promoters are
isolated from graminaceous monocots such as sugarcane, rice, wheat,
sorghum, barley, rye and maize; more preferred are those isolated
from maize plants.
[0100] In a preferred embodiment, the pith-preferred promoter is
isolated from a plant TrpA gene; in a most preferred embodiment, it
is isolated from a maize TrpA gene. That is, the promoter in its
native state is operatively associated with a maize tryptophan
synthase-alpha subunit gene (hereinafter "TrpA"). The encoded
protein has a molecular mass of about 38 kD. Together with another
alpha subnit and two beta subunits, TrpA forms a multimeric enzyme,
tryptophan synthase. Each subunit can operate separately, but they
function more efficiently together. TrpA catalyzes the conversion
of indole glycerol phosphate to indole. Neither the maize TrpA gene
nor the encoded protein had been isolated from any plant before
Applicants' invention. The Arabidopsis thaliana tryptophan synthase
beta subunit gene has been cloned as described Wright et al., The
Plant Cell, 4:711-719 (1992). The instant maize TrpA gene has no
homology to the beta subunit encoding gene. The present invention
also provides purified pollen-specific promoters obtainable from a
plant calcium-dependent protein kinase (CDPK) gene. That is, in its
native state, the promoter is operably linked to a plant CDPK gene.
In a preferred embodiment, the promoter is isolated from a maize
CDPK gene. By "pollen-specific," it is meant that the expression of
an operatively associated structural gene of interest is
substantially exclusively (i.e. essentially entirely) in the pollen
of a plant, and is negligible in all other plant parts. By "CDPK,"
it is meant a plant protein kinase which has a high affinity for
calcium, but not calmodulin, and requires calcium, but not
calmodulin, for its catalytic activity.
[0101] To obtain tissue-preferred or tissue specific promoters,
genes encoding tissue specific messenger RNA (mRNA) can be obtained
by differential screening of a cDNA library. For example, a
pith-preferred cDNA can be obtained by subjecting a pith cDNA
library to differential screening using cDNA probes obtained from
pith and seed mRNA. See, Molecular Cloning, A Laboratory Manual,
Sambrook et al. eds. Cold Spring Harbor Press: New York (1989).
[0102] Alternately, tissue specific promoters may be obtained by
obtaining tissue specific proteins, sequencing the N-terminus,
synthesizing oligonucleotide probes and using the probes to screen
a cDNA library. Such procedures are exemplified in the Experimental
section for the isolation of a pollen specific promoter.
[0103] The scope of the present invention in regard to the
pith-preferred and pollen-specific promoters encompasses
functionally active fragments of a full-length promoter that also
are able to direct pith-preferred or pollen-specific transcription,
respectively, of associated structural genes. Functionally active
fragments of a promoter DNA sequence may be derived from a promoter
DNA sequence, by several art-recognized procedures, such as, for
example, by cleaving the promoter DNA sequence using restriction
enzymes, synthesizing in accordance with the sequence of the
promoter DNA sequence, or may be obtained through the use of PCR
technology. See, e.g. Mullis et al., Meth. Enzymol. 155:335-350
(1987); Erlich (ed.), PCR Technology, Stockton Press (New York
1989).
[0104] Further included within the scope of the instant invention
are pith-preferred and pollen-specific promoters "equivalent" to
the full-length promoters. That is, different nucleotides, or
groups of nucleotides may be modified, added or deleted in a manner
that does not abolish promoter activity in accordance with known
procedures.
[0105] A pith-preferred promoter obtained from a maize TrpA gene is
shown in FIG. 24 (SEQ ID NO:18). Those skilled in the art, with
this sequence information in hand, will recognize that
pith-preferred promoters included within the scope of the present
invention can be obtained from other plants by probing pith
libraries from these plants with probes derived from the maize TrpA
structural gene. Probes designed from sequences that are highly
conserved among TrpA subunit genes of various species, as discussed
generally in Example 17, are preferred. Other pollen-specific
promoters which in their native state are linked to plant CDPK
genes other than maize, can be isolated in similar fashion using
probes derived from the conserved regions of the maize CDPK gene to
probe pollen libraries.
[0106] In another embodiment of the present invention, the
pith-preferred or pollen-specific promoter is operably linked to a
DNA sequence, i.e. structural gene, encoding a protein of interest,
to form a recombinant DNA molecule or chimeric gene. The phrase
"operably linked to" has an art-recognized meaning; it may be used
interchangeably with "operatively associated with," "linked to," or
"fused to".
[0107] The structural gene may be homologous or heterologous with
respect to origin of the promoter and/or a target plant into which
it is transformed. Regardless of relative origin, the associated
DNA sequence will be expressed in the transformed plant in
accordance with the expression properties of the promoter to which
it is linked. Thus, the choice of associated DNA sequence should
flow from a desire to have the sequence expressed in this fashion.
Examples of heterologous DNA sequences include those which encode
insecticidal proteins, e.g. proteins or polypeptides toxic or
inhibitory to insects or other plant parasitic arthropods, or plant
pathogens such as fungi, bacteria and nematodes. These heterologous
DNA sequences encode proteins such as magainins, Zasloff, PNAS USA,
84:5449-5453 (1987); cecropins, Hultmark et al., Eur. J. Biochem.
127:207-217 (1982); attacins, Hultmark et al., EMBO J. 2:571-576
(1983); melittin, gramicidin S, Katsu et al., Biochem. Biophys.
Acta, 939:57-63 (1988); sodium channel proteins and synthetic
fragments, Oiki et al., PNAS USA. 85:2395-2397 (1988); the alpha
toxin of Staphylococcus aureusm Tobkes et al., Biochem.,
24:1915-1920 (1985); apolipoproteins and fragments thereof, Knott
et al., Science 230:37 (1985); Nakagawa et al., J. Am. Chem. Soc.,
107:7087 (1985); alamethicin and a variety of synthetic amphipathic
peptides, Kaiser et al., Ann. Rev. Biophys. Biophys. Chem.
16:561-581 (1987); lectins, Lis et al., Ann. Rev. Biochem.,
55:35-68 (1986); protease and amylase inhibitors; and insecticidal
proteins from Bacillus thuringiensis, particularly the
delta-endotoxins from B. thuringiensis; and from other bacteria or
fungi.
[0108] In a preferred embodiment of the invention, a pith-preferred
promoter obtained from a maize TrpA subunit gene or pollen-specific
promoter obtained from a maize CDPK gene is operably linked to a
heterologous DNA sequence encoding a Bacillus thuringiensis
("B.t.") insecticidal protein. These proteins and the corresponding
structural genes are well known in the art. See, Hofte and
Whiteley, Microbiol. Reviews, 53:242-255 (1989).
[0109] While it is recognized that any promoter capable of
directing expression can be utilized, it may be preferable to use
heterologous promoters rather than the native promoter of the
protein of interest. In this manner, chimeric nucleotide sequences
can be constructed which can be determined based on the plant to be
transformed as well as the insect pest. For example, to control
insect pests in maize, a monocot or maize promoter can be operably
linked to a Bt protein. The maize promoter can be selected from
tissue-preferred and tissue-specific promoters such as
pith-preferred and pollen-specific promoters, respectively as
disclosed herein.
[0110] In some instances, it may be preferred to transform the
plant cell with more than one chimeric gene construct. Thus, for
example, a single plant could be transformed with a pith-preferred
promoter operably linked to a Bt protein as well as a
pollen-specific promoter operably linked to a Bt protein. The
transformed plants would express Bt proteins in the plant pith and
pollen and to a lesser extent the roots, outer sheath and brace
roots.
[0111] For various other reasons, particularly management of
potential insect resistance developing to plant expressed
insecticidal proteins, it is beneficial to express more than one
insecticidal protein (IP) in the same plant. One could express two
different genes (such as two different Bacillus thuringiensis
derived delta-endotoxins which bind different receptors in the
target insect's midgut) in the same tissues, or one can selectively
express the two toxins in different tissues of the same plant using
tissue specific promoters. Expressing two Bt genes (or any two
insecticidal genes) in the same plant using three different tissue
specific promoters presents a problem for production of a plant
expressing the desired phenotype. Three different promoters driving
two different genes yields six different insecticidal genes that
need to be introduced into the plant at the same time. Also needed
for the transformation is a selectable marker to aid in
identification of transformed plants. This means introducing seven
different genes into the plant at the same time. It is most desired
that all genes, especially the insecticidal genes, integrate into
the plant genome at the same locus so they will behave as a single
gene trait and not as a multiple gene trait that will be harder to
track during breeding of commercial hybrids. The total number of
genes can be reduced by using differential tissue specific
expression of the different insecticidal proteins.
[0112] For example, by fusing cryIA(b) with the pollen and PEP
carboxylase promoters, one would obtain expression of this gene in
green tissues and pollen. Fusing a pith-preferred promoter with the
cryIB delta endotoxin from Bacillus thuringiensis would produce
expression of this insecticidal protein most abundantly in the pith
of a transformed plant, but not in seed tissues. Transformation of
a plant with three genes, PEP carboxylase/cryIA(b),
pollen/cryIA(b), and pith/cryIB produces a plant expressing two
different Bt insecticidal endotoxins in different tissues of the
same plant. CryIA(b) would be expressed in the "outside" tissues of
a plant (particularly maize), that is, in those tissues which
European corn borer feeds on first after hatching. Should ECB prove
resistant to cryIA(b) and be able to burrow into the stalk of the
plant after feeding on leaf tissue and/or pollen, it would then
encounter the cryIB delta-endotoxin and be exposed to a second
insecticidal component. In this manner, one can differentially
express two different insecticidal components in the same plant and
decrease the total number of genes necessary to introduce as a
single genetic unit while at the same time providing protection
against development of resistance to a single insecticidal
component.
[0113] Likewise, a plant may be transformed with constructs
encoding more than one type of insecticidal protein to control
various insects. Thus, a number of variations may be constructed by
one of skill in the art.
[0114] The recombinant DNA molecules of the invention may be
prepared by manipulating the various elements to place them in
proper orientation. Thus, adapters or linkers may be employed to
join the DNA fragments. Other manipulations may be performed to
provide for convenient restriction sites, removal of restriction
sites or superfluous DNA. These manipulations can be performed by
art-recognized methods. See, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, second
edition, 1989. For example, methods such as restriction, chewing
back or filling in overhangs to provide blunt ends, ligation of
linkers, complementary ends of the DNA fragments can be provided
for joining and ligation. See, Sambrook et al., supra.
[0115] Other functional DNA sequences may be included in the
recombinant DNA molecule, depending upon the way in which the
molecule is to be incorporated into the target plant genome. For
instance, in the case of Agrobacterium-mediated transformation, if
Ti- or the Ri-plasmid is used to transform the plant cells, the
right and left borders of the T-DNA of the Ti- and Ri-plasmid will
be joined as flanking regions to the expression cassette.
Agrobacterium tumefaciens-mediated transformation of plants has
been described in Horsch et al., Science, 225:1229 (1985); Marton,
Cell Culture Somatic Cell Genetics of Plants, 1:514-521 (1984);
Hoekema, In: The Binary Plant Vector System Offset-Drukkerij
Kanters B. V., Alblasserdam, 1985, Chapter V Fraley, et al., Crit.
Rev. Plant Sci., 4:146; and An et al., EMBO J., 4:277-284
(1985).
[0116] The recombinant DNA molecules of the invention also can
include a marker gene to facilitate selection in recombinant plant
cells. Examples of markers include resistance to a biocide such as
an antibiotic, e.g. kanamycin, hygromycin, chloramphenicol,
paramomycin, methotrexate and bleomycin, or a herbicide such as
imidazolones, sulfonylureas, glyphosate, phosphinothricin, or
bialaphos. Marker genes are well known in the art.
[0117] In another embodiment of the present invention, plants
stably transformed with a recombinant DNA molecule or chimeric gene
as described hereinabove are provided. The resultant transgenic
plant contains the transformed gene stably incorporated into its
genome, and will express the structural gene operably associated to
the promoter in the respective fashion.
[0118] Transgenic plants encompassed by the instant invention
include both monocots and dicots. Representative examples include
maize, tobacco, tomato, cotton, rape seed, soybean, wheat, rice,
alfalfa, potato and sunflower. Preferred plants include maize,
particularly inbred maize plants.
[0119] All transformed plants encompassed by the instant invention
may be prepared by several methods known in the art. A.
tumefaciens-mediated transformation has been disclosed above. Other
methods include direct gene transfer into protoplasts, Paszkowski
et al., EMBO J., 12:2717 (1984); Loerz et al., Mol. Gen. &
Genet., 1199:178 (1985); Fromm et al., Nature 319:719 (1986);
microprojectile bombardment, Klein et al., Bio/Technology,
6:559-563 (1988); injection into protoplasts, cultured cells and
tissues, Reich et al., Bio/Technology, 4:1001-104 (1986); or
injection into meristematic tissues or seedlings and plants as
described by De La Pena et al., Nature, 325:274-276 (1987); Graves
et al., Plant Mol. Biol., 7:43-50 (1986); Hooykaas-Van Slogteren et
al., Nature, 311:763-764 (1984); Grimsley et al., Bio/Technology,
6:185 (1988); and Grimsley et al., Nature, 325:177 (1988); and
electroporation, WO92/09696.
[0120] The expression pattern of a structural gene operatively
associated with an instant tissue-preferred or tissue-specific
promoter in a transformed plant containing the same is critical in
the case where the structural gene encodes an insecticidal protein.
For example, the instantly disclosed pith-preferred expression
pattern will allow the transgenic plant to tolerate and withstand
pathogens and herbivores that attack primarily the pith, but also
the brace roots, outer sheath and leaves of the plant since the
protein will be expressed to a lesser extent but still in an insect
controlling amount in these plant parts, but yet in the case of
both types of promoters, will leave the seed of the plant
unaffected.
EXAMPLES
[0121] The following examples further describe the materials and
methods used in carrying out the invention. They are offered by way
of illustration, and not by way of limitation.
Example 1
General Methods
[0122] DNA manipulations were done using procedures that are
standard in the art. These procedures can often be modified and/or
substituted without substantively changing the result. Except where
other references are identified, most of these procedures are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, second edition,
1989.
Synthesis of DNA Oligomers:
[0123] DNA oligomers which are from about twenty to about ninety,
preferably from about sixty to about eighty nucleotides in length,
are synthesized using an Applied Biosystems model 380B DNA
synthesizer and standard procedures. The oligomers are made using
the updated SSCAF3 cycle on a 0.2 .mu.mole, wide pore, small scale
ABI column. The end procedure is run trityl off and the oligomer is
cleaved from the column using the 380B's automatic cleavage cycle.
The oligomers are then deblocked in excess ammonium hydroxide
(NH.sub.4OH) at 55.degree. C. for 8-12 hours. The oligomers are
then dried in an evaporator using nitrogen gas. After completion,
the oligomers are resuspended in 0.25-0.5 ml of deionized
water.
Purification of Synthetic Oligomers:
[0124] An aliquot of each oligomer is mixed with an equal volume of
blue dye\formamide mix with the final solution containing 0.05%
bromophenol blue, 0.05% xylene cyanol FF, and 25% formamide. This
mixture is heated at 95.degree. C. for 10 minutes to denature the
oligomers. Samples are then applied to a 12% polyacrylamide-urea
gel containing 7 M urea (Sambrook et al.). After electrophoresis at
300-400 volts for 3-4 hours using a Vertical Slab Gel Unit (Hoefer
Scientific Instruments, San Francisco, Calif.), UV shadowing is
used to locate the correct sized fragment in the gel which was then
excised using a razor blade. The purified gel fragment is minced
and incubated in 0.4 M LiCl, 1 mM EDTA (pH 8) buffer overnight at
37.degree. C.
[0125] Either of two methods is used to separate the oligomers from
the polyacrylamide gel remnants: Gene\X 25 .mu.M porous
polyethylene filter units or Millipore's ultrafree-MC 0.45 .mu.M
filter units. The purified oligomers are ethanol precipitated,
recovered by centrifuging in a microfuge for 20 min at 4.degree.
C., and finally resuspended in TE (10 mM Tris, 1 mM EDTA, pH 8.0).
Concentrations are adjusted to 50 ng\|H 25 l based on absorption
readings at 260 nm.
Kinasing Oligomers for Size Determinations:
[0126] To check the size of some of the oligomers on a sequencing
gel, kinase labeling reactions are carried out using purified
synthetic oligomers of each representative size: 40mers, 60mers,
70mers, 80mers, and 90mers. In each 20 .mu.l kinasing reaction, one
pmole of purified oligomer is used in a buffer of 7.0 mM Tris pH
7.5, 10 mM KCl, 1 mM MgCl2), 0.5 mM DTT, 50 .mu.g/ml BSA, 3000
.mu.Ci (3 pmoles) of 32P-gammaATP, and 8 units of T4 polynucleotide
kinase. The kinase reaction is incubated for 1 hour at 37.degree.
C., followed by a phenol\chloroform extraction and three ethanol
precipitations with glycogen as carrier (Tracy, Prep. Biochem.
11:251-268 (1981).
[0127] Two gel loadings (one containing 1000 cpm, the other
containing 2000 cpm) of each reaction are prepared with 25%
formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol FF. The
kinased oligomers are boiled for 5 minutes before loading on a 6%
polyacrylamide, 7 M urea sequencing gel (BRL Gel Mix TM6, BRL,
Gaithersburg, Md.). A sequencing reaction of plasmid pUC18 is run
on the same gel to provide size markers. After electrophoresis, the
gel is dried and exposed to diagnostic X-ray film (Kodak, X-OMAT
AR). The resulting autoradiograph shows all purified oligomers
tested to be of the correct size. Oligomers which had not been
sized directly on the sequencing gel are run on a 6%
polyacrylamide, 7 M urea gel (BRL Gel Mix TM6), using the sized
oligomers as size markers. All oligomers are denatured first with
25% formamide at 100.degree. C. for 5 minutes before loading on the
gel. Ethidium bromide staining of the polyacrylamide gel allows all
the oligomers to be visualized for size determination.
Hybridizing Oligomers for Direct Cloning:
[0128] Oligomers to be hybridized are pooled together (from 1 .mu.g
to 20 .mu.g total DNA) and kinased at 37.degree. C. for 1 hour in
1.times. Promega ligation buffer containing 30 mM Tris-HCl pH 7.8,
10 mM MgCl2, 10 mM DTT, and 1 mM dATP. One to 20 units of T4
polynucleotide kinase is used in the reaction, depending on the
amount of total DNA present. The kinasing reactions are stopped by
placing the reaction in a boiling water bath for five minutes.
Oligomers to form the 5' termini of the hybridized molecules are
not kinased but are added to the kinased oligomers along with
additional hybridization buffer after heating. The pooled oligomers
are in a volume of 50-100 ul with added hybridization buffer used
to adjust the final salt conditions to 100 mM NaCl, 120 mM Tris pH
7.5, and 10 mM MgCl2. The kinased and non-kinased oligomers are
pooled together and heated in a boiling water bath for five minutes
and allowed to slowly cool to room temperature over a period of
about four hours. The hybridized oligomers are then
phenol\chloroform extracted, ethanol precipitated, and resuspended
in 17 .mu.l of TE (10 mM Tris, 1 mM EDTA, pH 8.0). Using this 17
.mu.l, a ligation reaction with a final volume of 20 .mu.l is
assembled (final conditions=30 mM Tris-HCl pH 7.8, 10 mM MgCl2, 10
mM DTT, 1 mM ATP, and 3 units of T4 DNA ligase (Promega, Madison
Wis.). The ligation is allowed to incubate for about 2 hours at
room temperature. The hybridized\ligated fragments are generally
purified on 2% Nusieve gels before and\or after cutting with
restriction enzymes prior to cloning into vectors. A 20 .mu.l
volume ligation reaction is assembled using 100 ng to 500 ng of
each fragment with approximate equimolar amounts of DNA in 30 mM
Tris-HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, and 3 units of
T4 DNA ligase (Promega, Madison, Wis.). Ligations are incubated at
room temperature for 2 hours. After ligation, DNA is transformed
into frozen competent E. coli cells using standard procedures
(Sambrook et al.) and transformants are selected on LB-agar
(Sambrook et al.) containing 100 .mu.g/ml ampicillin (see
below).
PCR Reactions for Screening Clones in E. coli:
[0129] E. coli colonies which contain the correct DNA insert are
identified using PCR (see generally, Sandhu et al., BioTechniques
7:689-690 (1989)). Using a toothpick, colonies are scraped from an
overnight plate and added to a 20 .mu.l to 45 .mu.l PCR reaction
mix containing about 50 pmoles of each hybridizing primer (see
example using primers MK23A28 and MK25A28 to select orientation of
SacII fragment in pHYB2#6), 200 .mu.m to 400 mM of each dNTP, and
1.times. reaction buffer (Perkin Elmer Cetus, Norwalk, Conn.).
After boiling the E. coli\PCR mix in a boiling water bath for 10
minutes, 5 .mu.l of Taq polymerase (0.5 units) (Perkin Elmer Cetus,
Norwalk, Conn.) in 1.times. reaction buffer is added. The PCR
reaction parameters are generally set with a denaturing step of
94.degree. C. for 30 seconds, annealing at 55.degree. C. for 45
seconds, and extension at 72.degree. C. for 45 seconds for 30 to 36
cycles. PCR reaction products are run on agarose or Nusieve agarose
(FMC) gels to detect the correct fragment size amplified
Ligations:
[0130] Restriction enzyme digested fragments are either purified in
1% LGT (low gelling temperature agarose, FMC), 2% Nusieve (FMC), or
0.75% agarose using techniques standard in the art. DNA bands are
visualized with ethidium bromide and bands are recovered from gels
by excision with a razor blade. Fragments isolated from LGT are
ligated directly in the LGT. Ten microliters of each recovered DNA
fragment is used to assemble the ligation reactions, producing
final ligation reaction volumes of about 23 .mu.l. After excision
with a razor blade, the recovered gel bands containing the desired
DNA fragments are melted and brought to 1.times. ligase buffer and
3 units of T4 DNA ligase (Promega) are added as described above.
Fragments isolated from either regular agarose or Nusieve agarose
are purified from the agarose using ultrafree-MC 0.45 .mu.M filter
units (Millipore) and the fragments are ligated as described above.
Ligation reactions are incubated at room temperature for two hours
before transforming into frozen competent E. coli cells using
standard procedures (Sambrook et al.).
Transformations:
[0131] Frozen competent E. coli cells of the strain DH5alpha or
HB101 are prepared and transformed using standard procedures
(Sambrook et al.). E. Coli "SURE" competent cells are obtained from
Stratagene (La Jolla, Calif.). For ligations carried out in LGT
agarose, after ligation reactions are complete, 50 mM CaCl2 is
added to a final volume of about 150 .mu.l and the solution heated
at approximately 65.degree. C. for about 10 minutes to completely
melt the agarose. The solution is then mixed and chilled on ice for
about 10 minutes before the addition of about 200 .mu.l of
competent cells which had been thawed on ice. This mixture is
allowed to incubate for 30 minutes on ice. The mixture is next heat
shocked at 42.degree. C. for 60 seconds before chilling on ice for
two minutes. Next, 800 .mu.l of SOC media (20% tryptone, 0.5% yeast
extract, 10 mM NaCl, 2.5 mM KCl, adjusted to pH 8 with 5 N NaOH, 20
mM MgCl2:MgSO4 mix, and 20 mM glucose; Sambrook et al.) is added
and the cells are incubated at 37.degree. C. with shaking for about
one hour before plating on selective media plates. Plates typically
are L-agar (Sambrook et al.) containing 100 .mu.g/ml
ampicillin.
[0132] When ligations are carried out in a solution without
agarose, typically 200 .mu.l of frozen competent E. coli cells
(strain DH5alpha (BRL Gaithersburg, Md. or Sure cells, Stratagene,
La Jolla, Calif.) are thawed on ice and 5 .mu.l of the ligation
mixture added. The reaction is incubated on ice for about 45 to 60
minutes, the cells are then heat shocked at 42.degree. for about 90
seconds. After recovery at room temperature for about 10 minutes,
800 .mu.l of SOC medium is added and the cells are then incubated 1
hour at 37.degree. C. with shaking and plated as above.
[0133] When screening for inserts into the beta-galactosidase gene
in some of the standard vectors used, 200 .mu.l of the recovered
transformation mixture is plated on LB-agar plates containing
0.008% X-gal, 80 .mu.M IPTG, and 100 .mu.g/ml ampicillin (Sambrook
et al.). The plates are incubated at 37.degree. overnight to allow
selection and growth of transformants.
Miniscreening DNA:
[0134] Transformants from the selective media plates are grown and
their plasmid structure is examined and confirmed using standard
plasmid mini-screen procedures (Sambrook et al.). Typically, the
"boiling" procedure is used to produce small amounts of plasmid DNA
for analysis (Sambrook et al.). Alternatively, an ammonium acetate
procedure is used in some cases. This procedure is a modification
of that reported by Shing-yi Lee et al., Biotechniques 9:676-679
(1990).
[0135] 1) Inoculate a single bacterial colony from the overnight
selection plates into 5 ml (can be scaled down to 1 ml) of TB
(Sambrook et al.) medium and grow in the presence of the
appropriate antibiotic.
[0136] 2) Incubate on a roller at 37.degree. C. overnight.
[0137] 3) Collect 5 ml of bacterial cells in a plastic Oakridge
tube and spin for 5 min. at 5000 rpm in a Sorvall SS-34 rotor at
4.degree. C.
[0138] 4) Remove the supernatant.
[0139] 5) Resuspend the pellet in 1 ml of lysis buffer (50 mM
glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA and 5 mg/ml lysozyme),
vortex for 5 seconds, and incubate at room temperature for 5
min.
[0140] 6) Add 2 ml of freshly prepared alkaline solution (0.2 N
NaOH, 1% sodium dodecyl sulfate), tightly secure lid, mix by
inverting 5 times and place tube in an ice-water bath for 5
min.
[0141] 7) Add 1.5 ml of ice-cold 7.5 M ammonium acetate (pH 7.6) to
the solution, mix by inverting the tube gently 5 times and place on
an ice-water bath for 5 min.
[0142] 8) Centrifuge mixture at 9000 rpm for 10 min. at room
temperature.
[0143] 9) Transfer clear supernatant to a 15 ml Corex tube and add
0.6 volumes of isopropanol (approx. 2.5 ml). Let sit at room
temperature for 10 min.
[0144] 10) Centrifuge the mixture at 9000 rpm for 10 min. at room
temperature and discard the supernatant.
[0145] 11) Resuspend the pellet in 300 ul of TE buffer. Add 6 ul of
a stock of RNase A & T1 (made as a 200 ul solution by adding
180 ul of RNase A (3254 Units/mg protein, 5.6 mg protein/ml) and 20
ul of RNase T1 (481 Units/ug protein, 1.2 mg protein/ml)). These
stocks may be purchased from USB (US Biochemical). Transfer to a
microcentrifuge tube and incubate at 37.degree. C. for 15 min.
[0146] 12) Add 75 ul of distilled water and 100 ul of 7.5 M
ammonium acetate and incubate in an ice-water bath for 10 min.
[0147] 13) Centrifuge the mixture at 14,000 rpm for 10 min. in a
Beckman microfuge at 4.degree. C.
[0148] 14) Precipitate by adding 2.5 volumes of 100% EtOH (approx.
1 ml) and incubate in an ice-water bath for 10 min.
[0149] 15) Spin at 14,000 rpm for 10 min. in a microfuge.
[0150] 16) Wash pellet with 70% ethanol (using 0.5 ml-1 ml). Dry
the pellet and resuspend in 100 .mu.l of 1.times. New England
Biolabs restriction enzyme Buffer 4 (20 mM Tris-HCl (pH 7.9), 10 mM
magnesium acetate, 50 mM potassium acetate, 1 mM DTT). Measure
concentration and check purity by spectrophotometry at absorbances
260 and 280 nm.
[0151] For a more rapid determination as to whether or not a
particular bacterial colony harbored a recombinant plasmid, a PCR
miniscreen procedure is carried out using a modification of the
method described by (Sandhu, G. S. et al., 1989, BioTechniques,
7:689-690). Briefly, the following mixture is prepared:
[0152] 100 .mu.l primer mix above, 20 .mu.M each primer,
[0153] 100 .mu.l dNTP mix (2.5 mM each)
[0154] 100 .mu.l 10.times. AmpliTaq buffer (Perkin-Elmer Cetus,
1.times. buffer=10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and
0.01% gelatin)
[0155] 700 .mu.l deionized water.
[0156] 20 .mu.l of the above mixture is put into a a 0.5 ml
polyproplyene PCR tube. A transformed bacterial colony is picked
with a toothpick and resuspended in the mixture. The tube is put in
a boiling water bath for 10 minutes and then cooled to room
temperature before adding 5 .mu.l of the mix described below:
[0157] 265 .mu.l deionized water
[0158] 30 .mu.l 10.times. Amplitaq buffer (Perkin-Elmer Cetus,
1.times. buffer=10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and
0.01% gelatin)
[0159] 7.5 .mu.l Taq polymerase
[0160] The samples are overlaid with 50 .mu.l of mineral oil and
PCR is carried out for 30 cycles using the following
parameters:
[0161] denature: 94.degree. for 1 min
[0162] anneal: 55.degree. for 1 min
[0163] extend: 72.degree. for 45 seconds.
[0164] After PCR amplification, 1 .mu.l of loading dye (30%
glycerol, 0.25% Bromophenol blue, 0.25% xylene cyanol) is added to
the whole reaction and 20 .mu.l of the mixture is loaded on a 2%
Nusieve, 1% agarose gel to see if there is a PCR product of the
expected size.
[0165] This procedure is used as an initial screen. Minipreps are
subsequently carried out to confirm the structure of the plasmid
and its insert prior to sequencing.
Example 2
Amplification and Assembly of Each Quarter
Cloning Fragments of the Synthetic Bt cryIA(b) Gene:
[0166] The synthetic gene was designed to be cloned in four pieces,
each roughly one quarter of the gene. The oligomers for each
quarter were pooled to either be assembled by PCR, hybridization,
or a combination of hybridization followed by PCR amplification as
described elsewhere. Synthetic quarters were pieced together with
overlapping restriction sites Aat II, NcoI, and Apa I between the
1st and 2nd, 2nd and 3rd, and 3rd and 4th quarters
respectively.
[0167] Each quarter of the gene (representing about 500 bp) was
assembled by hybridizing the appropriate oligomers and amplifying
the desired fragment using PCR primers specific for the ends of
that quarter. Two different sets of PCR reactions employing two
sets of slightly different primers were used. The PCR products of
the two reactions were designed to be identical except that in the
first reaction there was an additional AATT sequence at the 5' end
of the coding region and in the second reaction there was an AGCT
sequence at the 3' end of a given quarter. When the products of the
two reactions for a particular quarter were mixed (after removing
the polymerase, primers and incomplete products), denatured, and
subsequently re-annealed, a certain ratio (theoretically 50%) of
the annealed product should have non-homologous overhanging ends.
These ends were designed to correspond to the "sticky ends" formed
during restriction digestion with EcoRI at the 5' end and Hind III
at the 3' end of the molecule. The resulting molecules were
phosphorylated, ligated into an EcoRI/HindIII digested and
phosphatased Bluescript vector, and transformed into frozen
competent E. coli strain DH5alpha. After selection, the E. coli
colonies containing the desired fragment are identified by
restriction digest patterns of the DNA. Inserts representing parts
of the synthetic gene are subsequently purified and sequenced using
standard procedures. In all cases, clones from multiple PCR
reactions are generated and sequenced. The quarters are then joined
together using the unique restriction sites at the junctions to
obtain the complete gene.
[0168] Cloned quarters are identified by mini-screen procedures and
the gene fragment sequenced. It is found that errors are frequently
introduced into the sequence, most probably during the PCR
amplification steps. To correct such errors in clones that contain
only a few such errors, hybridized oligomers are used. Hybridized
fragments are digested at restriction enzyme recognition sites
within the fragment and cloned to replace the mutated region in the
synthetic gene. Hybridized fragments range from 90 bp in length
(e.g. the region that replaces the fragment between the Sac II
sites in the 2nd quarter) to the about 350 bp 4th quarter fragment
that replaces two PCR induced mutations in the 4th quarter of the
gene.
[0169] Due to the high error rate of PCR, a plasmid is designed and
constructed which allows the selection of a cloned gene fragment
that contains an open reading frame. This plasmid is designed in
such a manner that if an open reading frame is introduced into the
cloning sites, the transformed bacteria could grow in the presence
of kanamycin. The construction of this vector is described in
detail below. This selection system greatly expedites the progress
by allowing one to rapidly identify clones with open reading frames
without having to sequence a large number of independent clones.
The synthetic quarters are assembled in various plasmids, including
BSSK (Stratagene; La Jolla, Calif.), pUC18 (Sambrook et al.), and
the Km-expression vector. Other suitable plasmids, including pUC
based plasmids, are known in the art and may also be used. Complete
sequencing of cloned fragments, western blot analysis of cloned
gene products, and insect bioassays using European corn borer as
the test insect verify that fully functional synthetic Bt cryIA(b)
genes have been obtained.
Construction of the Km-Expression Vector to Select Open Reading
Frames:
[0170] The Km-expression vector is designed to select for fragments
of the synthetic gene which contain open-reading frames. PCR
oligomers are designed which allow the fusion of the NPTII gene
from Tn5 starting at nucleotide 13 (Reiss et al., EMBO J.
3:3317-3322 (1984)) with pUC18 and introduce useful restriction
sites between the DNA segments. The polylinker region contains
restriction sites to allow cloning various synthetic Bt IP
fragments in-frame with the Km gene. The 88 bp 5' oligomer
containing the polylinker region is purified on a 6% polyacrylamide
gel as described above for the oligomer PAGE purification. A PCR
reaction is assembled with a 1 Kb Bgl II\Sma I template fragment
which contains the NPT II gene derived from Tn5. The PCR reaction
mix contains 100 ng of template with 100 pmols of oligomers KE72A28
and KE74A28 (see sequences below), 200 nM dNTP, and 2.5 Units of
Taq polymerase all in a 50 .mu.l volume with an equal volume of
mineral oil overlaid. Sequences of the primers are: TABLE-US-00002
KE74A28 (SEQ ID NO:29) 5'-GCAGATCTGG ATCCATGCAC GCCGTGAAGG
GCCCTTCTAG AAGGCCTATC GATAAAGAGC TCCCCGGGGA TGGATTGCAC GCAGGTTC-3'
KE72A28 (SEQ ID NO:30) 5'-GCGTTAACAT GTCGACTCAG AAGAACTCGT
CAAGAAGGCG-3'
[0171] The PCR parameters used are: 94.degree. C. for 45 seconds
(sec), 55.degree. C. for 45 sec, and 72.degree. C. for 55 sec with
the extension at step 3 for 3 sec for 20 cycles. All PCR reactions
are carried out in a Perkin-Elmer Cetus thermocycler. The amplified
PCR product is 800 bp and contains the polylinker region with a
translational start site followed by unique restriction sites fused
in-frame with the Km gene from base #13 running through the
translational terminator. pUC:KM74 is the Km-expression cassette
that was assembled from the 800 bp Bgl II\Sal I polylinker/Km
fragment cloned in the PUC18 vector. The lacZ promoter allows the
Km gene to be expressed in E. coli. pUC:KM74 derivatives has to
first be plated on LB-agar plates containing 100 .mu.g/ml
ampicillin to select transformants which can subsequently be
screened on LB-agar plates containing 25 .mu.g/ml kanamycin/IPTG.
Synthetic Bt IP gene fragments are assembled from each quarter in
the Km-cassette to verify cloning of open-reading-frame containing
fragments pieces. The first ECB active synthetic Bt IP gene
fragment, pBt:Km#6, is a Bt IP gene that shows Km resistance. This
fragment is subsequently discovered to contain mutations in the 3rd
and 4th quarter which are later repaired.
Example 2A
Synthesis and Cloning of the First Quarter of the Synthetic Gene
(Base Pairs 1 to 550)
[0172] The following procedures are followed in order to clone the
first quarter of the synthetic DNA sequence encoding a synthetic Bt
cryIA(b) gene. The same procedures are essentially followed for
synthesis and cloning of the other quarters, except as noted for
primers and restriction sites.
Template for Quarter 1: Mixture of Equal Amounts of Purified
Oligomers U1-U7 and L1 to L7
[0173] PCR Primers: TABLE-US-00003 Forward: P1 (a): 5'-GTCGACAAGG
ATCCAACAAT GG-3' (SEQ ID NO:31) P1 (b): 5'-AATTGTCGAC AAGGATCCAA
CAATGG-3' (SEQ ID NO:32) Reverse: P2 (a): 5'-ACACGCTGAC GTCGCGCAGC
ACG-3' (SEQ ID NO:33) P2 (b): 5'-AGCTACACGC TGACGTCGCG CAG-3' (SEQ
ID NO:34)
[0174] Primer pair A1: P1(b)+P2(a) [0175] Primer pair A2:
P1(a)+P2(b)
[0176] The PCR reaction containing the oligomers comprising the
first quarter of the synthetic maize-optimized Bt IP gene is set up
as follows:
[0177] 200 ng oligo mix (all oligos for the quarter mixed in equal
amounts based on weight)
[0178] 10 .mu.l of primer mix (1:1 mix of each at 20 .mu.M; primers
are described above)
[0179] 5 .mu.l of 10.times.PCR buffer
[0180] PCR buffer used may be either
[0181] (a) 1.times. concentration=10 mM KCl, 10 mM (NH4)2SO4, 20 mM
Tris-HCl, pH 8.0, 2 mM MgSO4, and 0.1% Triton X-100), or
[0182] (b) 1.times. concentration=10 mM Tris-HCl pH 8.3, 50 mM KCl
1.5 mM MgCl2, 0.01% wt/vol gelatin.
[0183] Components are mixed, heated in a boiling water bath for 5
minutes, and incubated at 65.degree. C. for 10 minutes.
[0184] Next, the following reagents are added:
[0185] 8 .mu.l of dNTPs mixture (final concentration in the
reaction=0.2 mM each)
[0186] 5 units polymerase.
The final reaction volume is 50 microliters.
[0187] Oligomers are then incubated for 3 min at 72.degree. C. and
then a PCR cycle is run. The PCR reaction is run in a Perkin Elmer
thermocycler on a step cycle protocol as follows:
[0188] denaturation cycle: 94.degree. for 1 minute
[0189] annealing cycle: 60.degree. for 1 minute
[0190] extension cycle: 72.degree. for 45 seconds (+3 sec per
cycle)
[0191] number of cycles: 15
[0192] After the reaction is complete, 10 .mu.l of the PCR reaction
is loaded on a 2% Nusieve-GTG (FMC), 1% agarose analytical gel to
monitor the reaction. The remaining 40 .mu.l is used to clone the
gene fragments as described below.
PCR Products
[0193] The termini of the double stranded PCR product corresponding
to the various primer pairs are shown (only upper strand):
TABLE-US-00004 (SEQ ID NO:35) A1
AATTGTCGAC.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub-
.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--_GCGTGT (554
bp) first qtr. (SEQ ID NO:36) A2
GTCGAC.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.---
.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--_GCGTGTAGCT (554
bp) first qtr.
Hybridization
[0194] 40 .mu.l of each of the PCR reactions described above is
purified using a chromaspin 400 column (Clonetech, Palo Alto,
Calif.) according to manufacturers directions. Five .mu.g of
carrier DNA was added to the reactions before loading on the
column. (This is done for most of the cloning. However, in some
reactions the PCR reactions are phenol:chloroform extracted using
standard procedures (Sambrook et al.) to remove the Taq polymerase
and the PCR generated DNA is recovered from the aqueous phase using
a standard ethanol precipitation procedure.) The carrier DNA does
not elute with the PCR generated fragments. The A1 and A2 reaction
counterparts for each quarter are mixed, heated in a boiling water
bath for 10 minutes and then incubated at 65.degree. C. overnight.
The reactions are then removed from the 65.degree. bath and ethanol
precipitated with 1 .mu.l (20 .mu.g) of nuclease free glycogen
(Tracy, Prep. Biochem. 11:251-268 (1981) as carrier. The pellet is
resuspended in 40 .mu.l of deionized water.
Phosphorylation Reaction
[0195] The phosphorylation reaction is carried out as follows:
[0196] 40 .mu.l DNA
[0197] 2.5 .mu.l 20 mM ATP
[0198] 0.5 .mu.l 10.times.BSA/DTT (1.times.=5 mM DTT, 0.5 mg/ml
BSA)
[0199] 1.0 .mu.l 10.times. polynucleotide kinase buffer
(1.times.=70 mM Tris.HCl, pH 7.6, 0.1 M KCl, 10 mM MgCl2)
[0200] 2.0 .mu.l polynucleotide kinase (New England Biolabs, 20
units).
[0201] Incubation is for 2 hours at 37.degree. C.
[0202] The reaction is then extracted one time with a 1:1
phenol:chloroform mixture, then once with chloroform and the
aqueous phase ethanol precipitated using standard procedures. The
pellet is resuspended in 10 .mu.l of TE.
Restriction Digests
[0203] 20 .mu.g of Bluescript vector (BSSK+, Stratagene, La Jolla,
Calif.)
[0204] 10 .mu.l 10.times. restriction buffer (1.times.=20 mM
Tris-HCl pH 8.0, 10 mM MgCl2, 100 mM NaCl)
[0205] 5 .mu.l Eco RI (New England Biolabs) 100 units
[0206] 5 .mu.l Hind III (New England Biolabs) 100 units
[0207] Final reaction volume is 100 .mu.l.
[0208] Incubation is for 3 hours at 37.degree..
[0209] When completed, the reaction is extracted with an equal
volume of phenol saturated with TE (10 mM Tris.HCl pH 8.0 and 10 mM
EDTA). After centrifugation, the aqueous phase was extracted with
an equal volume of 1:1 mixture of (TE saturated) phenol:chloroform
(the "chloroform" is mixed in a ratio of 24:1 chloroform:isoamyl
alcohol), and finally the aqueous phase from this extraction is
extracted with an equal volume of chloroform. The final aqueous
phase is ethanol precipitated (by adding 10 .mu.l of 3 M sodium
acetate and 250 .mu.l of absolute ethanol, left at 4.degree. for 10
min and centrifuged in a microfuge at maximum speed for 10 minutes.
The pellet is rinsed in 70% ethanol and dried at room temperature
for 5-10 minutes and resuspended in 100 .mu.l of 10 mM Tris.HCl (pH
8.3).
Phosphatase Reaction
[0210] Vector DNA is routinely treated with phosphatase to reduce
the number of colonies obtained without an insert. Calf intestinal
alkaline phosphatase is typically used (Sambrook et al.), but other
phosphatase enzymes can also be used for this step.
[0211] Typical phosphatase reactions are set up as below:
[0212] 90 .mu.l of digested DNA described above
[0213] 10 .mu.l of 10.times. Calf intestinal alkaline phosphatase
buffer (1.times.=50 mM Tris-HCl (pH 8.3), 10 mM MgCl2, 1 mM ZnCl2,
10 mM spermidine)
[0214] 1 .mu.l (1 unit) of calf intestinal alkaline phosphatase
(CIP, Boehringer Mannheim, Indianapolis, Ind.)
[0215] Incubation is at 37.degree. C. for 1 hour.
[0216] The DNA is then gel purified (on a 1% low gelling
temperature (LGT) agarose gel) and the pellet resuspended in 50
.mu.l TE. After electrophoresis, the appropriate band is excised
from the gel using a razor blade, melted at 65.degree. for 5
minutes and diluted 1:1 with TE. This solution is extracted twice
with phenol, once with the above phenol:chloroform mixture, and
once with chloroform. The final aqueous phase is ethanol
precipitated and resuspended in TE buffer.
Ligation:
[0217] To ligate fragments of the synthetic gene into vectors, the
following conditions are typically used.
[0218] 5 .mu.l of phosphorylated insert DNA
[0219] 2 .mu.l of phosphatased Eco RI/Hind III digested Bluescript
vector heated at 650 for 5 minutes, then cooled
[0220] 1 .mu.l 10.times. ligase buffer (1.times. buffer=30 mM
Tris.HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, 1 mM ATP)
[0221] 1 .mu.l BSA (1 mg/ml)
[0222] 1 .mu.l ligase (3 units, Promega, Madison, Wis.)
[0223] Ligase reactions are typically incubated at 16.degree.
overnight or at room temperature for two hours.
Transformation:
[0224] Transformation of ligated DNA fragments into E. coli is
performed using standard procedures (Sambrook et al.) as described
above.
Identification of Recombinants
[0225] White or light blue colonies resulting from overnight
incubation of transformation plates are selected. Plasmids in the
transformants are characterized using standard mini-screen
procedures (Sambrook et al.) or as described above. One of the
three procedures listed below are typically employed:
[0226] (1) boiling DNA miniprep method
[0227] (2) PCR miniscreen
[0228] (3) Ammonium acetate miniprep.
[0229] The restriction digest of recombinant plasmids believed to
contain the first quarter is set up as follows:
[0230] (a) Bam HI/Aat II digest: 10 .mu.l DNA+10 .mu.l 1.times. New
England Biolabs restriction enzyme Buffer 4
[0231] 0.5 .mu.l Bam HI (10 units)
[0232] 0.5 .mu.l Aat II (5 units)
[0233] Incubation is for about 2 hours at 37.degree. C.
[0234] Clones identified as having the desired restriction pattern
are next digested with Pvu II and with Bgl II in separate
reactions. Only clones with the desired restriction patterns with
all three enzyme digestions are carried further for sequencing.
Sequencing of Cloned Gene Fragments:
[0235] Sequencing is performed using a modification of Sanger's
dideoxy chain termination method (Sambrook et al.) using double
stranded DNA with the Sequenase 2 kit (United States Biochemical
Corp., Cleveland, Ohio). In all, six first quarter clones are
sequenced. Of the clones sequenced, only two clones designated pQA1
and pQA5 are found to contain only one deletion each. These
deletions are of one base pair each located at position 452 in pQA1
and position 297 in pQA5.
[0236] Plasmid pQA1 is used with pP1-8 (as described below) to
obtain a first quarter with the expected sequence.
Example 2B
Synthesis and Cloning of the Second Quarter (Base Pairs 531 to
1050)
Template: Oligomers U8-U14 and L8-L14
[0237] PCR Primers: TABLE-US-00005 forward: P3 (a): 5'-GCTGCGCGAC
GTCAGCGTGT TCGG-3' (SEQ ID NO:37) P3 (b): 5'-AATTGCTGCG CGACGTCAGC
GTG-3' (SEQ ID NO:38) Reverse: P4 (a): 5'-GGCGTTGCCC ATGGTGCCGT
ACAGG-3' (SEQ ID NO:39) P4 (b): 5'-AGCTGGCGT TGCCCATGGT GCCG-3'
(SEQ ID NO:40)
[0238] Primer pair B1: P3(b)+P4(a) [0239] Primer pair B2:
P3(a)+P4(b)
[0240] PCR Products TABLE-US-00006 (SEQ ID NO:41) B1
AATTGCTGCG.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub-
.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--_AACGCC (524
bp) second quarter (SEQ ID NO:42) B2
GCTGCG.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.---
.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--_AACGCCAGCT (524
bp)
[0241] Hybridization, PCR amplification, spin column size
fractionation, and cloning of this gene fragment in Bluescript
digested with Eco RI/Hind III are performed as described above for
the first quarter (Example 2A). The PCR product for this quarter is
about 529 bp in size representing the second quarter of the gene
(nucleotides 531 to 1050). Transformation is into frozen competent
E. coli cells (DH5alpha) using standard procedures described above
(Sambrook et al.)
Miniscreen of pQB Clones:
[0242] Miniprep DNA is prepared as described above and digested
with (a) Aat II/Nco I, (b) Pvu II and (c) with Bgl I to confirm the
structure insert in the vector before sequencing.
[0243] Sequencing is performed as described above using the dideoxy
method of Sanger (Sambrook et al.).
[0244] A total of thirteen clones for this quarter are sequenced.
The second quarter consistently contains one or more deletions
between position 884 and 887. In most cases the G at position 884
is deleted.
[0245] Plasmid pQB5 had only one deletion at position 884. This
region lies between two Sac II sites (positions 859 and 949).
Correction of this deletion is described in Example 3.
Clones of the First Half (1-1050 bp).
[0246] A fragment for cloning the first half (quarters 1 and 2) of
the synthetic Bt maize gene as a single DNA fragment is obtained by
restriction digestion of the product of a PCR reaction comprising
the first quarter and the second quarter. Restriction endonuclease
Aat II is used to cut the DNA (following phenol extraction and
ethanol precipitation) in a 20 .mu.l reaction. 15 .mu.l of each of
the Aat II digested quarters is mixed and ligated (in a 50 .mu.l
volume by adding 51 .mu.l of 10.times. ligase buffer, (1.times.=30
mM Tris-HCl pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP) 14 .mu.l of
deionized water and 1 .mu.l of T4 DNA ligase, 3 units, Promega,
Madison, Wis.) at room temperature for 2 hr. The result is an about
1 kb fragment as judged by electrophoresis on a 1% agarose gel run
using standard conditions (Sambrook et al.) Ten .mu.l of the
ligation product is amplified by PCR using conditions described
previously except that only 5 cycles were run. [0247] Primer Pair:
HA=P1(a)+P4(b) [0248] Primer Pair: HB=P1(b)+P4(a)
[0249] The product of these reactions is cloned into Bluescript
(Stratagene, La Jolla, Calif.) as described for the individual
quarters. This procedure is only done once i.e., all insert DNA is
obtained in a particular region from a single PCR reaction.
[0250] Thirty-six colonies are miniscreened with Sal I digests and
Pvu II digests. All except 4 contain an insert of approximately 1
kb in size of which at least 20 contain the correct Pvu II
digestion pattern. Eight of these clones are selected for sequence
analysis. One of the clones, P1-8, has the desired sequence between
the Eco NI site (396 bp) and the Dra III site (640 bp). This clone
is used to obtain a plasmid with the desired sequence up to the Dra
III site (640 bp) in the second quarter with pQA1 (first quarter
with a deletion at position 452 bp described previously.)
Example 2C
Cloning and Synthesis of Third Quarter (Base Pairs 1021 to
1500)
Template: Oligos U15-U20 and L15-L21
[0251] PCR primers: TABLE-US-00007 forward P5 (a): (SEQ ID NO:43)
5'-TTCCCCCTGT ACGGCACCAT GGGCAACGCC GC-3' P5 (b): (SEQ ID NO:44)
5'-AATTGTACGG CACCATGGGC AAC-3' reverse P6 (a): (SEQ ID NO:45)
5'-GAAGCCGGGG CCCTTCACCA CGCTGG-3' P6 (b): (SEQ ID NO:46)
5'-AGCTGAAGCC GGGGCCCTTC ACC-3'
[0252] Primer pair C1: P5(b)+P6(a) [0253] Primer pair C2:
P5(a)+P6(b)
[0254] PCR Product: TABLE-US-00008 (SEQ ID NO:47) C1
AATTGTACGG.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.su-
b.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--_GGCTTC (475
bp) 3d qtr (SEQ ID NO:48) C2
TTCCCCTGTACGG.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.-
sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--_GGCTTCAGCT
(SEQ ID NO:49) (484 bp) 3d qtr
[0255] PCR reactions, spin column recovery of the correct sized DNA
fragment, and ligation into vectors are performed as described
above (Example 2A) using a Bluescript vector cut with Eco RI and
Hind III. The approximately 479 base pair PCR product represents
the third quarter of the synthetic gene (NT 1021-1500).
[0256] Transformation into frozen competent E. coli strain DH5alpha
cells, selection and identification of transformants,
characterization of transformants by mini-screen procedures, and
sequencing of the synthetic gene fragment in the vector are all as
described above.
Mini Screen of pQC Clones:
[0257] The third quarter is miniscreened using standard procedures
(Sambrook et al.). Miniprep DNA is cut with (a) Nco I/Apa I and (b)
with Pvu II. Clones containing the correct restriction digest
patterns are sequenced using standard procedures. A total of 22
clones of the third quarter are sequenced. Three major deletion
"hotspots" in the third quarter are identified (a) at position 1083
(b) between position 1290-1397 and (c) between positions 1356-1362.
In all clones except one, pQC8, there is also consistently an
insertion of a C at position 1365. In addition to these mutations,
the third quarter clones contain a large number of other apparently
random deletions. The common factor to the three mutational
"hotspots" in the third quarter and the one in the second quarter
is that these regions are all flanked on either side by sequences
that are about 80% C+G. Other regions containing 5 to 9 C-Gs in a
row are not affected. The oligomers in U15, U16, U18, U19, L15,
L16, L18 and L19 are redesigned to reduce the C+G content in these
regions. Five clones each from PCR reaction using the modified
oligomers are sequenced.
[0258] Plasmid pQCN103 has the correct sequence for the third
quarter except for a change at position 1326. This change, which
substitutes a G for a C, results in the substitution of one amino
acid (leucine) for the original (phenylalanine).
Example 2D
Synthesis and Cloning of Fourth Quarter (Base Pairs 1480 to
1960)
[0259] The fourth quarter of the gene is obtained from a clone
which is originally designed to comprise the third and fourth
quarters of the gene. The "second half" of the synthetic gene is
obtained from PCR reactions to fuse the third and fourth quarters.
These reactions are run with PCR primers P5(a) and P6(a) described
above for the third quarter and primers P7(a) and P8(a) (described
below). The reverse primer is modified to include a Sac I site and
a termination codon. Separate reactions for each quarter are run
for 30 cycles using the conditions described above. The two
quarters are joined together by overlapping PCR and subsequently
digested with restriction enzymes Nco I and Sac I. The resulting
953 bp fragment is cloned directionally into pCIB3054, which has
been cut with Nco I/Sac I and treated with alkaline
phosphatase.
[0260] pCIB3054 is constructed by inserting intron #9 of PEP
carboxylase (PEPC ivs #9) in the unique Hpa I site of pCIB246
(described in detail in Example 4) pCIB246 is cut with HpaI and
phosphatased with CIP using standard procedures described in
Example 2A. PEPC ivs #9 is obtained by PCR using pPEP-10 as the
template. pPEP-10 is a genomic subclone containing the entire maize
PEP carboxylase gene encoding the C4 photosynthetic enzyme, plus
about 2.2 Kb of 5'-flanking and 1.8 Kb of 3'-flanking DNA. The 10
Kb DNA is ligated in the HindIII site of pUC18. (Hudspeth et al.,
Plant Molecular Biology, 12: 576-589 (1989). The forward PCR primer
used to obtain the PEPCivs#9 is GTACAAAAACCAGCAACTC (SEQ ID NO:50)
and the reverse primer is CTGCACAAAGTGGAGTAGT (SEQ ID NO:51). The
PCR product is a 108 bp fragment containing only the PEP
carboxylase intron #9 sequences. The PCR reaction is extracted with
phenol and chloroform, ethanol precipitated phosphorylated with
polynucleotide kinase and treated with T4 polymerase to fill in the
3' nontemplated base addition found in PCR products (Clark, J. M.,
Nucleic Acid Research, 16: 9677-9686 (1988)) using standard
procedures. The kinased fragment is blunt-end cloned into the HpaI
site of pCIB246, using standard procedures described earlier.
Amplification and Assembly of the Fourth Quarter
Template: U21-U26 and L22-L28
[0261] PCR Primers TABLE-US-00009 FORWARD P7 (a): (SEQ ID NO:52)
5'-TGGTGAAGGG CCCCGGCTTC ACCGG-3' REVERSE P8 (a): (SEQ ID NO:53)
5'-ATCATCGATG AGCTCCTACA CCTGATCGAT GTGGTA-3'
[0262] PRIMER PAIR 4: P7(A)+P8(a) [0263] PRIMER PAIR 3: P5(A)+P6(a)
[0264] Primer pair for overlapping PCR: P7(a)+P8(a)
[0265] PCR Product TABLE-US-00010 fourth quarter: (SEQ ID NO:54)
GGTGAA.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub-
.--.sub.--.sub.--.sub.--.sub.--.sub.--.sub.--_ATCAGGAGCTCATCGATGAT
(484 bp) third quarter: (SEQ ID NO:55) TTCCCCCTGTA-------TTCACCGG
(484 bp) second half: GGTGAA-------CATGATGAT (953 bp)
[0266] Four positive clones are identified by plasmid miniscreen
and are subsequently sequenced using standard procedures.
[0267] Plasmid BLP2 #1 contains approximately the correct fourth
quarter sequence except for two mutations. These mutations are at
position 1523 (substituting an A for a G, resulting in an amino
acid change which substitutes a His for an Arg) and at position
1634 (substituting a T for a C, resulting in an amino acid
substitution of a Ser for a Thr).
[0268] Plasmid Bt.P2 #1 is used in the construction of pCIB4414
described below. (The mistakes are ultimately corrected by
hybridizing all the oligos of the fourth quarter, digesting with
Apa I/Bst E II and replacing that region in pCIB4414. Therefore,
only sequences from position 1842-1960 remain of Bt.P2#1 in the
final construct.)
Example 3
Assembly and Repair of the Final Synthetic Gene
[0269] The synthetic maize optimized Bt cryIA(b) gene is designed
to be cloned in quarters. Using the PCR technique, however, results
in mutations, which in most cases are deletions resulting in
frameshift mutations. Plasmids containing individual quarters are
therefore sequenced and the correct parts ligated together using
standard procedures.
[0270] After obtaining first and second quarter clones with almost
the desired sequence, plasmids pEB1Q#4 and pEB1Q#5 are constructed
to obtain the desired sequence of the synthetic Bt gene up to the
Dra III site at the base pair position 634 (this mutation destroys
the Dra III site). The pEB1Q constructs are made by ligating a 3.9
Kb Eco NI\Bam HI fragment from pP1-8 with a 400 bp fragment from
pQA1. pEB1Q#5 has the desired sequence up to the Dra III site, but
pEB1Q#4 has a mutation at base pair position 378.
[0271] Plasmids p1H1M4 and p1H1M5 are constructed to repair the Dra
III site in pEB1Q#4 and pEB1Q#5. Plasmids p1H1M#4 and #5 are made
by ligating a 3.5 Kb Nco I\Aat II fragment from pEB1Q#4 and #5
respectively, with a 500 bp Nco I\Aat II fragment from pQB5.
Plasmid p1H1M5 contains a mutation between the Sac II sites at
position 884 in the second quarter of the synthetic Bt gene.
Plasmid p1H1M4 contains the additional mutation as described in its
precursor construct pEB1Q#4.
[0272] The Sac II site in the Bluescript vector region of p1H1M4 is
deleted by cutting p1H1M4 with Not I and Sac I and converting these
sites to blunt ends using T4 DNA polymerase under standard
conditions before ligating this 3.9 Kb fragment to make p1H1M4 S.
Deleting the Sac II site in the vector region allows the 90 bp Sac
II fragment with the mutation at position 884 in the 2nd quarter of
p1H1M4 S to be removed prior to replacement with a 90 bp Sac II
fragment. Oligomers U\L 12 and 13 are kinased and hybridized
(described above) before cutting with Sac II and isolating a 90 bp
fragment on a 2% Nusieve gel. The Sac II fragment is ligated into
the about 3.8 Kb Sac II cut p1H1M4 S vector which has been
phosphatased with CIP. The repaired Sac II construct is called
pHYB2#6. The orientation of the Sac II fragment in pHYB2#6 is
detected by PCR screening as described earlier using the following
primers: TABLE-US-00011 MK23A28 = 5'-GGGGCTGCGGATGCTGCCCT-3' (SEQ
ID NO:56) MK25A28 = 5'-GAGCTGACCCTGACCGTGCT-3' (SEQ ID NO:57)
MK26A28 = 5'-CACCTGATGGACATCCTGAA-3' (SEQ ID NO:58)
[0273] Running the PCR reactions with 50 pmoles of primers MK3A28
and MK25A28 produces an approximate 180 bp fragment, indicating the
inserted fragment bounded by the Sac II sites in pHYB2#6 is in the
correct orientation. Using primers MK25A28 and MK6A28 in the PCR
screening acts as the negative control producing an approximate 180
bp fragment only in constructs containing the Sac II bounded
fragment in the wrong orientation. pHYB2#6 sequence is determined
using standard procedures.
[0274] pHYB2#6 has one mutation at position 378 which needed to be
repaired to obtain a first quarter containing the desired
sequence.
[0275] Plasmid p1HG#6 contains the desired sequence for the entire
first half of the synthetic Bt gene. p1HG#6 is made from a 3.4 Kb
Aat II\Nco I fragment of p1H1M5#2 ligated to a 500 bp Aat II\Nco I
fragment from pHYB2#6.
[0276] To identify clones or partial clones of the synthetic gene
which contain open reading frames, the kanamycin selection vector
(described above) is used. The fourth quarter of the synthetic Bt
gene is the first put into the kanamycin cassette. pKM74-4 contains
the approximately 500 bp Apa I\Cla I fragment from plasmid BtP2
(which had been previously transformed into a dam--E. coli strain
(PO-100) to be able to cut with Cla I), ligated to pUC:KM74 cut
with Apa I\Cla L Plasmid pKM744 displays kanamycin resistance but
is later found to contain two substitution mutations at positions
1523 and 1634 (mutations are described above in the section on
cloning the fourth quarter, they are substitutions, not deletions
or insertions). The correct first half of the synthetic Bt gene
from plasmid p1HG#6 is inserted into plasmid pKM744. The resulting
plasmid, called pKm124, is made from the about 3.9 Kb Apa I\Bam HI
fragment derived from pKM74-4 ligated to 1 Kb Apa I\Bam HI fragment
from p1HG#6. pKm124 shows kanamycin resistance. This plasmid
contains the first, second, and fourth quarters of the synthetic
gene forming a single open reading frame.
[0277] The third quarter of the synthetic gene is next cloned into
pKM124. The first functional clone, in plasmid pBt:Km#6, is a
functional copy of the truncated synthetic cryIA(b) gene in the
Km-cassette which displays kanamycin resistance but which contains
deletion mutations between the third and fourth quarters. Plasmid
pBt:Km#6 is made from the approximately 5 Kb Apa I\Nco I-pKM124
vector fragment ligated to the approximately 500 bp Apa I\Nco I
fragment from pQCN103 (pQCN103 contains a mismatch mutation at
position 1326 which is repaired later). Contaminating nuclease
activity appears to have deleted the Apa I site between the third
and fourth quarters in pBt:Km#6. The Bt gene encoded by the
synthetic gene in plasmid pBT:Km#6 has about 50-60% of the native
proteins' activity against ECB. The 2 Kb Sma I\Bam HI fragment from
pBt:Km#6 is inserted into a 35S:expression cassette to make a
plasmid called 35S:Bt6.
[0278] Two functional synthetic Bt clones, each with mutations, are
initially obtained: plasmids pBT:KM#6 and pCIB4414. pCIB4414, which
is 100% active in insect bioassays against European corn borer
compared with the native gene, contains substitution mutations in
the third and fourth quarters at positions 1323, 1523, and
1634.
[0279] pCIB4414 is constructed from two plasmids, MG3.G4#18 and 1HG
which is described above. MG3.G4#18 is obtained by cloning the Apa
I/Kpn I fragment in plasmid Bt.P2#1 into pQCN103 (using those same
restriction sites). This produces a plasmid containing the third
and fourth quarters of the gene. The first half of the synthetic
gene from plasmid 1HG is cut with Bam HI and Nco I and moved into
MG3.G4#18 (containing the third and fourth quarters of the gene).
The resulting plasmid, pCIB4414, contains a functional version of
the synthetic gene. While being functional, the synthetic gene in
this plasmid contains three errors; position 1326 (G substituted
for a C), position 1523 (substitute A for a G), and at position
1634 (substitution of a T for a C).
[0280] The fourth quarter in pCIB4414 is replaced with a 354 bp
fourth quarter Apa I\Bst E II fragment obtained from hybridizing,
ligating, and restriction cleaving fourth quarter oligomers as
described earlier, and isolating the fragment from a 2% Nusieve
agarose gel. pCIB4408 is a synthetic Bt gene clone obtained by
replacing the fourth quarter fragment in pCIB4414 with the
hybridized fourth quarter fragment To insert the CaMV 35S promoter
in front of the synthetic Bt gene, pCIB4406 is made from a 4 Kb Eco
NI\Kpn I fragment from plasmid p35SBt6 and 1.8 Kb Eco NI\Kpn I
fragment from pCIB4408.
[0281] pCIB4406 is 100% active (as compared with the protein from
the native gene) against ECB but contains the substitution mutation
in the third quarter of the synthetic gene at position 1323
resulting in an amino acid substitution of a leucine for a
phenylalanine. Plasmid pBS123#13 is used to repair this
mutation.
[0282] The third quarter fragment in plasmid pBS123#13 is made from
an approximately 479 bp hybridized oligomer generated fragment.
Third quarter oligomers U15-U20 and L15-L21 are kinased,
hybridized, and ligated as described above. PCR reactions are
carried out as described above with primers P5(a) and P6(b) for 15
cycles. The PCR product is treated with proteinase K at a final
concentration of about 50 .mu.g\ml in an approximate 95 .mu.l
volume for 30 minutes at 37.degree. C. followed by 10 minutes at
65.degree. C. (Crowe et al., Nucleic Acid Research 19:184, 1991.)
Subsequently, the product is phenol\chloroform extracted and
ethanol precipitated using standard procedures before cutting with
restriction enzymes Apa I and Nco I.
[0283] The approximate 450 bp Apa I\Nco I PCR fragment is ligated
to the 3.8 Kb Apa I\Nco I vector fragment from p1HG#6 to make
pBS123#13. Plasmid pBS123#13 contains the desired sequence for the
third quarter of the maize optimized cryIA(b) gene from position
1319 at the Nsp I site through the Apa I site at position 1493.
This 170 bp Nsp I\Apa I fragment from pBS123#13 is used in the
fully active synthetic cryIA(b) gene in plasmid pCIB4418.
Western Blot Analysis:
[0284] Western blot analyses of various transformants are performed
using crude extracts obtained from E. coli grown on selective
plates. Using a toothpick, cultures are scraped from fresh plates
containing the transformants of interest which have been grown
overnight at 37.degree. C. The positive control for expression of
the Bt gene in E. coli was a construct called pCIB3069 which
contains the native Bt-k gene fused with the plant expressible CaMV
35S promoter. pCIB3069 also contains the 35S promoter operably
linked to the hygromycin resistance gene, 35S promoter, with Adh
intron #1 operably linked to the GUS gene, and 35S promoter
operably linked to a gene coding for the production of the native
Bt cryIA(b) IP. A negative control of E. coli which does not
contain a Bt gene is also included in the analyses. Cultures are
resuspended in 100 .mu.l of loading buffer containing 62 mM
Tris-HCl pH 6.8, 1% SDS, 0.0025% bromophenol blue, 10% glycerol and
7.5% mercaptoethanol. After heating the mixtures at 95.degree. C.
for 10 minutes, the preparations are sonicated for 1-3 seconds. The
debris is centrifuged in a microfuge at room temperature for about
5 minutes and 10 to 15 .mu.l of each sample is loaded onto an
acrylamide gel with a 10% running gel below a 6% stacking gel
(Laemmli, Nature 227; 680-685 (1970)). After electrophoresis
overnight at 10 mAmps, proteins are transferred from the gel to an
Immobilon membrane (Millipore). The transfer is done using an
electrophoretic Blotting Unit (American BioNuclear, Emeryville,
Calif.) in transfer buffer (20 mM Tris, 150 mM glycine, and 20%
methanol) for 1.5 hours at 450 mAmps.
Buffers for Western Blotting Included:
[0285] Blocking Buffer: [0286] 2% Tween-20 [0287] 30 mM Tris-HCl pH
10.2 [0288] 150 mM NaCl
[0289] Wash Buffer: [0290] 0.05% Tween-20 [0291] 30 mM Tris-HCl pH
10.2 [0292] 150 mM NaCl
[0293] Developing Buffer: [0294] 100 mM Tris-HCl pH 9.6 [0295] 100
mM NaCl [0296] 10 mM MgCl2
[0297] After transfer is complete, the membrane is incubated for
about ten minutes in the blocking buffer. Three 15 minute washes
with wash buffer are done before the first antibody treatment. The
first antibody is an immunoaffinity purified rabbit or goat
antibody prepared using the CryIA(b) protein as the antigen
(Ciba-Geigy, RTP, N.C.; Rockland Inc., Gilbertsville, Pa.; and
Berkeley Antibody CO., Richmond, Calif.). The cryIA(b) specific
antibody is treated immediately before use with E. coli lysate from
Bio-Rad in a 1 ml volume with 5 .mu.g of antibody, 50 .mu.l of E.
coli lysate in the wash buffer solution. This mixture is incubated
for 1 hour at room temperature before diluting it 1 to 30 for a
final dilution of 1:6000 with wash buffer. Incubation of the
membrane with the first antibody is at room temperature for 1.5
hours.
[0298] Three 10 minute washes are done between the 1st and 2nd
antibody treatments. The second antibody is either rabbit anti-goat
or goat anti-rabbit/alkaline phosphatase conjugate (Sigma, St.
Louis, Mo.). Incubation with the alkaline phosphatase conjugate is
carried out at room temperature for one hour using a 1 to 6000
dilution in wash buffer. Six 10 minute washes are done between the
second antibody treatment and developing the western blot. The
western blot is developed in 100 ml of developing buffer with 440
.mu.l of nitroblue tetrazolium in 70% o dimethyl formamide (75
mg\ml), and 330 .mu.l of 5-bromo-4-chloro-indolyl-phosphate in 100%
dimethyl formamide (50 mg\ml). After developing for 15 to 30
minutes, the membrane is washed in water and air dried.
Example 4
Construction of Transformation Vectors
Construction of pCIB710 and Derivatives.
[0299] CaMV 35S Promoter Cassette Plasmids pCIB709 and pCIB710 are
constructed as shown in Rothstein et al., Gene 53:153-161 (1987).
pCIB710 contains CaMV promoter and transcription termination
sequences for the 35S RNA transcript (Covey et al., Nucl. Acids.
Res., 9:6735-6747 (1981)). A 1149 bp BglII restriction fragment of
CaMV DNA (bp 6494-7643 in Hohn et al., Current Topics in
Microbiology and Immunology, 96:194-220 and Appendices A to G
(1982)) is isolated from CaMV DNA by preparative agarose gel
electrophoresis as described earlier The fragment is mixed with
BamHI-cleaved plasmid pUC19 DNA, treated with T4 DNA ligase, and
transformed into E. coli. (Note the BamHI restriction site in the
resulting plasmid is destroyed by ligation of the Bg1II cohesive
ends to the BamHI cohesive ends.)
[0300] The resulting plasmid, called pUC19/35S, is then used in
oligonucleotide-directed in-vitro mutagenesis to insert the BamHI
recognition sequence GGATCC immediately following CaMV nucleotide
7483 in the Hohn reference. The resulting plasmid, pCIB710,
contains the CaMV 35S promoter region and transcription termination
region separated by a BamHI restriction site. DNA sequences
inserted into this BamHI site will be expressed in plants by these
CaMV transcription regulation sequences. (Also note that pCIB710
does not contain any ATG translation initiation codons between the
start of transcription and the BamHI site).
[0301] pCIB710 is modified to produce pCIB709 by inserting a Bam HI
fragment containing the coding sequence for hygromycin
phosphotransferase from pLG90 (Rothstein et al., Gene, 53:153-161
(1987)) in the Bam HI site.
[0302] pCIB709 is modified to produce pCIB996 by removing the ATG
just upstream from the initiation codon of the hygromycin
phosphotranserase gene using standard mutagenesis techniques while
inserting a Bgl II restriction site at this location. The resulting
plasmid, pCIB996, is further modified to remove the Bam HI, Sma I
and Bgl II sites in the 5' untranslated leader region located 5' of
the initiation codon for the initiation codon. The result is a
change of DNA base sequence from -TATAAGGATC CCGGGGGCA AGATCTGAGA
TATG (SEQ ID NO:59)-Hyg to -TATAAGGATC TGAGATATG (SEQ ID NO:59 with
nucleotides 11-24 deleted)-Hyg. The resulting plasmid is known as
pCIB3073.
[0303] Alternatively, pCIB710 is modified to produce pCIB900, by
inserting the Bam HI-Bcl I fragment of pCIB10/35SBt, which contains
the 645 amino acid Bt coding sequence, described in Part C4 below,
into the Bam HI site of pCIB710 to create pCIB710/35SBt. To
introduce an antibiotic resistance marker, pCIB709 is cut with Sal
I, a Kpn I/Sal I adaptor is ligated and the resulting ligation
product is cut with Kpn I. The Kpn fragment of pCIB709 containing
the 35S/hygromycin resistance gene is inserted into the Kpn I site
of pCIB710/35SBt to produce pCIB900.
[0304] Genes useful as the selectable marker gene include the
hygromycin resistance gene described in Rothstein et al., Gene 53:
153-161 (1987). The hygromycin gene described in this reference is
moved into a pUC plasmid such as pCIB710 or pCIB709 and the "extra"
ATG upstream from the hygromycin phosphotransferase coding sequence
is removed to create pCIB996. This modified pCIB996 gene is further
modified to remove a BglII, BamHI and SmaI sites from the 5' region
of the gene using standard techniques of molecular biology to make
pCIB3073.
[0305] pCIB932 is a pUC19-based plasmid containing the chimeric
gene Pep-C:promoter\Bt\Pep-C:terminator. It is composed of
fragments derived from pPEP-10, a HindIII subclone of a genomic
clone, H1-lambda-14, PNAS USA, 83:2884-2888 (1986), of the maize
gene encoding the PEP carboxylase enzyme active in photosynthesis,
and from pCIB930, which is a BamHI fragment containing the 645
amino acid truncated form of the the cryIAb endotoxin gene in the
BamHI site of pUC18.
[0306] The 2.6 kb EcoRI-XhoI fragment from pPEP-10, containing the
polyA addition site from the PEP carboxylase gene, is isolated and
digested with PstI and HincII. The restriction digest is ligated
with PstI/HincII digested pUC18, transformed into E. coli and
transformants screened for those containing a 412 bp PstI-HincII
insert in pUC18 and the insert verified by sequencing. The
resulting plasmid is called pCIB931.
[0307] The nuclear gene encoding the phosphoenolpyruvate
carboxylase isozyme ("Pep-C") is described in Hudspeth et al.,
Plant Molecular Biology, 12: 579-589 (1989). pCIB932 is constructed
by the ligation of three fragments. The first fragment, containing
the PEP-C transcription terminator, is produced by digesting
pCIB931 to completion with HindIII, partially with SphI and the
3098 bp fragment isolated. The second fragment, containing the Bt
endotoxin coding sequence, is produced by digesting pCIB930 with
NcoI and SphI and isolating the 1950 bp fragment. The third
fragment, containing the PEP-C promoter, is produced by digesting
pPEP-10 to completion with HindIII, partially with NcoI and
isolating the 2.3 kb fragment. The ligation mix is transformed into
E. coli, transformants with the correct insertion identified and
the insert verified by sequencing.
[0308] pCIB932 is cut with PvuII to generate a 4.9 Kb fragment
containing the maize Pep-C:promoter\Bt\Pep-C:terminator and
purified on a 1% LGT agarose gel in 1.times.TAE. The linearized
pCIB3079 vector and the 4.9 Kb insert from pCIB932 are ligated
using T4 DNA ligase in LGT to make pCIB4401. pCIB4401 is a maize
transformation vector containing the chimeric genes:
35S:promoter\PAT\35S:terminator, Pep-C:promoter\Bt\Pep-C:
terminator, and 35S:promoter\AdhI #1 intron\GUS\35S:
terminator.
Construction of pCIB246 (35S-GUS-35S)
[0309] A CaMV 35S promoter cassette, pCIB246, is constructed as
follows.
[0310] The DdeI restriction site at nucleotide position 7482 of the
CaMV genome (Franck et al., Cell, 21:285-294 (1980)) is modified by
insertion of a 48 bp oligonucleotide containing several restriction
enzyme sites including an NcoI (CCATGG) site, a SalI (GTCGAC) site,
and an SstI (GAGCTC) site. This altered CaMV 35S promoter is
inserted into a pUC19 vector that had been modified to destroy the
vector's SstI and SalI sites. Thus, the CaMV 35S promoter of
pCIB1500 contains unique SstI and SalI sites for cloning.
[0311] pCIB1500 is digested with SstI/NcoI and ligated with the GUS
gene obtained from pBI221 (Clontech Laboratories, Inc., Palo Alto,
Calif.). The NcoI site is fused to the GUS gene such that the ATG
of the NcoI site functions as the start codon for the translation
of the GUS gene. The CaMV 35S polyadenylation and termination
signals are used for the 3' end of the chimeric gene.
Construction of pCIB3069 (35S-Adh1-GUS-35S)
[0312] pCIB246 is modified by adding the maize alcohol
dehydrogenase gene Adh1 intron number 1 (Adh1) (Dennis et al.,
Nucleic Acids Research, 12:3983-4000 (1984)) into the Sal I site of
pCIB246 to produce plasmid pCIB3007. The Adh1 intron is excised
from the maize Adh1 gene as a Bal I/Pst I fragment and subcloned
into pUC18 that was cut with Sma I/Pst I to make a plasmid called
Adh 1026. Adh 1026 is cut with Pvu II/Sac II, the fragments are
made blunt ended with T4 DNA polymerase, Sal I linkers are added
using standard procedures and a fragment of about 560 bp is
recovered from a 3% NuSeive gel and ligated into Sal I
cut/phosphatase treated pUC18. The Sal I linkered Adh intron #1 in
the resulting plasmid is cut out with Sal I, gel purified, and
ligated into Sal I cut/phosphatase treated pCIB246 to make plasmid
pCIB3007.
[0313] pCIB3007 is cut with PstI and the ends made blunt by using
T4 DNA polymerase (NEW England Biolabs) according to the suppliers'
specifications. The resulting blunt ended molecules are cut with
Sph I and the approximately 5.8 Kb fragment with one blunt end and
one Sph I end is purified on a low gelling temperature (LGT)
agarose gel using standard procedures. pCIB900 is cut with Sma
I/Sph I and the fragment containing the 35S/Bt gene is purified on
a LGT agarose gel. The two gel purified fragments are ligated in
LGT agarose using T4 DNA ligase according to standard conditions.
The resulting ligated fragments are transformed into E. coli using
standard procedures and the resulting plasmid is called pCIB3062.
There are two versions of pCIB3062. pCIB3062#1 has a Sma I site
regenerated where the Sma I site and the T4 polymerase blunted ends
are ligated. This most likely results from the T4 polymerase
nibbling a few base pairs from the Pst I site during the blunting
reaction. pCIB3062#3 does not have this SmaI site.
[0314] pCIB3062#3 is cut with KpnI and made blunt-ended using T4
DNA polymerase, and subsequently cut with Pvu II to yield a 6.4 Kb
fragment with blunt ends containing the 35S/GUS and 35S/Bt genes.
This blunt-end fragment is ligated into Sma I cut pCIB3073 to
produce pCIB3063 or pCIB3069. pCIB3069 contains the same fragment
used to make pCIB3063, but the chimeric genes in pCIB3069 are all
in the same relative orientation, unlike those in pCIB3063. These
plasmids contain a) a 35S promoter operably linked to the
hygromycin resistance gene; b) a 35S promoter, with Adh intron #1,
operably linked to the GUS gene; and c) a 35S promoter operably
linked to a gene coding for the production of the synthetic
cryIA(b) insecticidal protein from Bacillus thuringiensis, as
described above.
GUS Assays:
[0315] GUS assays are done essentially as described in Jefferson,
Plant Mol. Bio. Reporter, 5:387-405 (1987). As shown above, plasmid
pCIB246 contains a CaMV 35S promoter fused with the GUS gene. The
5' untranslated leader of this chimeric gene contains a copy of the
maize Adh1 intron #1. It is used here as a transformation control.
Although the same amount of pCIB246 is added to each
transformation, the calculated activity varied among Bt constructs
tested. The values reported below are averages of 3 replicates.
pCIB4407 was tested twice. TABLE-US-00012 pCIB3069 28 nM MU/ug/min
pCIB4407 0.7 nM MU/ug/min, 2.3 nM MU/ug/min
Example 5A
Assay of Synthetic cryIA(b) Gene for Insecticidal Activity Against
European Corn Borer
[0316] The synthetic cryIA(b) gene in pCIB4414 in E. coli is
assayed for insecticidal activity against European corn borer
according to the following protocol.
[0317] Molten artifical insect diet is poured into a 60 mm Gellman
snap-cap petri dish. After solidification, E. coli cells, suspended
in 0.1% Triton X-100, are spread over the surface at a
concentration of 3.times.107 cells/cm2. The plates are air dried.
Ten first instar European corn borer, Ostrinia nubilalis, which are
less than 12 hours old are then placed onto the diet surface. The
test is incubated at 30 C in complete darkness for 2-5 days. At the
end of the test percent mortality is recorded. A positive clone has
been defined as one giving 50% or higher mortality when control E.
coli cells give 0-10% background mortality.
[0318] For comparison, the native cryIA(b) gene in pCIB3069 is
tested at the same concentration. Clones are tested at
3.times.10.sup.7 cells/cm.sup.2 diet; 20 insects per clone.
[0319] The following results are observed: TABLE-US-00013 Clone
Percent Mortality Control 0 pCIB3069 100 pCIB4414 100
[0320] These results indicate that the insecticidal crystal protein
produced by the synthetic cryIA(b) gene demonstrates activity
against European corn borer comparable to that of the IP produced
by the native cryIA(b). Other plasmids containing a synthetic
cryIA(b) gene were assayed in a similar manner.
Example 5B
Assay of CryIA(b) Protein for Insecticidal Activity Against
Sugarcane Borer
[0321] CryIA(b) was expressed in E. coli and assayed for
insecticidal activity against Sugarcane borer (Diatrea saccharalis)
according to the same protocol used for European corn borer,
described immediately above. The results are summarized in the
Table. TABLE-US-00014 TABLE SUGARCANE BORER ASSAY WITH Bt PROTEIN
FROM E. COLI Protein Concentration (ng/g) Percent Mortality
CryIA(b) 10 0 25 0 50 7 100 13 250 40 500 53 1000 80 LC50 380 95%
Cl 249-646
[0322] The results indicate that the insecticidal protein produced
by a maize optimized Bt gene is effective against Sugarcane borer.
The upper concentrations of CryIA(b) protein, 250 ng/g-1000 ng/g,
are achievable in transgenic maize plants produced in accordance
with the instant invention.
Example 6
Maize Protoplast Isolation and Transformation with the Synthetic Bt
Gene
[0323] Expression of the synthetic Bt gene is assayed in
transiently transformed maize protoplasts.
Protoplast Isolation Procedure:
[0324] 1. The contents of 10 two day old maize 2717 Line 6
suspension cultures are pipetted into 50 ml sterile tubes and
allowed to settle. All culture media is then removed and
discarded.
[0325] 2. Cells (3-5 ml Packed Cell Volume) are resuspended in 30
ml protoplast enzyme solution. Recipe follows:
[0326] 3% Cellulase RS
[0327] 1% Macerozyme R10 in KMC Buffer
[0328] KMC Buffer (Recipe for 1 Liter) TABLE-US-00015 KCl 8.65 g
MgCl.sub.2--6H.sub.2O 16.47 g CaCl.sub.2--2H.sub.2O 12.50 g MES 5.0
g
[0329] pH 5.6, filter sterilize
[0330] 3. Mix cells well and aliquot into 100.times.25 mm petri
dishes, about 15 ml per plate. Shake on a gyratory shaker for 4
hours to digest.
[0331] 4. Pipette 10 ml KMC through each 100 micron sieve to be
used. Filter contents of dishes through sieve. Wash sieve with an
equal volume KMC.
[0332] 5. Pipette sieved protoplasts carefully into 50 ml tubes and
spin in a Beckman TJ-6 centrifuge for 10 minutes at 1000 rpm
(500.times.g).
[0333] 6. Remove supernatant and resuspend pellet carefully in 10
ml KMC. Combine contents of 3 tubes into one and bring volume to 50
ml with KMC.
[0334] 7. Spin and wash again by repeating the above step.
[0335] 8. Resuspend all washed protoplasts in 50 ml KMC. Count in a
hemocytometer. Spin protoplasts and resuspend at
8.times.10.sup.6/ml in resuspending buffer (RS Buffer).
RS Buffer (Recipe for 500 ml)
[0336] mannitol 27.33 g
[0337] CaCl.sub.2 (0.1 M stock) 75 ml
[0338] MES 0.5 g
[0339] pH 5.8, filter sterilize
Protoplast Transformation Procedure:
[0340] 1. Aliquot 50 .mu.g plasmid DNA (Bt IP constructs, both
synthetic (pCIB4407) and native (pCIB3069)) to 15 ml polystyrene
culture tubes. Also aliquot 25 .mu.g GUS-containing plasmid DNA
(which does not contain Bt IP (pCIB246) to all tubes. 3
replications are used per construct to be tested, with 1 rep
containing no DNA as a control. TABLE-US-00016 Bt constructs: GUS
construct: pCIB3069 pCIB246 pCIB4407
[0341] 2. Gently mix protoplasts well and aliquot 0.5 ml per
tube.
[0342] 3. Add 0.5 ml PEG-40 to each tube. PEG-40:
[0343] 0.4 M mannitol
[0344] 0.1 M Ca(NO.sub.3).sub.2-4H.sub.2O
[0345] pH 8.0, filter sterilize
[0346] 4. Mix gently to combine protoplasts with PEG. Wait 30
minutes.
[0347] 5. Sequentially add 1 ml, 2 ml, and 5 ml W5 solution at 5
minute intervals.
W5 Solution:
[0348] 154 mM NaCl
[0349] 125 mM CaCl.sub.2-H20
[0350] 5 mM KCl
[0351] 5 mM glucose
[0352] pH 7.0, filter sterilize
[0353] 6. Spin for 10 minutes in a Beckman TJ-6 centrifuge at about
1000 rpm (500 g). Remove supernatant.
[0354] 7. Gently resuspend pellet in 1.5 ml FW media and plate
carefully in 35.times.10 mm petri dishes.
[0355] FW Media (Recipe for 1 Liter): TABLE-US-00017 MS salts 4.3 g
200X B5 vits. 5 ml sucrose 30 g proline 1.5 g mannitol 54 g 2,4 D 3
mg
[0356] pH 5.7, filter sterilize
[0357] 8. Incubate overnight in the dark at room temperature.
[0358] 9. Perform GUS assays, insect bioassays, and ELISA's on
protoplast extracts as described below.
Example 7
Construction of a Full-Length Synthetic Maize Optimized cryIA(b)
Gene
[0359] SEQ ID NO:4 shows the synthetic maize optimized sequence
encoding the full-length cryIA(b) insecticidal protein from B.
thuringiensis. The truncated version described above represents the
first approximately 2 Kb of this gene. The remainder of the
full-length gene is cloned using the procedures described above.
Briefly, this procedure entails synthesizing DINA oligomers of 40
to 90 NT in length, typically using 80 mers as an average size. The
oligomers are purified using standard procedures of HPLC or
recovery from a polyacrylamide gel. Purified oligomers are kinased
and hybridized to form fragments of about 500 bp. The hybridized
oligomers can be amplified using PCR under standard conditions. The
500 bp fragments, either directly from hybridizations, from PCR
amplification, or recovered from agarose gels after either
hybridization or PCR amplification, are then cloned into a plasmid
and transformed into E. coli using standard procedures. Recombinant
plasmids containing the desired inserts are identified, as
described above, using PCR and/or standard miniscreen procedures.
Inserts that appear correct based upon their PCR and/or restriction
enzyme profile are then sequenced to identify those clones
containing the desired open reading frame. The fragments are then
ligated together with the approximately 2 Kb synthetic sequence
described in Example 2 to produce a full-length maize optimized
synthetic cryIA(b) gene useful for expression of high levels of
CryIA(b) protein in maize.
[0360] G+C Content of Native and Synthetic Bt Genes: TABLE-US-00018
Full-length native 38.8% Truncated native 37.2% Full-length
synthetic 64.8% Truncated synthetic 64.6%
% homology of the final truncated version of the Bt gene relative
to a "pure" maize codon usage gene: 98.25%
Example 8
Construction of a Plant Expressible, Full-Length, Hybrid Partially
Maize Optimized cryIA(b) Gene
[0361] pCIB4434 contains a full length CryIA(b) gene (SEQ ID NO:8)
comprised of about 2 Kb of the synthetic maize optimized cryIA(b)
gene with the remainder (COOH terminal encoding portion) of the
gene derived from the native gene. Thus, the coding region is a
chimera between the synthetic gene and the native gene, but the
resulting protein is identical to the native cryIA(b) protein. The
synthetic region is from nucleotide 1-1938 (amino acids 1 to 646)
and the native coding sequence is from nucleotide 1939-3468 (amino
acids 647 to 1155). The sequence of this gene is set forth in FIG.
7. A map of pCIB4434 is shown in FIG. 8.
[0362] The following oligos were designed to make pCIB4434:
TABLE-US-00019 (SEQ ID NO:60) KE134A28 = 5'-CGTGACCGAC TACCACATCG
ATCAAGTATC CAATTTAGTT GAGT-3' (SEQ ID NO:61) KE135A28 =
5'-ACTCAACTAA ATTGGATACT TGATCGATGT GGTAGTCGGTC ACG-3' (SEQ ID
NO:62) KE136A28 = 5'-GCAGATCTGA GCTCTTAGGT ACCCAATAGC GTAACGT-3'
(SEQ ID NO:63) KE137A28 = 5'-GCTGATTATG CATCAGCCTAT-3' (SEQ ID
NO:64) KE138A28 = 5'-GCAGATCTGA GCTCTTATTC CTCCATAAGA AGTAATTC-3'
(SEQ ID NO:65) MK05A28 = 5'-CAAAGGTACC CAATAGCGTA ACG-3' (SEQ ID
NO:66) MK35A28 = 5'-AACGAGGTGT ACATCGACCG-3'
[0363] pCIB4434 is made using a four-way ligation with a 5.7 kb
fragment from pCIB4418, a 346 bp Bst E II\Kpn I PCR-generated
synthetic:native fusion fragment, a 108 bp Kpn I\Nsi I native
CryIA(b) fragment from pCIB1315, and a 224 bp Nsi I\Bgl II
PCR-generated fragment. Standard conditions for ligation and
transformation are as described previously.
[0364] A synthetic:native gene fusion fragment is made in two steps
using PCR. The first 253 bp of the PCR fusion fragment is made
using 100 pmols of oligos KE134A28 and MK04A28 with approximately
200 ng of native cryIA(b) template in a 100 ul volume with 200 nm
of each dNTP, 1.times.PCR buffer (Perkin Elmer Cetus), 20%
glycerol, and 5 units of Taq polymerase (Perkin Elmer Cetus). The
PCR reaction is run with the following parameters: 1 minute at
94.degree. C., 1 minute at 55.degree. C., 45 seconds at 72.degree.
C., with extension 3 for 3 seconds for 25 cycles. A fraction (1%)
of this first PCR reaction is used as a template along with 200 ng
of the synthetic cryIA(b) DNA to make the complete 351 bp
synthetic:native fusion fragment. Oligos used as PCR primers in
this second PCR reaction are 50 pmols of MK5A28, 50 pmols of
MK04A28, and 25 pmols of KE135A28. The PCR reaction mix and
parameters are the same as those listed above. The resultant 351 bp
synthetic:native fusion fragment is treated with Proteinase K at 50
ug\ml total concentration and phenol\chloroform extraction followed
by ethanol precipitation before cutting with Bst E II\Kpn I using
standard conditions.
[0365] The 224 bp Nsi I\Bgl II PCR fragment used in making pCIB4434
is made using 100 pmols of oligos KE137A28 and KE138A28 and 200 ng
of the native cryIA(b) gene as template in 100 ul volume with the
same PCR reaction mix and parameters as listed above. The 230 bp
PCR native cryIA(b) fragment is treated with Proteinase K,
phenol\chloroform extracted, and ethanol precipitated as described
above, before cutting with Nsi I\Bgl II.
[0366] pCIB4434 was transformed into maize protoplasts as described
above. Line 6 2717 protoplasts were used with pCIB4434 and pCIB4419
as a control for comparison. The results are shown below:
TABLE-US-00020 ng Bt/mg protein 4419(35S) 14,400 .+-. 2,100
4434(full-length) 2,200 .+-. 900
Background=13 ng Bt/mg protein for untransformed protoplasts
[0367] The results indicate that pCIB4434 expresses at a level of
about 15% of pCIB4419.
[0368] Western blot analysis shows at least one-third of the
cryIA(b) protein produced by pCIB4434 in this system is about 130
kD in size. Therefore, a significant amount of full-length cryIA(b)
protein is produced in maize cells from the expression of
pCIB4434.
Example 8A
Construction of a Full-Length, cryIA(b) Genes Encoding a
Temperature-Stable cryIA(b) Protein
[0369] Constructs pCIB5511-5515, each containing a full-length,
cryIA(b) gene are described below. In these sequences, the 26 amino
acid deletion between amino acids 793 and 794,
KCGEPNRCAPHLEWNPDLDCSCRDGE (see: SEQ ID NOS:8, 10, 12, 14, 16),
present in cryIA(a) and cryIA(c) but not in cryIA(b), has been
repaired. The gene in pCIB5513 is synthetic; the other four genes
are hybrids, and thus are partially maize optimized.
Construction of pCIB5511
[0370] This plasmid is a derivative of pCIB4434. A map of pCIB5511
is shown in FIG. 10. A 435 bp segment of DNA between bp 2165 and
2590 was constructed by hybridization of synthetic oligomers
designed to represent the upper and lower strand as described above
for the construction of the truncated cryIA(b) gene. This segment
of synthetic DNA is synthesized using standard techniques known in
the art and includes the 26 amino acid deletion found to occur
naturally in the cryIA(b) protein in Bacillus thuringiensis
kurstaki HD-1. The entire inserted segment of DNA uses maize
optimized codon preferences to encode amino acids. The 26 amino
acids used to repair the naturally occurring deletion are contained
within this fragment. They are inserted starting at position 2387
between the KpnI site at nt 2170 and the XbaI site at nt 2508 (2586
in pCIB5511) of pCIB4434. pCIB5511 is constructed via a three way
ligation using a 3.2 Kb fragment obtained by restriction digestion
of pCIB4434 with SphI and KpnI, a 3.8 Kb fragment obtained by
digestion of pCIB4434 with SphI and XbaI, and a 416 bp fragment
obtained by digestion of the synthetic DNA described above, with
KpnI and XbaI. Enzymatic reactions are carried out under standard
conditions. After ligation, the DNA mixture is transformed into
competent E. coli cells using standard procedures. Transformants
are selected on L-agar containing 100 .mu.g/ml ampicillin. Plasmids
in transformants are characterized using standard mini-screen
procedures. The sequence of the repaired cryIA(b) gene encoding the
cryIA(b) temperature (heat) stable protein is set forth in FIG. 9
(SEQ ID NO:10).
Construction of pCIB5512
[0371] This plasmid construct is a derivative of pCIB4434. A map of
pCIB5512 is shown in FIG. 12. DNA to repair the 26 amino acid
deletion is prepared using standard techniques of DNA synthesis and
enzymatic reaction. Three double stranded DNA cassettes, pGFcas1,
pGFcas2 and pGFcas3, each about 300 bp in size, are prepared. These
cassettes are designed to contain the maize optimized codons while
maintaining 100% amino acid identity with the insecticidal protein.
These cassettes are used to replace the region between restriction
site BstEII at position 1824 and XbaI at position 2508 and include
the insertion of the additional 78 bp which encode the missing 26
amino acids (described above for pCIB5511 in pCIB4434). Each of
these cassettes is cloned into the EcoRV site of the vector
Bluescript (Stratagene) by standard techniques. The three cassettes
are designed to contain overlapping restriction sites. Cassette 1
has restriction sites BstEII at the 5' end and EcoRV at the 3' end:
cassette 2 has EcoRV at the 5' end and ClaI at the 3' end and
cassette 3 has ClaI at the 5' end and Xba I at the 3' end. They are
cloned individually in Bluescript and the the complete 762 bp
fragment is subsequently assembled by ligation using standard
techniques. pCIB5512 is assembled using this 762 bp fragment and
ligating it with a 6.65 Kb fragment obtained by a complete
digestion of pCIB4434 with BstEII and a partial digestion with
XbaI. Alternatively, a four way ligation using the same vector and
the three cassettes digested with the specific enzymes can be
employed. Enzymatic reactions are carried out under standard
conditions. After ligation, the DNA mixture is transformed into
competent E. coli cells using standard procedures. Transformants
are selected on L-agar containing 100 .mu.g/ml ampicillin. Plasmids
in transformants are characterized using standard mini-screen
procedures. The resulting plasmid is pCIB5512. The sequence of the
repaired cryIA(b) gene is illustrated in FIG. 11 (SEQ ID NO:12).
This repaired cryIA(b) differs from that carried in pCIB5511 in
that a larger region of the cryIA(b) coding region is optimized for
maize expression by using maize preferred codons.
Construction of pCIB5513
[0372] This plasmid contains a repaired cryIA(b) gene derived from
pCIB5512. A map of pCIB5513 is shown in FIG. 14. The region 3' from
the XbaI site at position 2586 to the end of the gene (BglII site
at position 3572) is replaced entirely with maize optimized codons.
This region is synthesized, using standard techniques of DNA
synthesis and enzymatic reaction, well known in the art, as four
double stranded DNA cassettes (cassettes # 4, 5, 6, 7). Adjacent
cassettes have overlapping restriction sites to facilitate assembly
between cassettes. These are XbaI and XhoI at the 5' and 3' ends of
cassette 4; XhoI and SacI at the 5' and 3' ends, respectively, of
cassette 5; SacI and BstXI at the 5' and 3' ends, respectively, of
cassette 6; and BstXI and BglII at the 5' and 3' ends,
respectively, of cassette 7. As described for pCIB5512, the
cassettes are cloned into the blunt-end EcoRV site of the
Bluescript vector (Stratagene) and the full-length "repaired"
cryIA(b) gene cloned either by sequential assembly of the above
cassettes in Bluescript followed by ligation of the complete 967 bp
synthetic region with a 6448 bp fragment obtained by a complete
digestion of pCIB5512 with BglII and a partial digestion with XbaI.
Alternately, the plasmid containing the full-length genes is
obtained by a 5-way ligation of each of the four cassettes (after
cleavage with the appropriate enzymes) and the same vector as
above. The sequence of the full-length, "repaired" cryIA(b) gene is
set forth in FIG. 13 (SEQ ID NO:14). The protein encoded by the
various synthetic and synthetic/native coding region chimeras
encode the same protein. This protein is the heat-stable version of
cryIA(b) produced by repairing the naturally occurring 26 amino
acid deletion found in the cryIA(b) gene from Bacillus
thuringiensis kurstaki HD-1 when the homologous region is compared
with either cryIA(a) or cryIA(c) Bacillus thuringiensis
delta-endotoxins.
Construction of pCIB5514
[0373] This plasmid is a derivative of pCIB4434. A map of pCIB5514
is shown in FIG. 16. It is made using synthetic DNA cassette #3
(see above) which contains a maize optimized sequence of the region
between the ClaI site (position 2396) found in the 26 amino acid
thermostable region and the XbaI site at position 2508 in pCIB4434
(2586 in pCIB5511). The region between nt 2113 of pCIB4434 and the
junction of the thermostable region is PCR amplified by using
pCIB4434 as template with the following primers: TABLE-US-00021
forward: (SEQ ID NO:67)
5'GCACCGATATCACCATCCAAGGAGGCGATGACGTATTCAAAG-3' reverse: (SEQ ID
NO:68) 5'-AGCGCATCGATTCGGCTCCCCGCACTTGCCGATTGGACTTGGGGCTG
AAAG-3'.
[0374] The PCR product is then digested with restriction enzymes
KpnI and ClaI and ligated in a four part reaction with a 189 bp
fragment obtained by digestion of cassette 3 with ClaI and XbaI, a
3.2 Kb fragment of pCIB4434 digested with SphI and KpnI, and a 3.8
Kb fragment of pCIB4434 obtained by digestion with SphI and Xba.
Enzymatic reactions are carried out under standard conditions. The
ligation product is transformed into competent E coli cells,
selected with ampicillin and screened using standard procedures
described above. The sequence of the repaired cryIA(b) gene
contained in pCIB5514 is shown in FIG. 15 (SEQ ID NO:16).
[0375] pCIB4434 was modified by adding the 78 bp Geiser
thermostable element (Geiser TSE), described above, between the Kpn
I site (2170 bp) and the Xba I site (2508 bp) in the native Btk
region. The exact insertion site starts at the nucleotide #2379.
The region containing the Geiser TSE was amplified by two sets of
PCR reactions, i.e. the Kpn I-Geiser TSE fragment and the Geiser
TSE-Xba I fragment. TABLE-US-00022 PCR primer#1: (Kpn I site) (SEQ
ID NO:69) 5'- ATTACGTTAC GCTATTGGGT ACCTTTGATG -3' PCR primer#2:
(Geiser TSE bottom) (SEQ ID NO:70) 5'- TCCCCGTCCC TGCAGCTGCA
GTCTAGGTCC GGGTTCCACT CCAGGTGCGG AGCGCATCGA TTCGGCTCCC CGCACTTGCC
GATTGGACTT GGGGCTGA -3' PCR primer#3: (Geiser TSE top) (SEQ ID
NO:71) 5'- CAAGTGCGGG GAGCCGAATC GATGCGCTCC GCACCTGGAG TGGAACCCGG
ACCTAGACTG CAGCTGCAGG GACGGGGAAA AATGTGCCCA TCATTCCC -3' PCR
primer#4: (Xba I site) (SEQ ID NO:72) 5'- TGGTTTCTCT TCGAGAAATT
CTAGATTTCC -3'
[0376] After the amplification, the PCR fragments were digested
with (Kpn I+Cla I) and (Cla I+Xba I), respectively. These two
fragments were ligated to the Kpn I and Xba I digested pCIB4434.
The resulting construct pCIB5515 is pCIB4434 with a Geiser TSE and
an extra Cla I site flanked by Kpn I and Xba I. A map of pCIB5515
is illustrated in FIG. 38. The cryIA(b) gene contained herein,
which encodes a temperature stable cryIA(b) protein, is shown in
FIG. 37 (SEQ ID NO:27).
[0377] Examples 9-20 set forth below are directed to the isolation
and characterization of a pith-preferred promoter.
Example 9
RNA Isolation and Northern Blots
[0378] All RNA was isolated from plants grown under greenhouse
conditions. Total RNA was isolated as described in Kramer et al.,
Plant Physiol., 90:1214-1220 (1990) from the following tissues of
Funk maize line 5N984: 8, 11, 15, 25, 35, 40, and 60 day old green
leaves; 8, 11, 15, 25, 35, 39, 46, 60 and 70 day old pith; 60 and
70 day old brace roots from Funk maize line 5N984; 60 and 70 day
5N984 sheath and ear stock. RNA was also isolated from 14 day 211D
roots and from developing seed at weekly intervals for weeks one
through five post-pollenation. Poly A+ RNA was isolated using
oligo-dT as described by Sambrook et al., Molecular Cloning: A
Laboratory Manual (2nd ed.), 1989, and Northern blots were carried
out, also as per Sambrook et al. using either total RNA (30 .mu.g)
or poly A+ RNA (2-10 Ag). After electrophoresis, RNA was blotted
onto Nitroplus 2000 membranes (Micron Separations Inc). The RNA was
linked to the filter using the Stratalinker (Stratagene) at 0.2
mJoules. The northerns were probed with the 1200 bp EcoRI pith
(TRpA) 8-2 cDNA fragment, isolated by using 0.8% low melting
temperature agarose in a TBE buffer system. Northerns were
hybridized and washed and the filters exposed to film as described
in Isolation of cDNA clones.
Example 10
Isolation of cDNA Clones
[0379] First strand cDNA synthesis was carried out using the BRL
AMV reverse transcriptase system I using conditions specified by
the supplier (Life Technologies, Inc., Gaithersburg, Md.).
Specifically, 25 .mu.l reactions containing 50 mM Tris-HCl pH 8.3,
20 mM KCl, 1 mM DTT, 6 mM MgCl2, 1 mM each of each dNTP, 0.1 mM
oligo (dT)12-18, 2 .mu.g pith poly(A+) RNA, 100 .mu.g/ml BSA, 50
.mu.g/ml actinomycin D, 8 units placental RNase inhibitor, 1 .mu.l
(10 mM Ci/ml) 32P dCTP >3000 mCi/mM as tracer, and 30 units AMV
reverse transcriptase were incubated at 42.degree. C. for 30 min.
Additional KCl was added to a concentration of 50 mM and incubation
continued a further 30 min. at 42.degree. C. KCl was added again to
yield a final concentration of 100 mM. Additional AMV reverse
transcriptase reaction buffer was added to maintain starting
concentrations of the other components plus an additional 10 units,
and the incubation continued at 42.degree. C. for another 30 min.
Second strand synthesis was completed using the Riboclone cDNA
synthesis system with Eco RI linkers (Promega, Madison, Wis.).
Double stranded cDNA was sized on an 1% agarose gel using
Tris-borate-EDTA buffer as disclosed in Sambrook et al., and showed
an average size of about 1.2 Kb. The cDNA was size fractionated
using NA45 DEAE membrane so as to retain those molecules of about
1000 bp or larger using conditions specified by the supplier
(Schleicher and Schuell). Size fractionated cDNA was ligated into
the Lambda ZapII vector (Stratagene, La Jolla, Calif.) and packaged
into lambda particles using Gigapack II Plus (Stratagene, La Jolla,
Calif.). The unamplified library had a titer of 315,000 pfu while
the amplified library had a titer of 3.5 billion/ml using PLK-F
cells.
[0380] Recombinant phage were plated at a density of 5000 pfu on
150.times.15 mm L-agar plates. A total of 50,000 phage were
screened using duplicate lifts from each plate and probes of first
strand cDNA generated from either pith derived mRNA or seed derived
mRNA. The lifts were done as described in Sambrook et al. using
nitrocellulose filters. DNA was fixed to the filters by UV
crosslinking using a Stratalinker (Stratagene, La Jolla, Calif.) at
0.2 mJoule. Prehybridization and hybridization of the filter were
carried out in a solution of 10.times. Denhardts solution, 150
.mu.g/ml sheared salmon sperm DNA, 1% SDS, 50 mM sodium phosphate
pH 7, 5 mM EDTA, 6.times.SSC, 0.05% sodium pyrophosphate.
Prehybridization was at 62.degree. C. for 4 hours and hybridization
was at 62.degree. C. for 18 hours (overnight) with 1 million cpm/ml
in a volume of 40 ml. Filters were washed in 500 ml of 2.times.SSC,
0.5% SDS at room temperature for 15 min. then at 63.degree. C. in
0.1.times.SSC, 0.5% SDS for 30 min. for each wash. Radiolabeled DNA
probes were made using a BRL random prime labeling system and
unincorporated counts removed using Nick Columns (Pharmacia).
Filters were exposed overnight to Kodak X-Omat AR X-ray film with
(DuPont) Cronex Lightning Plus intensifying screens at -80.degree.
C. Plaques showing hybridization with the pith-derived probe and
not the seed-derived probe were plaque purified for further
characterization.
Example 11
Isolation of Genomic Clones
[0381] Genomic DNA from Funk inbred maize line 211D was isolated as
described by Shure et al., Cell, 35:225-233 (1988). The DNA was
partially digested with Sau 3A and subsequently size fractionated
on 10-40% sucrose gradients centrifuged in a Beckman SW40 rotor at
22,000 rpm for 20 hours at 20.degree. C. Fractions in the range of
9-23 Kb were pooled and ethanol precipitated. Lambda Dash II
(Stratagene) cut with Bam HI was used as described by the supplier.
The library was screened unamplified and a total of 300,000 pfu
were screened using the conditions described above. The library was
probed using pith-specific (TrpA) cDNA clone 8-2, pCIB5600 which
was identified in the differential screen of the cDNA library.
Isolated clones were plaque purified and a large scale phage
preparation was made using Lambdasorb (Promega) as described by the
supplier. Isolated genomic clones were digested with Eco RI and the
4.8 kb EcoRI fragment was subcloned into Bluescript vector
(Stratagene).
Example 12
DNA Sequence and Computer Analysis
[0382] Nucleotide sequencing was performed using the dideoxy
chain-termination method disclosed in Sanger et al., PNAS,
74:5463-5467 (1977). Sequencing primers were synthesized on an
Applied Biosystems model 380B DNA synthesizer using standard
conditions. Sequencing reactions were carried out using the
Sequenase system (US Biochemical Corp.). Gel analysis was performed
on 40 cm gels of 6% polyacrylamide with 7 M urea in
Tris-Borate-EDTA buffer (BRL Gel-Mix 6). Analysis of sequences and
comparison with sequences in GenBank were done using the U. of
Wisconsin Genetic Computer Group Sequence Analysis Software
(UWGCG).
Example 13
Mapping the Transcriptional Start Site
[0383] Primer extension was carried according to the procedure of
Metraux et al., PNAS, 86:896-900 (1988). Briefly, 30 .mu.g of maize
pith total RNA were annealed with the primer in 50 mM Tris pH 7.5,
40 mM KCl, 3 mM MgCl2 (RT buffer) by heating to 80.degree. C. for
10 minutes and slow cooling to 42.degree. C. The RNA/primer mix was
allowed to hybridize overnight. Additional RT buffer, DTT to 6 mM,
BSA to 0.1 mg/ml, RNAsin at 4 U/ml and dNTP's at 1 mM each were
added. Then 8 units AMV reverse transcriptase were added and
reaction placed at 37.degree. C. for one hour. The primer used was
5'-CGTTCGTTC CTCCTTCGTC GAGG-3' (SEQ ID NO:73), which starts at +90
bp relative to the transcription start. See FIG. 29A. A sequencing
ladder using the same primer as in the primer extension reaction
was generated using the 4.8 Kb genomic clone to allow determination
of the transcriptional start site. The sequencing reaction was
carried out as described in Example 12.
[0384] RNase protection was used to determine if the the 371 bp
sequence from +2 bp to +373 bp (start of cDNA) was contiguous or if
it contained one or more introns. A 385 bp SphI-NcoI fragment
spanning +2 bp to +387 bp relative to transcriptional start see
FIG. 29B was cloned into pGEM-5Zf(+) (Promega) and transcribed
using the Riboprobe Gemini system (Promega) from the SP6 promoter
to generate radioactive antisense RNA probes as described by the
supplier. RNase protection was carried out as described in Sambrook
et al. pBR322 (cut with HpaII and end labelled with 32P-dCTP) and
Klenow fragment were used molecular weight markers. Gels were 6%
acrylamide/7M urea (BRL Gel-Mix 6) and were run at 60 watts
constant power.
Example 14
Genomic Southern Blots
[0385] Genomic DNA was isolated from maize line 211D using the
procedure of Shure et al., supra. 8 .mu.g of genomic DNA were used
for each restriction enzyme digest. The following enzymes were used
in the buffer suggested by the supplier: BamHI, EcoRI, EcoRV,
HindIII, and SacI. Pith cDNA clone number 8-2 was used for
estimating gene copy number. The digested DNA was run on a 0.7%
agarose gel using Tris-Borate-EDTA buffer system. The gel was
pretreated with 250 mM HCl for 15 min. to facilitate transfer of
high molecular weight DNA. The DNA was transferred to Nitroplus
2000 membrane and subsequently probed with the pith cDNA 8-2. The
blot was washed as described in Example 10.
Example 15
PCR Material and Methods
[0386] PCR reactions were preformed using the GeneAmp DNA
Amplication reagent kit and AmpliTaq recombinant Taq DNA polmerase
(Perkin Elmer Cetus). Reaction condition were as follows: 0.1 to
0.5 uM of each of the two primers used per reaction, 25 ng of the
pith 4.8 Kb EcoRI fragment in Bluescript, plus the PCR reaction mix
described by the supplier for a total volume of 50 uL in 0.5 mL
GeneAmp reaction tube (Perkin Elmer Cetus). The DNA Thermal Cycler
(Perkin Elmer Cetus) using the Step-Cycle program set to denature
at 94.degree. C. for 60 s, anneal at 55.degree. C. for 60 s, and
extend at 72.degree. C. for 45 s followed by a 3-s-per-cycle
extension for a total of 30 cycles. The following primer sets were
used: I. 83.times.84, 429 bp to -2 bp; II. 49.times.73, -69 bp to
+91 bp; III. 38.times.41, +136 bp to +258 bp; and IV. 40.times.75,
+239 bp to +372 bp. These are marked on FIG. 24.
Example 16
Isolation of a Pith-Preferred Gene
[0387] A cDNA library derived from pith mRNA cloned into Lambda Zap
and screened using first strand cDNA derived from either pith or
seed mRNA. Clones which hybridized with only the pith probe were
plaque purified and again screened. Clones passing the second
screen were used as probes in northern blots containing RNA from
various maize tissues.
Example 17
Gene Structure and Sequence Analysis
[0388] The 1.2 Kb insert of the cDNA clone 8-2 was sequenced using
the dideoxy method of Sanger et al., supra. Likewise, the genomic
equivalent contained on a 4.8 Kb EcoRI fragment in Bluescript
denoted as pCIB5601, was sequenced. This information revealed that
the genomic copy of the coding region spans 1.7 Kb and contains
five introns. The mRNA transcript represents six exons. This is
shown in FIG. 24. The exons range in size from 43 bp to 313 bp and
the introns vary in size from 76 bp to 130 bp. The entire sequence
of the gene and its corresponding deduced amino acid sequence are
shown in FIG. 24 (SEQ ID NOS:18 and 19).
[0389] This gene encodes a protein of 346 amino acids with a
molecular mass of about 38 kD. As illustrated in Table 1, the
predicted protein shows 62% similarity and 41% identity with the
subunit protein of Pseudomonas aeruginosa and has high homology
with trpA proteins from other organisms. TABLE-US-00023 TABLE 1
Conservation of TrpA sequences between a maize TrpA gene and other
organisms. Organisms % amino acid % amino acid compared Similarity
Identity Haloferax volancii 56.4 36.1 Methanococcus voltae 58.1
35.1 Pseudomonas aeruginosa 62.5 41.8 Neurospora crassa 61.4 39.3
Saccharomyces cerevisiae 56.7 36.1
Similarity groupings, I=L=M=V, D=E, F=Y, K=R, N=Q, S=T Similarities
and indentities were done using the GAP program from UWGCG.
[0390] Crawford et al., Ann. Rev. Microbiol., 43:567-600 (1989),
incorporated herein by reference, found regions of conserved amino
acids in bacterial trpA genes. These are amino acids 49 to 58,
amino acids 181 to 184, and amino acids 213 to 216, with the rest
of the gene showing greater variability than is seen in the TrpB
sequence. An alignment of known trpA proteins with the maize TrpA
protein (not shown) illustrates that the homology between the maize
gene and other trpA proteins is considerable. Also, it is
comparable to the level of homology observed when other TrpA
proteins are compared to each other as described in Crawford et
al., supra.
[0391] To determine the location of the transcription start site
and whether or not there were introns present in this region, four
polymerase chain reaction (PCR) generated fragments of about 122 bp
to 427 bp from the region -429 bp to +372 bp were used for northern
analysis. The results of the northerns showed that PCR probes II,
III, IV hybridized to pith total RNA and PCR probe I did not
hybridize. This indicated that the transcription start was in the
-69 bp to +90 bp region. To more precisely locate the
transcriptional start site, primer extension was employed. FIG. 28A
shows that when a primer (#73) located at +90 bp relative to the
transcriptional start is used for primer extension, the
transcriptional start site is located at +1, 1726 bp on the genomic
sequence.
[0392] The first ATG from the transcriptional start site is at +114
bp. This is the ATG that would be expected to serve as the site for
translational initiation. This ATG begins an open reading that runs
into the open reading frame found in the cDNA clone. The first 60
amino acids of this predicted open reading frame strongly resemble
a chloroplast transit peptide. See Berlyn et al. PNAS, 86:4604-4608
(1989) and Neumann-Karlin et al., EMBO J., 5:9-13 (1986). This
result suggests that this protein is targeted to a plastid and is
likely processed to yield the active protein. Transient expression
assays in a maize mesophyll protoplast system using a maize
optimized B.t. gene driven by the trpA promoter showed that when
the ATG at +114 bp is used as the fusion point, the highest levels
of expression are obtained. Using either of the next two ATGs in
the sequence substantially reduces the level of expression of the
reporter gene. The ATG at +390 bp gave some activity, but at a much
lower level than the +114 ATG, and the ATG at +201 bp gave no
activity.
[0393] Athough a number of TATA like boxes are located upstream of
the upstream of the transcriptional start site at +1 bp, the TATAAT
at -132 bp is most like the plant consensus of TATAAA. See Joshi,
Nuc. Acids Res., 15:6643-6653 (1987). The presumptive CCAAT like
box was found at -231 bp. The nucleotide sequence surrounding the
ATG start (GCGACATGGC; see SEQ ID NO:18) has homology to other
maize translation starts as described in Messing et al., Genetic
Engineering of Plants: An Agricultural Perspective, Plenum Press,
pp. 211-227 (1983), but differs from that considered a consensus
sequence in plants (ANNATGGC). See, Joshi, above. The presumptive
poly(A) addition signal is located at 3719 bp (AATAAA) on the
genomic sequence, 52 bp from the end of the cDNA. The sequence
matches known sequences for maize as described in Dean et al., Nuc.
Acids Res., 14:2229-2240 (1986), and is located 346 bp downstream
from the end of protein translation. See Dean et al., Nuc. Acids
Res., 14:2229-2240 (1986). The 3' untranslated sequence of the cDNA
ends at 3775 bp on the genomic sequence.
[0394] FIG. 27 shows a Southern blot of maize 211D genomic DNA with
the approximate gene copy number as reconstructed using pith gene
8-2 cDNA. From the restriction digests and reconstruction there
appear to be 1-2 copies of the gene present per haploid genome.
There do not appear to be other genes with lower levels of homology
with this gene. Therefore, this represents a unique or small member
gene family in maize.
Example 18
RNase Protection
[0395] The structure of the 5' end of the mRNA was determined using
RNase protection. The RNase protection was carried out using a
probe representing 385 nt from +2 bp to +387 bp. This region from
the genomic clone was placed in the RNA transcription vector
pGEM-5Zf(+) and a 32P labelled RNA probe generated using SP6
polymerase. The probe and the extra bases from the multiple cloning
site produce a transcript of 461 nt. The probe was hybridized with
total pith RNA and subsequently digested with a mixture of RNase A
and T1 and the protected fragments analyzed on denaturing
polyacrylamide gels. Analysis of the gels shows a protected
fragment of about 355 nt and another fragment of about 160 nt. See
FIG. 28B.
[0396] The fact that primer extension using a primer (#73) at +80
bp produces a product of 90 NT in length argues that the 5' end of
the transcript is located at position +1 bp. Primer extension from
a primer in this region produces a product, so one would expect
this also to be detected by the RNase protection assay. This primer
is located in the 5' region of the RNase protection probe. The cDNA
clone contains sequences present in the 3' end of the RNase
protection probe and hence were expected to be protected in this
assay. Since only one band is present on the gel which could
account for both of these sequences, we are confident that the
protected fragment is indeed the larger band and that the smaller
single band is an artifact. If there were an intron in this region,
fragments from each end would be present in the probe, and hence
would be detectable on the gel. Of the two bands seen, one of them
appears to represent the entire 5' region, therefore we do not
believe that there is an intron located in this region.
Example 19
Complementation of E. coli TrpA Mutant with the Pith cDNA 8-2
[0397] E. coli strain CGSC strain 5531 from the E. coli Genetic
Stock Center, Yale University (O. H. Smith lab strain designation,
#M5004) with chromosomal markers glnA3, TrpA9825, 1-,
IN(rrnD-rrnE), thi-1 as described in Mayer et al., Mol. Gen.
Gentet., 137:131-142 (1975), was transformed with either the pith
(TRpA) cDNA 8-2 or Bluescript plasmid (Stratagene) as described in
Sambrook et al., supra. The transformants containing the TrpA cDNA
8-2 had the ability to grow without the presence of tryptophan on
minimal medium whereas the transformants with the Bluescript
(Stratagene) plasmid or untransformed control were not able to grow
without tryptophan. The cells transformed with the maize TrpA gene
grew very slowly with colonies visible after seven days growth at
room temperature. All strains were grown on M9 minimal medium
supplemented with 200 ug/ml glutamine, 0.01 ug/ml thiamine and with
or without 20 ug/ml tryptophan. All transformants were checked for
the presence of the appropriate plasmid by restriction enzyme
analysis. Colonies growing in the absence of tryptophan all
contained clone 8-2 containing the cDNA for the putative maize TrpA
gene, as confirmed by Southern hybridization (data not shown).
These results support the conclusion that this is the maize
tryptophan synthase subunit A protein.
Example 20
Gene Expression
[0398] The expression pattern of the pith-preferential gene
throughout the plant was examined. Different maize genotypes were
also examined for patterns of expression of this gene. The
following tissues were used as the source of RNA for these studies:
upper, middle, and lower pith, brace roots, ear shank, cob in
genotype 5N984; upper, middle, lower pith, 10 day old leaves, 14
day old roots and pith from the entire plant in genotype 211D, and
seed from genotype 211D which had been harvested at weekly
intervals one to five weeks post-pollination. Lower pith is derived
from, i.e. constitutes the two internodes above brace roots; middle
pith is derived from the next three internodes; upper pith
represents the last two internodes before the tassel in 60 and 70
day plants. Only two internodes were present in 39 day old plants
and three internodes for 46 day old plants. Northern blot analysis
shows that transcripts hybridizing with a probe derived from the
pith cDNA accumulate rapidly in young pith and young leaf. As the
age of the plant increases and one moves up the stalk, there is a
significant decrease in the amount of transcript detected. See
FIGS. 25A-D. At no time is message from this gene detected in seed
derived RNA, either total RNA or poly A+ RNA. See FIG. 26.
Transcript is also detected in root, earshank, and sheath but not
at the high levels detected in the pith and young leaf tissues. See
FIGS. 25B, 25C. Some message is detected in brace roots, but only
at a very low level. See FIG. 25D. Six maize undifferentiated
callus lines were analyzed by northern blot analysis and no
expression was found for this gene (data not shown) in any callus
sample. The level of expression of this gene is extremely high
since a very strong signal to a probe from TrpA gene 8-2 can be
detected in pith and leaf as little as two hours after exposure of
the blot to film (FIG. 25A). The amount of mRNA made is comparable
to that derived from the maize phosphoenolpyruvate carboxylase gene
disclosed in Hudspeth et al., Plant Mol. Biology, 12:579-589
(1989), another highly expressed maize gene. Hudspeth is
incorporated herein by reference.
[0399] The expression pattern of this gene is not temporally
constant. Expression is very high in the lower and middle pith of
plants less than 60 days old and decreases rapidly near the top of
the plant. As the plant reaches maturity, e.g. over 70 days old,
the expression drops to nearly undetectable levels except in the
lower pith and earshank. The accumulation of transcript in young
leaf is nearly as high as that seen in lower pith but expression
decreases rapidly and is undetectable in leaves over 40 days of
age. Expression in leaf was found to be variable depending on the
season when it is grown.
[0400] Examples 21-39 set forth below are directed to the
isolation, characterization and expression analysis of a
pollen-specific promoter according to the present invention.
Identification of Pollen-Specific Proteins
Example 21
Maize Plant Growth
[0401] Maize plants (Zea mays Funk inbred 211D) were grown from
seed in a vermiculite/sand mixture in a greenhouse under a 16 hour
light/8 hour dark regime.
Example 22
Total Pollen Protein Isolation
[0402] Mature pollen was isolated from maize plants at the time of
maximum pollen shed. It was sieved to remove debris, frozen in
liquid nitrogen, and a 3-4 ml volume of frozen pollen was ground in
a mortar and pestle with an equal volume of 75-150 .mu.m glass
beads. 40 ml of grinding buffer (2 mM EDTA, 5 mM DTT, 0.1% SDS, 100
mM Hepes pH 8) was added and the mixture was ground again. The
glass beads and intact pollen grains were pelleted by low speed
centrifugation, and mixture was clarified by centrifugation at
10,000 g for 15 minutes. Protein was precipitated from the
supernatant by addition of acetone to 90%.
Example 23
Pollen Exine Protein Isolation
[0403] Exine Protein was isolated from maize 211D shed pollen as
described in Matousek and Tupy, J., Plant Physiology 119:169-178
(1985).
Example 24
Leaf Protein Isolation
[0404] Young leaves (about 60% expanded) were cut from the maize
plant the midrib removed. Total protein was isolated as for pollen,
except that the material was not frozen and grinding was in a
Waring blender without glass beads.
Example 25
Kernel Protein Isolation
[0405] Ears with fully developed, but still moist kernels were
removed from the plant and the kernels cut off with a scalpel.
Total protein was isolated as for leaves.
Example 26
Gel Electrophoresis of Maize Proteins
[0406] Pollen, leaf and kernel proteins were separated on SDS
polyacrylamide gels as described in Sambrook et al, Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press:
New York (1989). Following staining by Coomasie blue, protein bands
from pollen, leaf and kernel were compared and abundant proteins of
approximately 10 kD, 13 kD, 20 kD, 45 kD, 55 kD and 57 kD were
determined to be pollen specific.
Identification of Pollen-Specific cDNA Clones
Example 27
Partial Sequence Determination of Pollen-Specific Proteins
[0407] Protein bands determined to be pollen-specific were purified
by electroblotting from the polyacrylamide gel onto PVDF membrane
(Matsudaira, P., J. Biol. Chem. 261:10035-10038 (1987)) or by
reverse phase HPLC. N-terminal sequence of the purified proteins
was determined by automated Edman egradation with an Applied
Biosystems 470A gas-phase sequencer. Phenylthiohydantoin (PTH)
amino acids were identified using an Applied Biosystems 120A PTH
analyzer. To obtain internal sequence, proteins were digested with
endoproteinase Lys-C (Boehringer Mannheim) in 0.1 M Tris-HCl, pH
8.5, for 24 hours at room temperature using an enzyme:substrate
ratio of 1:10. Resulting peptides were isolated by HPLC using an
Aquapore C-8 column eluted with a linear acetonitrile/isopropanol
(1:1 ratio) gradient (0 to 60%) in 0.1% TFA. Sequence of isolated
Lys-C peptides was determined as above. The following sequences
were determined for the 13 kD pollen-specific protein:
TABLE-US-00024 N-terminus: TTPLTFQVGKGSKPGHLILTPNVATI (SEQ ID
NO:74) LysC 61: KPGHLILTPNVATISDVVIK (SEQ ID NO:75) LysC 54:
SGGTRIADDVIPADFK (SEQ ID NO:76) LysC 49: EHGGDDFSFTLK (SEQ ID
NO:77) LysC 43: EGPTGTWTLDTK (SEQ ID NO:78)
Example 28
Synthesis of Oligonucleotide Probes for Pollen-Specific cDNAs
[0408] Regions of peptide sequence in the 13 kD protein with low
codon redundancy were selected, and suitable oligonucleotide probes
for the gene encoding these regions were synthesized on an Applied
Biosystems 380A synthesizer. The following oligonucleotides were
synthesized: TABLE-US-00025 Oligo #51 5'-AA RTC RTC ABC ACC RTG
YTC-3' (SEQ ID NO:79) Oligo #58 5'-CC YTT NCC CAC YTG RAA-3' (SEQ
ID NO:80)
where the columns of nucleotides represent bases that were
incorporated randomly in equal proportions at the indicated
position in the oligo. Oligo #51 encodes the amino acid sequence
EHGGDDF (amino acids 1 to 7 of SEQ ID NO:77) found in peptide LysC
49, and Oligo #58 encodes the amino acid sequence FQVGKG (amino
acids 6 to 11 of SEQ ID NO:74) found in peptide N-terminus. Use of
these mixed oligonucleotides to screen a cDNA library for the
pollen-specific gene will be described below.
Example 29
Construction of a Maize Pollen cDNA Library
[0409] Total maize RNA from maize 211D shed pollen was isolated as
described in Glisen et al, Biochemistry 13:2633-2637 (1974). Poly
A+mRNA was purified from total RNA as described in Sambrook et al.
Using this mRNA, cDNA was prepared using a cDNA synthesis kit
purchased from Promega, following protocols supplied with the kit.
The EcoRI linkers were added to the cDNA and it was ligated into
arms of the cloning vector lambda Zap, purchased from Stratagene
and using the protocol supplied by the manufacturer. The ligation
product was packaged in a lambda packaging extract also purchased
from Stratagene, and used to infect E. coli BB4 cells.
Example 30
Isolation of Pollen-Specific cDNA Clones
[0410] The maize pollen cDNA library was probed using the synthetic
oligonucleotides probes specific for the 13 kD protein gene, as
described in Sambrook et al. Briefly, about 100,000 phage plaques
of the pollen cDNA library were plated and lifted to nitrocellulose
filters. The filters were probed using oligonucleotides #51 and #58
which had been 32P end-labeled using polynucleotide kinase. The
probes were hybridized to the filters at low stringency (50 degrees
C. in 1M NaCl, 10% dextran sulfate, 0.5% SDS), washed 30 minutes at
room temperature and then 30 minutes at 45 degrees C. in
6.times.SSC, 0.1% SDS, and exposed to X-ray film to identify
positive clones. Putative clones were purified through four rounds
of plaque hybridization. Three classes of cDNA clones were
isolated. Type I contained EcoRI fragments of 0.2 kb and 1.8 kb.
Type II contained EcoRI fragments of 0.6 kb, 0.5 kb and 1.0 kb, and
Type III contained an EcoRI fragment of 2.3 kb.
Example 31
Characterization of Pollen-Specific cDNA Clones
[0411] The EcoRI fragments of the Type II cDNA clone were subcloned
into the plasmid vector pBluescript SK+, purchased from Stratagene.
See FIG. 29. The 0.6 kb fragment in pBluescript was named II-.6,
the 0.5 kb fragment in pBluescript was named II-.5 (later renamed
pCIB3169) and the 1.0 kb fragment in pBluescript was named II-1.0
(later renamed pCIB3168). As will be described below, the 0.5 kb
and 1.0 kb fragments encode the maize pollen-specific CDPK gene.
RNA from anthers, pollen, leaf, root and silk was denatured with
glyoxal, electrophoresed on a 1% agarose gel, transferred to
nitrocellulose, and probed separately with the three EcoRI
fragments that had been labeled with 32P by random primer extension
as described in Sambrook et al, Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory Press: New York (1989). The
blots were exposed to X-ray film, and an mRNA band of approximately
1.5 kb was identified with the 0.6 kb fragment probe, while the 0.5
and 1.0 kb fragments hybridized to an approximately 2.0 kb mRNA. In
all cases hybridization was only seen in the pollen RNA lane, with
the exception that the 0.6 kb fragment showed a slight signal in
anther mRNA. The conclusion from these data was that the original
cDNA clone was a fusion cDNA molecules derived from two different
mRNAs. The 0.6 kb fragment was a partial cDNA of a 1.5 kb
pollen-specific mRNA, and this mRNA encodes the peptides LysC 49
and N-terminus. The 1.0 and 0.5 kb fragments comprise a partial
cDNA of a 2.0 kb pollen-specific mRNA unrelated to the peptides and
oligonucleotide probes used for probes. This conclusion was
verified when the fragments were sequenced using the dideoxy chain
termination method as described in Sambrook et al. The cDNA
sequence is shown in FIG. 30 (SEQ ID NO:20).
Example 32
Determination of Specificity of mRNA Expression
[0412] To determine if the 2.0 kb RNA represented by cDNA clones
pCIB3169 and pCIB3168 were present only in pollen, total RNA was
isolated from maize 211D roots, leaves, pollen, anthers or silks.
The RNAs were denatured with glyoxal, electrophoresed on a 1%
agarose gel, transferred to nitrocellulose, and probed with
32P-labeled EcoRI insert from plasmid pCIB3168 or pCIB3169, all
using standard techniques as described in Sambrook et al, Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press:
New York (1989). Exposure of this blot to photographic film
demonstrates that the gene represented by these two clones is only
transcriptionally active in the pollen (FIG. 31).
Identification of a Pollen-Specific Promoter
Example 33
Construction of a Maize Genomic DNA Library
[0413] Genomic DNA from maize line 211D young shoots was isolated
as described in Shure et 1, Cell 35:225-233 (1983). The DNA was
provided to Stratagene, where a genomic DNA library was constructed
by cloning Sau3AI partially digested DNA into Stratagene's Lambda
Dash cloning vector.
Example 34
Genomic DNA Blot Hybridization to Determine Gene Copy Number
[0414] Genomic DNA from maize line 211D was digested with a number
of restriction enzymes, the individual digests electrophoresed on
an agarose gel, transferred to nitrocellulose, and probed with
32P-labeled EcoRI insert from plasmid pCIB3168 (1.0 kb fragment),
pCIB3169 (0.5 kb fragment) or clone II-.6 using standard techniques
described in Sambrook et al. More than 10 bands were detected by
the II-.6 probe on most digests, indicating that this cDNA is
derived from a large, multigene family. Probing with the 1.0 kb
fragment detected from 3 to 6 bands, and probing with the 0.5 kb
fragment detected only from 1 to 3 bands which were a subset of
those detected by the 1.0 kb fragment. Due to the smaller gene
family size detected by the 1.0 kb and 0.5 kb fragments, it was
decided to attempt to isolate the genomic clone corresponding to
them.
Example 35
Isolation of a Pollen-Specific Genomic Clone
[0415] The Stratagene maize 211D genomic library was screened by
probing plaque lifts with 32P labeled inserts from plasmid pCIB3168
(1.0 kb fragment) and pCIB3169 (0.5 kb fragment) using standard
procedures as described in the Stratagene manual accompanying the
library. Using this strategy, Lambda clone MG14 was isolated, and
it hybridized to both probes. The 9.0 kb BamHI fragment of MG14,
which also hybridized to both probes, was subcloned into the BamHI
site of pBluescript SK+ to create plasmid pCIB379. 1800 bp of
pCIB379, in the region corresponding to the cDNA sequence, was
sequenced as described above. Comparison of the cDNA and genomic
sequences showed only 91% identity. pCIB379 insert represents a
related pollen-specific gene.
[0416] A second maize 211D genomic library was constructed in the
vector lambda GEM-11, purchased from Promega, using the procedures
described in the Promega manual. Screening this un-amplified
library as above yielded clone GEM11-1, which hybridized to both
0.5 and 1.0 kb probes. The 20 kb HindIII fragment of GEM11-1, which
also hybridized to both probes, was subcloned into the HindIII site
of pBluescript SK+ to yield pCIB3166. The DNA sequence of 40.1 kb
of pCIB3166 was determined (FIG. 35; SEQ ID NO:26) and after
accounting for six introns in the genomic clone, was 100% identical
to the cDNA sequence of pCIB3168 and pCIB3169. Comparison of the
pCIB3166 sequence to the Genbank/EMBL database revealed that the 5'
portion, through the 3 exon, was 34.6% identical to rat
calmodulin-dependent protein kinase II at the amino acid level
(FIG. 32), while the fourth through seventh exons were 39.4%
identical to human calmodulin. See FIG. 33. No other
pollen-specific kinase has been described, and at the time this a
protein combining kinase and calmodulin domains was unknown.
Subsequently, Harper et al., Science 252:951-954 (1991) have
disclosed the cDNA sequence of a similar protein from soybean,
although this gene is not pollen-specific in expression. Comparison
of the soybean calcium-Dependent Protein Kinase (CDPK) and the
maize pollen CDPK reveals 38% identity at the amino acid level. See
FIG. 34.
Example 36
Identification of the Promoter's Transcriptional Start Site by
Primer Extension
[0417] Oligonucleotide PE51, with the following sequence was
synthesized as a primer.
[0418] 5'-TGGCCCATGGCTGCGGCGGGGAACGAGTGCGGC-3' (SEQ ID NO:81)
[0419] Primer extension analysis was carried out on polyA+ pollen
mRNA as described in Metraux et al., PNAS USA 86:896-890 (1989).
The transcription initiation site was determined to be between
bases 1415 and 1425 on the partial sequence of pCIB3166 shown in
FIG. 35.
Testing Promoter Function in Transgenic Plants
Example 37
Construction of Promoter Vectors for Plant Transformation
[0420] To demonstrate that the pollen CDPK promoter can drive
expression of a linked gene in transgenic plants, a gene fusion of
the pollen CDPK promoter to the Beta-glucuronidase gene of E. coli
was constructed as follows. The 10 kb BamHI fragment from lambda
GEM11-1 containing the first exon and part of the first intron of
the pollen CDPK gene plus 9 kb upstream of the gene was subcloned
into the BamHI site of pBluescript SK+ to create plasmid pCIB3167.
The 2.3 kb BamHI-HindIII fragment from pCIB3167 was subcloned into
the BamHI and HindIII sites of pBluescript SK+ to create plasmid
pSK105. The pSK105 was digested with AvaI and HindIII, and the 1.75
kb HindIII-AvaI fragment was isolated on an agarose gel. A PCR
reaction was run under standard conditions as described in Sambrook
et al. using intact pSK105 as a template and the following primers:
TABLE-US-00026 #42: (SEQ ID NO:82)
5'-AGCGGTCGACCTGCAGGCATGCGATCTGCACCTCCCGCCG-3' #43: (SEQ ID NO:83)
5'-ATGGGCAAGGAGCTCGGG-3
[0421] The PCR reaction products were digested with AvaI and SalI
and the resulting fragment isolated on an agarose gel. pBluescript
SK+ was digested with HindIII and SalI The 1.75 kb HindIII-AvaI
fragment, PCR derived AvaI-SalI fragment, and pBluescript vector
with HindIII and SalI ends were ligated in a three way ligation to
create plasmid pSK110.
[0422] A fusion of the promoter fragment in pSK110 to the
Beta-glucuronidase (GUS) gene was created by digesting PSK110 with
HindIII and SalI, isolating the 1.9 kb fragment on an agarose gel
and ligating it into HindIII and SalI sites of pCIB3054, to create
plasmid pKL2, a plasmid derived from pUC19 containing the GUS gene
followed by plant intron from the maize PEPC gene and a polyA
signal from cauliflower mosaic virus. This promoter fusion was
inactive in plants, probably due to the presence of out of frame
ATG codons in the leader sequence preceding the GUS gene ATG.
[0423] A function fusion of the promoter was created by digesting
pKL2 with XbaI and SalI to remove the previous fusion junction. A
new fusion junction was produced in a PCR reaction using pSK105 as
a template and the following primers: TABLE-US-00027 #SK50:
5'-CCCTTCAAAATCTAGAAACCT-3' (SEQ ID NO:84) #SK49:
5'-TAATGTCGACGAACGGCGAGAGATGGA-3' (SEQ ID NO:85)
[0424] The PCR product was digested with XbaI and SalI and purified
on an agarose gel. The purified fragment was ligated into the XbaI
and SalI sites of pKL2 to created plasmid pCIB3171. This plasmid
contains a functional fusion of pollen CDPK promoter and GUS which
directs expression the GUS gene exclusively in pollen.
[0425] To create a vector containing the pollen CDPK promoter-GUS
fusion suitable for use in Agrobacterium tumefaciens-mediated plant
transformation, the fusion gene was isolated from pCIB3171 by
digestion with HindIII and SalI. The resulting fragment was ligated
into the HindIII and SalI sites of pBI101 (purchased from Clontech)
to create plasmid pCIB3175.
Example 38
Production of Transgenic Plants
[0426] pCIB3175 was transformed into Agrobacterium tumefaciens
containing the helper plasmid pCIB542, and the resulting culture
used to transform leaf disks from tobacco shoot tip cultures as
described by Horsch et al., Science 227:1229-1231 (1985) except
that nurse cultures were omitted and selection was on 100 mg/l
kanamycin. Transgenic plants were regenerated and verified for
presence of the transgene by PCR.
Example 39
GUS Gene Expression Analysis
[0427] Pollen from primary transformants and their progeny were
analyzed histochemically for expression of the GUS gene as
described by Guerrero et al., Mol. Gen. Genet. 224:161-168 (1990).
The percentage of pollen grains expressing the GUS gene, as
demonstrated by blue staining in the X-gluc buffer, is shown in the
table below. TABLE-US-00028 Plant Number % Blue Pollen PP1-51 28%
PP1-54 54% PP1-55 none PP1-61 very few PP1-63 51% PP1-67 15% PP1-80
10% PP1-83 12%
[0428] Primary transformants in which a single pollen CDPK
promoter-GUS gene was integrated would produce a maximum 50% GUS
positive pollen due to segregation of the single gene. Flouometric
GUS assays were done on pollen, stem, root, leaf and pistil tissue
of selected plants to demonstrate the specificity of pollen CDPK
promoter expression. Assays were performed as described in
Jefferson, Plant Mol. Biol. 14:995-1006 (1990), and GUS activity
values are expressed as nmoles MU/ug protein/minute. TABLE-US-00029
Plant GUS Untransformed number Tissue Activity Plant GUS Activity
Net GUS Activity PP1-51 stem 0.01 0.02 0 leaf 0 0 0 root 0.15 0.10
0.05 pistil 0.02 0.01 0.01 pollen 0.24 0.02 0.22 PP1-54 stem 0.01
0.02 0 leaf 0 0 0 root 0.13 0.1 0.03 pistil 0.01 0.01 0 pollen 0.60
0.02 0.58 PP1-63 stem 0.01 0.02 0 leaf 0 0 0 root 0.07 0.1 0 pistil
0.01 0.01 0 pollen 0.57 0.02 0.55
Examples 40-50 are directed primarily to the preparation of
chimeric constructs, i.e. recombinant DNA molecules, containing
constitutive, tissue-preferred, or tissue-specific promoters
operably linked to an instant B.t. gene, insertion of same into
vectors, production of transgenic platns containing the vectors,
and analysis of expression levels of B.t. proteins of the
transgenic plants.
Example 40
Construction of Maize Optimized Bt Transformation Vectors
[0429] To demonstrate the effectiveness of the synthetic Bt
cryIA(b) gene in maize, the PepC and pith specific promoters are
fused to the synthetic Bt cryIA(b) gene using PCR. Oligomers
designed for the PCR fusions were: TABLE-US-00030 (PEPC) (SEQ ID
NO:86) KE99A28 = 5'-TGCGGTTACC GCCGATCACATG-3' (SEQ ID NO:87)
KE97A28 = 5'-GCGGTACCGC GTCGACGCGG ATCCCGCGGC GGGAAGCTAAG-3' (PITH)
(SEQ ID NQ:88) KE100A28 = 5'-GTCGTCGACC GCAACA-3' (SEQ ID NO:89)
KE98A28 = 5'-GCGGTACCGC GTTAACGCGG ATCCTGTCCG ACACCGGAC-3' (SEQ ID
NO:90) KE104A28 = 5'-GATGTCGTCG ACCGCAACAC-3' (SEQ ID NO: 91)
KE103A28 = 5'-GCGGTACCGC GGATCCTGTC CGACACCGGA CGGCT-3'
[0430] PCR primers are designed to replace the Nco I sites in the
5' untranslated leader region of each of these tissue specific
genes (containing ATG translational start sites) with Bam HI sites
to facilitate cloning of the synthetic cryIA(b) gene into this Bam
HI site. Subsequent construction of vectors containing the tissue
specific promoters fused to the synthetic cryIA(b) gene and also
containing the 35S:PAT:35S marker gene involves several
intermediate constructs.
1. pCIB4406 (35S:Synthetic-cryIA(b):pepC ivs#9:35S)
[0431] pCIB4406 contains the 2 Kb Bam HI\Cla I synthetic cryIA(b)
gene fused with the CaMV 35S promoter (Rothstein et al., Gene
53:153-161 (1987)). The gene also contains intron #6 derived from
the maize PEP carboxylase gene (ivs#9) in the 3' untranslated
region of the gene, which uses the CaMV 3' end. (PNAS USA,
83:2884-2888 (1986), Hudspeth et al., Plant Molecular Biology, 12:
579-589 (1989)). pCIB4406 is ligated and transformed into the
"SURE" strain of E. coli cells (Stratagene, La Jolla, Calif.) as
described above. One mutation is found in pCIB4406's cryIA(b) gene
at amino acid #436 which resulted in the desired Phe being changed
to a Leu. pCIB4406 is fully active against European corn borer when
tested in insect bioassays and produces a CryIA(b) protein of the
expected size as determined by western blot analysis.
2. pCIB4407 (35S:Synthetic-cryIA(b):pepC ivs#9:35S+35S:PAT:35S)
[0432] pCIB4407 is made from an approximately 4 Kb Hind III\Eco RI
fragment containing the 35S:PAT:35S gene, and the 3.1 Kb\Hind
III\Eco RI 35S:synthetic-cryIA(b):35S gene from pCIB4406. pCIB4407
is ligated and transformed into "SURE", DH5alpha, and HB101 strains
of E. coli using standard procedures (Sambrook et al.). The
synthetic cryIA(b) gene has the same properties as its precursor
pCIB4406.
3. pCIB4416 (35S:Synthetic-cryIA(b):pepC
ivs#9:35S+35S:PAT:35S+35S:Adh Intron:GUS:35S.)
[0433] pCIB4407 is cut with Eco RI and treated with calf intestinal
alkaline phosphatase (CIP) under standard conditions (Sambrook et
al.) to produce an about 7.2 Kb fragment that is ligated with a 3.4
Kb Eco RI 35S:Adh\GUS:35S fragment to produce pCIB4416. Ligations
and transformations into "SURE" cells is as described above. The
synthetic cryIA(b) gene in pCIB4416 has the same properties as the
gene in pCIB4406.
4. pCIB4418 (35S:Synthetic-cryIA(b):pepC ivs#9:35S)
[0434] pCIB4406 is digested with Apa I and Bam HI and treated with
CIP. pCIB4406 is digested with Bam HI and Nsp I. pBS123#13 is
digested with Nsp I and Apa I. A three-way ligation is made
consisting of a 4.3 Kb Apa I\Bam HI fragment from pCIB4406, a 1.3
Kb Bam HI\Nsp I fragment from pCIB4406, and a 170 bp Nsp I\Apa I
fragment from pBS123#13 to form pCIB4418. The host E. coli strain
for pCIB4418 is HB101.
5. pCIB4419 (35S:Synthetic-cryIA(b):pepC
ivs#9:35S+35S:PAT:35S+35S:Adh intron:GUS:35S.)
[0435] pCIB4416 and pCIB4418 are digested with Bst E II and Eco NI
and fragments of pCIB4416 are treated with CIP. A 9.1 Kb fragment
from pCIB4416 ligated to a 1.4 Kb fragment from pCIB4418 to form
pCIB4419. pCIB4419 transformed in HB101 competent E. coli cells
demonstrates full activity in insect bioassays against European
corn borer.
6. pCIB4420 (Pith:Synthetic-cryIA(b):PEPC
ivs#9:35S+35S:PAT:35S)
[0436] Intermediate constructs in making pCIB4420 are pBTin1,
pBtin2, p4420A and pBtin3. pBtin1 (pith promoter:second half of the
synthetic Bt gene +35S:PAT:35S) is made by ligating the 2.1 Kb Xba
I\Nco I pith promoter fragment from plasmid pith(3-1) with a 5.2 Kb
Xba I\Nco I fragment from pCIB4407. pBtin2 is an intermediate
construct containing the pith promoter modified with a 210 bp PCR
fragment made using primers KE100A28 and KE98A28 listed above. The
PCR reaction mix contains approximately 100 ng of a 2.1 Kb Bam
HI\Nco I pith promoter fragment with 100 pmol of each oligomer, 200
nM of each dNTP, 1.times. buffer (Cetus) and 2.5 units of thermal
stable polymerase. Since the Tm is relatively low (between
40.degree. and 50.degree. C.), PCR reactions are run with the
following parameters:
[0437] denaturation cycle: 94.degree. C. for 1 minute
[0438] annealing cycle: 37.degree. C. for 1 minute
[0439] extension cycle: 72.degree. C. for 45 seconds (+3 seconds
per cycle)
[0440] number of cycles: 25
[0441] PCR reactions are treated with proteinase K as described
above prior to cutting with Sal I\Kpn I followed by
phenol\chloroform extraction and ethanol precipitation as described
above. The 210 bp fragment is purified on a 2% Nusieve gel and
extracted from the gel using Millipore's filter units. The 210 bp
Sal I\Kpn I fragment is ligated to the 4.9 Kb Sal I\Kpn I fragment
from pith(3-1) to make pBtin2. p4420A (pith:synthetic-Bt:Pep
intron:35S+35S:PAT:35S) is made with a three-way ligation
consisting of a 700 bp Nsi I\Bam HI fragment from pBtin2, a 1.8 Kb
Bam HI\Bst E II fragment from pCIB4418, and a 5.9 Kb Bst E II\Nsi I
fragment from pBtin1. After p4420A is made three mutations are
discovered in pBtin2. A second PCR fragment is made to modify the
Nco I site in the pith leader using primers KE104A28 and KE103A28
with Tm values around 65.degree. C. The PCR reaction mix is
identical to that listed above with the addition of glycerol to 20%
to reduce mutations in G+C rich areas (Henry et al., Plant
Molecular Biology Reporter 9(2): 139-144, 1991). PCR parameters are
as follows:
File I:
[0442] 94.degree. C.: 3 minutes, 1 cycle File II: [0443] 60.degree.
C.: 1 minute [0444] 94.degree. C.: 1 minute [0445] 25 cycles File
III: [0446] 72.degree. C.: 5 minutes, 1 cycle
[0447] PCR reactions are treated as above and cut with restriction
endonucleases Sal I and Kpn I. The 210 bp Sal I\Kpn I PCR (glycerol
in the reaction) fragment is ligated to the 4.9 Kb Sal I\Kpn
I-fragment from plasmid pith(3-1) to make pBtin3. Sequence data on
pBtin3-G#1 shows this PCR generated fragment to be correct.
[0448] pBtin3-G#1 is used to make pCIB4420 (also called p4420B
"G#6"). pCIB4420 is constructed with a three-way ligation using the
700 bp Nsi I\Bam HI fragment from pBtin3-G#1, a 1.8 Kb Bam HI\Bst E
II fragment from pCIB4418, and a 5.9 Kb Bst E II\Nsi I fragment
from pBtin1. pCIB4420 is used in mesophyll protoplast experiments
and demonstrates full activity of the synthetic cryIA(b) gene
against European corn borer.
7. pCIB4413 (PEPC:Synthetic-Bt (Phe Mutation):PEPC Intron:35S.)
[0449] A fusion fragment is generated by PCR using primers KE99A28
and KE97A28 with a 2.3 KB Hind III\Sal I template from pGUS4.5. The
PCR mix contains the same concentration of primers, template,
dNTPs, salts, and thermal stable polymerase as described above. PCR
reaction parameters are:
[0450] denaturation cycle: 94.degree. C. for 1 minute
[0451] annealing cycle: 55.degree. C. for 1 minute
[0452] extension cycle: 72.degree. C. for 45 seconds (+3 seconds
per cycle)
[0453] number of cycles: 30
[0454] After completion, PCR reactions are treated with proteinase
K followed by phenol\chloroform extraction and ethanol
precipitation as described above prior to cutting with restriction
endonucleases Bam HI and Bst E II.
[0455] pCIB4413 is made with a three-way ligation using the 210 bp
Bam HI\Bst E II PCR fragment, a 4.7 Kb Bam HI\Hind III fragment
from pCIB4406, and a 2.2 Kb Hind III\Bst E II fragment from
pGUS4.5.
8. pCIB4421 (PEPC:Synthetic-cryIA(b):PEPC Intron:35S.)
[0456] pCIB4421 is made to replace the synthetic cryIA(b) gene
containing the Phe mutation in pCIB4413 with the synthetic cryIA(b)
gene from pCIB4419. pCIB4421 is made by ligating a 5.2 Kb Bam
HI\Sac I fragment from pCIB4413 with a 1.9 Kb Bam HI\Sac I fragment
from pCIB4419.
9. pCIB4423 (PEPC:Synthetic-cryIA(b):PepC
Intron:35S+35S:PAT:35S)
[0457] The 2.4 Kb Bam HI\Hind III PEPC promoter fragment from
pCIB4421 is ligated to the 6.2 Kb Bam HI\Hind III fragment in
pCIB4420 to make pCIB4423. The Hind III site is deleted by
exonucleases in the cloning of pCIB4423. pCIB4423 contains the
synthetic cryIA(b) gene under the control of the PEPC promoter, and
the PAT gene under the control of the 35S promoter.
10. Synthetic cryIA(b) Gene in Agrobacterium Strains:
[0458] Agrobacterium strains made with the synthetic cryIA(b) gene
allow transfer of this gene in a range of dicotyledenous plants.
Agrobacterium vector pCIB4417 contains the 3.3 Kb Hind III\Eco RI
35S:synthetic-CryIA(b):PepC:ivs#9:35S fragment from pCIB4406 (Phe
mutation) ligated to the 14 Kb Hind III\Eco RI fragment from pBI101
(Clontech). Using electroporation, pCIB4417 is transferred into the
A. tumefaciens strain LBA4404 (Diethard et al., Nucleic Acids
Research, Vol 17:#16:6747, 1989.).
[0459] 200 ng of pCIB4417 and 40 ul of thawed on ice LBA4404
competent cell are electroporated in a pre-cooled 0.2 cm
electroporation cuvette (Bio-Rad Laboratories Ltd.). Using Gene
Pulser-TM with the Pulse Controller unit (Bio-Rad), an electric
pulse is applied immediately with the voltage set at 2.5 kV, and
the capacity set at 25 uF. After the pulse, cells are immediately
transferred to 1 ml of YEB medium and shaken at 27 C for 3 hours
before plating 10 ul on ABmin:Km50 plates. After incubating at 28 C
for approximately 60 hours colonies are selected for miniscreen
preparation to do restriction enzyme analysis. The final
Agrobacterium strain is called pCIB4417:LBA4404.
Example 41
ELISA Analysis of Transformed Maize Protoplasts
[0460] The presence of the cryIA(b) toxin protein is detected by
utilizing enzyme-linked immunosorbent assay (ELISA). ELISAS are
very sensitive, specific assays for antigenic material. ELISA
assays are useful to determine the expression of polypeptide gene
products. Antiserum for these assays is produced in response to
immunizing rabbits with gradient-purified Bt crystals (Ang et al.,
Applied Environ. Microbiol., 36:625-626 (1978)) solubilized with
sodium dodecyl sulfate. ELISA analysis of extracts from transiently
transformed maize cells is carried out using standard procedures
(see for example Harlow, E., and Lane, D. in "Antibodies: A
Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988).
ELISA techniques are further described in Clark et al., Methods in
Enzymology, 118:742-766 (1986); and Bradford, Anal. Biochem.,
72:248 (1976). Thus, these procedures are well-known to those
skilled in the art. The disclosure of these references is hereby
incorporated herein by reference.
[0461] ELISA assays are performed to detect the production of
CryIA(b) protein in maize protoplasts. Protein produced is reported
below as ng of Bt per mg total protein (ng Bt/mg). Each construct
was tested twice.
pCIB3069
[0462] No detectable Bt (both tests) pCIB4407 [0463] 21,900 ng
Bt/mg total protein, [0464] 21,000 ng Bt/mg total protein
[0465] The transformed maize cells produce high levels, on the
order of approximately 20,000 ng of Bt CryIA(b) protein per mg
total soluble protein, of the Bt IP when transformed with the maize
optimized Bt gene. The level of detection of these ELISA based
assays is about 1 to 5 ng CryIA(b) protein per mg protein.
Therefore, the maize optimized Bt gene produces as much as
approximately a 20,000 fold increase in expression of this protein
in maize cells.
Example 42
Assay of Extract from Transformed Protoplasts for Insecticidal
Activity Against European Corn Borer
[0466] Western blot analysis is also performed using extracts
obtained from maize cells which had been transiently transformed
with DNA to express the maize optimized gene. When examined by
western blots, this protein appears identical with the protein
produced in E. coli. In contrast, as demonstrated in Example 6
above, no detectable Bt cryIA(b) insecticidal protein is produced
by maize cells transformed with comparable vectors attempting to
express the native Bt derived coding region.
[0467] Qualitative insect toxicity testing can be carried out using
harvested protoplasts. Suspensions are prepared for each replicate
tested in all bioassays. A replicate is considered positive if it
causes significantly higher mortality than the controls. For
example, replicates are tested for their activity against insects
in the order Lepidoptera by using the European corn borer, Ostrinia
nubilalis. One-hundred .mu.l of a protoplast suspension in 0.1%
Triton X-100 is pipetted onto the surface of artificial Black
cutworm diet, (Bioserv, Inc., Frenchtown, N.J.; F9240) in 50
mm.times.10 mm snap-cap petri dishes. After air drying 10 neonatal
larvae are added to each plate. Mortality is recorded after about 4
days. When this protein is fed to European corn borers, it produces
100% mortality.
Example 43
Expression of Synthetic Bt in Maize Mesophyll Protoplasts
[0468] The general procedure for the isolation of corn mesophyll
protoplasts is adapted from Sheen et al., The Plant Cell,
2:1027-1038 (1990). The protoplast transformation system used in
Sheen et al. is modified by using PEG mediated transformation,
rather than electroporation. That procedure, as well as changes
made in the isolation procedure, is described below. Maize
Mesophyll Protoplast Isolation/Transformation
[0469] 1. Sterilize and germinate corn seeds for leaf material.
Seedlings are grown in the light at 25 C.
[0470] 2. Surface sterilize leaf pieces of 10-12 day old seedlings
with 5% Clorox for 5 minutes followed by several washes with
sterile distilled water.
[0471] 3. Aliquot enzyme solution (see recipe below); 25 ml/dish
(100.times.25 mm petri dish).
[0472] 4. Remove any excess water from leaves and place 6-8 2 inch
pieces in each dish of enzyme. 14 plates are usually set up with
the leaf material from about 100 seedlings.
[0473] 5. Cut leaves in longitudinal strips as thin as possible
(2-5 mm).
[0474] 6. Shake slowly at 25 C for 6.5 to 7 hours. Cover plates so
that incubation takes place in the dark.
[0475] 7. Before filtering protoplasts, wash 100 um sieves with 10
ml 0.6 M mannitol. Pipet protoplasts slowly through sieves. Wash
plates with 0.6 M mannitol to gather any protoplasts left in the
dishes.
[0476] 8. Pipet filtered liquid carefully into 50 ml sterile tubes.
Add equal volumes of 0.6 M mannitol to dilute.
[0477] 9. Spin for 10 minutes at 1000 rpm/500 g in table-top
centrifuge (Beckman Model TJ-6).
[0478] 10. Remove enzyme solution and discard. Resuspend pellets
carefully in 5 ml mannitol. Pool several pellets. Bring volume to
50 ml with 0.6 M mannitol and spin.
[0479] 11. Resuspend to a known volume (50 ml) and count.
[0480] 12. After counting and pelleting, resuspend protoplasts at 2
million/ml in resuspending buffer (recipe below). Allow ppts to
incubate in the resuspending buffer for at least 30 min before
transformation.
Transformation:
[0481] 1. Aliquot plasmids to tubes (Fisherbrand polystyrene
17.times.100 mm Snap Cap culture tubes); at least three replicates
per treatment; use equimolar amounts of plasmids so that equal gene
copy numbers are compared.
[0482] 2. Add 0.5 ml protoplasts and 0.5 ml 40% PEG made with 0.6 M
mannitol.
[0483] 3. Shake gently to mix and incubate at 25 C for 30 min.
[0484] 4. Add protoplast culture media at 5 min intervals: 1, 2, 5
ml
[0485] 5. Spin for 10 min at 1000 rpm/500 g.
[0486] 6. Remove liquid from pellet and resuspend in 1 ml culture
media (BMV media)
[0487] 7. Incubate overnight at 25 C in the dark.
Recipes:
Enzyme Solution
[0488] 0.6 M mannitol [0489] 10 mM MES, pH 5.7 [0490] 1 mM
CaCL.sub.2 [0491] 1 mM MgCl2 [0492] 0.1% BSA [0493]
filter-sterilize To this solution, add the following enzymes:
[0494] 1% Cellulase RS, and 0.1% Macerozyme R10 [0495] Wash Buffer:
0.6 M mannitol, filter-sterilize [0496] Resuspending Buffer: 0.6 M
mannitol, 20 mM KCl, filter-sterilize [0497] Culture Media: BMV
media recipe from: [0498] Okuno et al., Phytopathology 67:610-615
(1977). [0499] 0.6 M mannitol [0500] 4 mM MES, pH 5.7 [0501] 0.2 mM
KH.sub.2PO.sub.4 [0502] 1 mM KNO.sub.3 [0503] 1 mM MgSO.sub.4
[0504] 10 mM CaCl.sub.2 [0505] 1.times.K3 micronutrients [0506]
filter-sterilize
[0507] ELISA analysis of transformed protoplasts is done one day
after transformation. ELISA's are done as previously described The
following three experiments are done with maize inbred line 211D.
Of course, other lines of maize may be used. 50 ug of plasmid
pCIB4419 and equimolar amounts of other plasmids are used. Total
soluble protein is determined using the BioRad protein assay.
(Bradford, Anal. Biochem, 72:248 (1976).
Transformation Experiment:
Constructs Tested:
[0508] 1. pCIB4419 (Construct contains synthetic Bt under control
of CaMV 35S promoter and 35S/PAT and 35S/GUS marker genes) [0509]
2. pCIB4420 (Construct contains synthetic Bt under control of Pith
promoter and PAT marker gene) [0510] 3. pCIB4421 (Construct
contains synthetic Bt under control of PEPC promoter) [0511] 4.
pCIB4423 (Construct contains synthetic Bt under control of PEPC
promoter and PAT marker gene) (PEPC:synthetic-cryIA(b):PepC
intron:35S+35S:PAT:35S)
[0512] In the following experiments, 10 or 11 day old 211D
seedlings are analyzed for production of the Bt CryIA(b) protein in
the Biorad protein assay:
[0513] Experiment 1 (11 Day Seedlings): TABLE-US-00031 pCIB4419
15,000 .+-. 3,000 ng Bt/mg protein pCIB4420 280 .+-. 65 ng Bt/mg
protein pCIB4421 9,000 .+-. 800 ng Bt/mg protein
[0514] Experiment 2 (10 Day Seedlings): TABLE-US-00032 pCIB4419
5,000 .+-. 270 ng Bt/mg protein pCIB4420 80 .+-. 14 ng Bt/mg
protein pCIB4421 1,600 .+-. 220 ng Bt/mg protein
[0515] Experiment 3 (11 Day Seedlings): TABLE-US-00033 pCIB4419
21,500 .+-. 1,800 ng Bt/mg protein pCIB4420 260 .+-. 50 ng Bt/mg
protein pCIB4421 11,900 .+-. 4,000 ng Bt/mg protein pCIB4423 7,200
.+-. 3,400 ng Bt/mg protein
[0516] The above experiments confirm that both the CaMV 35S and
PEPC promoters express the synthetic Bt CryIA(b) protein at very
high levels. The pith promoter, while less efficient, is also
effective for the expression of synthetic CryIA(b) protein.
Example 44
Stable Expression of Synthetic Bt in Lettuce
[0517] The synthetic Bt gene in the Agrobacterium vector pCIB4417
is transformed into Lactuca sativa cv. Redprize (lettuce). The
transformation procedure used is described in Enomoto et al., Plant
Cell Reports, 9:6-9 (1990).
Transformation Procedure:
[0518] Lettuce seeds are suface sterilized in 5% Clorox for 5
minutes followed by several washes in sterile distilled water.
Surface-sterilized seeds are plated on half strength MS media
(Murashige and Skoog, Physiol. Plant. 15:473-497 (1962)).
[0519] Cotyledons of 6-day-old Redprize seedlings, grown under
illumination of 3,000 lx 16 hr at 25 C, are used as the explants
for Agrobacterium infection. The base and tip of each cotyledon are
removed with a scalpel. The explants are soaked for 10 minutes in
the bacterial solution which have been cultured for 48 hours in AB
minimal media with the apropriate antibiotics at 28 C. After
blotting excess bacterial solution on sterile filter paper, the
explants are plated on MS media (0.1 mg/l BA and 0.1 mg/l NAA) for
2 days. Explants are then transferred to selective media containing
500 mg/l carbenicillin and 50 mg/l kanamycin. The explants are
subcultured to fresh media weekly. The growth chamber conditions
are 16 hour 2,000 lx light at 25 C. After approximately 4 weeks, an
ELISA is done on healthy looking callus from each of four plates
being subcultured. The ELISA procedure is the same as described
above for protoplasts; soluble protein is again determined by the
Biorad assay described above.
[0520] Results: TABLE-US-00034 pCIB3021 (kan control) 0 pCIB4417
(plate 1) 0 pCIB4417 (plate 2) 505 ng Bt/mg protein pCIB4417 (plate
3) 45 ngBt/mg protein pCIB4417 (plate 4) 1,200 ng Bt/mg protein
This example demonstrates that dicot plants can also show increased
expression of the optimized insecticidal gene.
Example 45
Construction of pCIB4429
[0521] pCIB4429 contains a preferred maize pollen-specific promoter
fused with the maize optimized cryIA(b) gene. The pollen-specific
maize promoter used in this construct was obtained from the plasmid
pKL2, described in Example 37. The maize optimized cryIA(b) gene
was obtained from plasmid pCIB4418, also described in Example
37.
[0522] pKL2 is a plasmid that contains a preferred maize
pollen-specific promoter fused with the E. coli beta-glucuronidase
gene. It was constructed from plasmids pSK110 and pCIB3054. pSK110
contains the pollen specific maize promoter. pCIB3054, a pUC19
derivative, contains the E. coli beta-glucuronidase (GUS) gene
fused with the cauliflower mosaic virus (CaMV) 35S promoter. It's
construction is described elsewhere in this application. This
promoter can be removed from this plasmid by cutting with
SalI/HindIII to yield a fragment containing the GUS gene, a
bacterial ampicillin resistance gene and a ColEI origin of
replication. A second fragment contains the CaMV 35S promoter.
[0523] pCIB3054 was cut with the restriction enzymes SalI and
HindIII, using standard conditions, for 2 hours at room
temperature. The reaction was then extracted with phenol/chloroform
using standard conditions and the DNA recovered by ethanol
precipitation using standard conditions. The recovered DNA was
resuspended in buffer appropriate for reaction with calf intestinal
alkaline phosphatase (CIP) and reacted with 2.5 units of CIP at
37.degree. C. overnight. After the CIP reaction, the DNA was
purified on an agarose gel using standard conditions described
elsewhere in this application. pSK110 was cut with SalI/HindIII
under standard conditions for 2 hours at room temperature and the
DNA subsequently purified on an agarose gel using standard
conditions. The recovered DNA fragments were ligated using standard
conditions for two hours at room temperature and subsequently
transformed into competent E. coli strain HB101 cells using
standard conditions. Transformants were selected on L-agar
containing 100 .mu.g ampicillin/ml. Transformants were
characterized for the desired plasmid construct using standard
plasmid mini-screen procedures. The correct construct was named
pKL2.
[0524] To make pCIB4429, a three way ligation was performed using
standard conditions known to those in the art. The three fragments
ligated were:
[0525] 1) a HindIII/BamHI fragment from pCIB4418, of about 4.7 kb
in size, containing the cryIA(b) gene, the bacterial ampicillin
resistance gene, and the ColEI origin of replication
[0526] 2) a HindIII/XbaI fragment from pKL2 of about 1.3 kb in size
and containing the pollen specific promoter from maize
[0527] 3) a PCR generated fragment derived from the pollen promoter
with a BamHI site introduced downstream from the start of
transcription. This fragment is approximately 120 bp and has ends
cut with the restriction enzymes XbaI/BamHI.
[0528] The PCR fragment was generated using a 100 .mu.l reaction
volume and standard conditions described above. The primers used
were: TABLE-US-00035 SK50: (SEQ ID NO:84) 5'-CCC TTC AAA ATC TAG
AAA CCT-3' KE127: (SEQ ID NO:92) 5'-GCG GAT CCG GCT GCG GCG GGG AAC
GA-3'
[0529] The above primers were mixed in a PCR reaction with plasmid
pSK105, a plasmid that contains the pollen specific promoter from
maize.
[0530] After the PCR reaction was complete, 10 .mu.l of the
reaction was run on an agarose gel, using standard condition, to
make sure the reaction produced the expected size product. The
remaining 90 .mu.l was treated with proteinase K at a final
concentration of 50 .mu.g/ml for 30 min. at 37.degree. C. The
reaction was then heated at 65.degree. C. for 10 min., then
phenol/chloroform extracted using standard procedures. The DNA was
recovered from the supernatant by precipitating with two volumes of
ethanol using standard conditions. After precipitation, the DNA was
recovered by centrifuging in a microfuge. The pellet was rinsed one
time with 70% ethanol (as is standard in the art), briefly dried to
remove all ethanol, and the pellet resuspended in 17 .mu.l TE
buffer. 2 .mu.l of 10.times. restriction enzyme buffer was added as
were 0.5 .mu.l BamHI and 0.5 .mu.l XbaI. The DNA was digested for 1
hour at 37.degree. C. to produce a DNA fragment cut with
XbaI/BamHI. After digestion with the restriction enzymes, this
fragment was purified on an agarose gel composed of 2% NuSieve
(FMC)/1% agarose gel. Millipore filter units were used to elute the
DNA from the agarose using the manufacturer's specifications. After
elution, the DNA was used in the three-way ligation described
above.
[0531] After ligation, the DNA was transformed into competent E.
coli strain HB101 cells using standard techniques. Transformants
were selected on L-agar plates containing ampicillin at 100
.mu.g/ml. Colonies that grew under selective conditions were
characterized for plasmid inserts using techniques standard in the
art.
Example 46
Construction of pCIB4431, a Vector for Tissue Specific Expression
of the Synthetic cryIA(b) Gene in Plants
[0532] pCIB4431 is a vector designed to transform maize. It
contains two chimeric Bt endotoxin genes expressible in maize.
These genes are the PEP carboxylase promoter/synthetic-cryIA(b) and
a pollen promoter/synthetic-cryIA(b). The PEP carboxylase/cryIA(b)
gene in this vector is derived from pCIB4421 described above. The
pollen promoter is also described above. FIG. 20 is a map of
plasmid pCIB4431. pCIB4431 was constructed via a three part
ligation using the about 3.5 Kb Kpn I/Hind III fragment (containing
pollen/synthetic-cryIA(b) from pCIB4429, the about 4.5 Kb Hind
III/Eco RI (PEPC/synthetic-cryIA(b) and the about 2.6 Kb Kpn I/Eco
RI fragment from the vector Bluescript.
[0533] Other vectors including the pollen promoter/synthetic
CryIA(b) chimeric gene include pCIB4428 and pCIB4430. See FIGS. 21
and 22. pCIB4430 also contains the PEPC/synthetic-Bt gene described
above.
Example 47
Production of Transgenic Maize Plants Containing the Synthetic
Maize Optimized CryIA(b) Gene
[0534] The example below utilizes Biolistics to introduce DNA
coated particles into maize cells, from which transformed plants
are generated.
Experiment KC-65
Production of Transgenic Maize Plants Expressing the Synthetic
cryIA(b) Gene Using a Tissue-Specific Promoter.
Tissue
[0535] Immature maize embryos, approximately 1.5-2.5 mm in length,
were excised from an ear of genotype 6N615 14-15 days after
pollination. The mother plant was grown in the greenhouse. Before
excision, the ear was surface sterilized with 20% Clorox for 20
minutes and rinse 3 times with sterile water. Individual embryos
were plated scutellum side in a 2 cm square area, 36 embryos to a
plate, on the callus initiation medium, 2DG4+5 chloramben medium
(N6 major salts, B5 minor salts, MS iron, 2% sucrose, with 5 mg/l
chloramben, 20 mg/l glucose, and 10 ml G4 additions (Table 1) added
after autoclaving. TABLE-US-00036 TABLE 1 G4 Additions Ingredient
per liter medium Casein hydrolysate 0.5 gm Proline 1.38 gm
Nicotinic acid .2 mg Pyridoxine-HCl .2 mg Thiamine-HCl .5 mg
Choline-HCl .1 mg Riboflavin .05 mg Biotin .1 mg Folic acid .05 mg
Ca pantothenate .1 mg p-aminobenzoic acid .05 mg B12 .136 .mu.g
Bombardment
[0536] Tissue was bombarded using the PDS-1000He Biolistics device.
The tissue was placed on the shelf 8 cm below the stopping screen
shelf. The tissue was shot one time with the DNA/gold microcarrier
solution, 10 .mu.l dried onto the macrocarrier. The stopping screen
used was hand punched at ABRU using 10.times.10 stainless steel
mesh screen. Rupture discs of 1550 psi value were used. After
bombardment, the embryos were cultured in the dark at 25.degree.
C.
Preparation of DNA for Delivery
[0537] The microcarrier was prepared essentially according to the
instructions supplied with the Biolistic device. While vortexing 50
.mu.l 1.0.mu. gold microcarrier, added 5 .mu.l pCIB4431 (1.23
.mu.g/.mu.l) (#898)+2 .mu.l pCIB3064 0.895 .mu.g/.mu.l) (#456)
followed by 50 .mu.l 2.5 M CaCl.sub.2, then 20 .mu.l 0.1 M
spermidine (free base, TC grade). The resulting mixture was
vortexed 3 minutes and microfuged for 10 sec. The supernatant was
removed and the icrocarriers washed 2 times with 250 .mu.l of 100%
EtOH(HPLC grade) by vortexing briefly, centrifuging and removing
the supernatant. The microcarriers are resuspended in 65 .mu.l 100%
EtOH.
Callus Formation
[0538] Embryos were transferred to callus initiation medium with 3
mg/l PPT 1 day after bombardment. Embryos were scored for callus
initiation at 2 and 3 weeks after bombardment. Any responses were
transferred to callus maintenance medium, 2DG4+0.5 2,4-D medium
with 3 mg/L PPT. Callus maintenance medium is N6 major salts, B5
minor salts, MS iron, 2% sucrose, with 0.5 mg/l 2,4-D, 20 mg/l
glucose, and 10 ml G4 additions added after autoclaving.
Embryogenic callus was subcultured every 2 weeks to fresh
maintenance medium containing 3 mg/L PPT. All callus was incubated
in the dark at 25.degree. C.
[0539] The Type I callus formation response was 15%. Every embryo
which produced callus was cultured as an individual event giving
rise to an individual line.
Regeneration
[0540] After 12 weeks on selection, the tissue was removed from
callus maintenance edium with PPT and was placed on regeneration
medium. Regeneration medium is 0.25MS3S5BA (0.25 mg/l 2,4 D, 5 mg/l
BAP, MS salts, 3% sucrose) for 2 weeks followed by subculture to
MS3S medium for regeneration of plants. After 4 to 10 weeks, plants
were removed and put into GA 7's. Our line KC65 0-6, which became
the #176 BT event, produced a total of 38 plants.
Assays
[0541] All plants, as they became established in the GA7's, were
tested by the chlorophenol red (CR) test for resistance to PPT as
described in U.S. patent application Ser. No. 07/759,243, filed
Sep. 13, 1991, the relevant portions of which are hereby
incorporated herein by reference This assay utilizes a pH sensitive
indicator dye to show which cells are growing in the presence of
PPT. Cells which grow produce a pH change in the media and turn the
indicator yellow (from red). Plants expressing the resistance gene
to PPT are easily seen in this test. (#176=8 positive/30 negative)
Plants positive by the CR test were assayed by PCR for the presence
of the synthetic BT gene. (#176=5 positive/2 negative/1 dead)
[0542] Plants positive by PCR for the syn-BT gene were sent to the
phytotron. Once established in the phytotron, they were
characterized using insect bioassays and ELISA analysis. Plants
were insect bioassayed using a standard European Corn Borer assay
(described in Example 5A) in which small pieces of leaf of clipped
from a plant and placed in a small petri dish with a number of ECB
neonate larvae. Plants are typically assayed at a height of about 6
inches. Plants showing 100% mortality to ECB in this assay are
characterized further. ELISA data are shown below. Positive plants
are moved to the greenhouse.
Greenhouse/Fertility
[0543] Plant number #176-11 was pollinated with wild-type 6N615
pollen. One tassel ear and one ear shoot were produced. All of the
embryos from the tassel ear (11) and 56 kernels from Ear 1 were
rescued. 294 kernels remained on the ear and dried down
naturally.
[0544] Pollen from #176-11 was outcrossed to various maize
genotypes 5N984, 5NA89, and 3N961. Embryos have been rescued from
all 3 outcrosses (5N984=45; 5NA89=30; 3N961=8). Most of the kernels
remained on the ears on the plants in the greenhouse and were dried
down naturally. DNA was isolated from plant #176-11 using standard
techniques and analysed by Southern blot analysis. It was found to
contain sequences which hybridize with probes generated from the
synthetic cryIA(b) gene and with a probe generated from the PAT
gene. These results showed integration of these genes into the
genome of maize.
Experiment KC-64
Production of Transgenic Maize Plants Expressing the Synthetic
cryIA(b) Gene Using a Constitutive Promoter.
Tissue
[0545] Immature maize embryos, approximately 1.5-2.5 mm in length,
were excised from an ear of genotype 6N615 14-15 days after
pollination. The mother plant was grown in the greenhouse. Before
excision, the ear was surface sterilized with 20% Clorox for 20
minutes and rinse 3 times with sterile water. Individual embryos
were plated scutellum side in a 2 cm square area, 36 embryos to a
plate, on the callus initiation medium, 2DG4+5 chloramben medium
(N6 major salts, B5 minor salts, MS iron, 2% sucrose, with 5 mg/l
chloramben, 20 mg/l glucose, and 10 ml G4 additions Table 1) added
after autoclaving. TABLE-US-00037 TABLE 1 G4 Additions Ingredient
per liter medium Casein hydrolysate 0.5 gm Proline 1.38 gm
Nicotinic acid .2 mg Pyridoxine-HCl .2 mg Thiamine-HCl .5 mg
Choline-HCl .1 mg Riboflavin .05 mg Biotin .1 mg Folic acid .05 mg
Ca pantothenate .1 mg p-aminobenzoic acid .05 mg B12 .136 .mu.g
Bombardment
[0546] Tissue was bombarded using the PDS-1000He Biolistics device.
The tissue was placed on the shelf 8 cm below the stopping screen
shelf. The tissue was shot one time with the DNA/gold microcarrier
solution, 10 .mu.l dried onto the macrocarrier. The stopping screen
used was hand punched at ABRU using 10.times.10 stainless steel
mesh screen. Rupture discs of 1550 psi value were used. After
bombardment, the embryos were cultured in the dark at 25.degree.
C.
Preparation of DNA for Delivery
[0547] The microcarrier was prepared essentially according to the
instructions supplied with the Biolistic device. While vortexing 50
.mu.l 1.0.mu. gold microcarrier, added 3.2 .mu.l pCIB4418 (0.85
.mu.g/.mu.l) (#905)+2 .mu.l pCIB3064 0.895 .mu.g/.mu.l) (#456)+1.6
.mu.l pCIB3007A (1.7 .mu.g/.mu.l (#152) followed by 50 .mu.l 2.5 M
CaCl.sub.2, then 20 .mu.l 0.1 M spermidine (free base, TC grade).
The resulting mixture was vortexed 3 minutes and microfuged for 10
sec. The supernatant was removed and the microcarriers washed 2
times with 250 .mu.l of 100% EtOH (HPLC grade) by vortexing
briefly, centrifuging and removing the supernatant. The
microcarriers are resuspended in 65 .mu.l 100% EtOH.
Callus Formation
[0548] Embryos were transferred to callus initiation medium with 3
mg/l PPT 1 day after bombardment. Embryos were scored for callus
initiation at 2 and 3 weeks after bombardment. Any responses were
transferred to callus maintenance medium, 2DG4+0.5 2,4-D medium
with 3 mg/L PPT. Callus maintenance medium is N6 major salts, B5
minor salts, MS iron, 2% sucrose, with 0.5 mg/l 2,4-D, 20 mg/l
glucose, and 10 ml G4 additions added after utoclaving. Embryogenic
callus was subcultured every 2 weeks to fresh maintenance medium
containing 3 mg/L PPT. All callus was incubated in the dark at
25.degree. C.
[0549] The Type I callus formation response was 18%. Every embryo
which produced callus was cultured as an individual event giving
rise to an individual line.
Regeneration
[0550] After 12 weeks on selection, the tissue was removed from
callus maintenance medium with PPT and was placed on regeneration
medium and incubated at 25.degree. C. using a 16 hour light (50
.mu.E .m-2. s-1)/8 hour dark photoperiod. Regeneration medium is
0.25MS3S5BA (025 mg/l 2,4 D, 5 mg/l BAP, MS salts, 3% sucrose) for
2 weeks followed by subculture to MS3S medium for regeneration of
plants. After 4 to 10 weeks, plants were removed and put into GA
7's. Our line KC64 0-1, which became the #170 BT event, produced 55
plants. Our line KC64 0-7, which became the #171 BT event, produced
a total of 33 plants.
Assays
[0551] Eleven plants, as they became established in the GA7's, were
tested by the chlorophenol red (CR) test for resistance to PPT as
per Shillito, et al, above. This assay utilizes a pH sensitive
indicator dye to show which cells are growing in the presence of
PPT. Cells which grow produce a pH change in the media and turn the
indicator yellow (from red). Plants expressing the resistance gene
to PPT are easily seen in this test. Plants positive by the CR test
were assayed by PCR for the presence of the synthetic BT gene.
(Event 170=37 positive/18 negative; #171=25 positive/8
negative).
[0552] Plants positive by PCR for the syn-Bt gene were sent to the
phytotron. Once established in the phytotron, they were
characterized using insect bioassays and ELISA analysis. Plants
were insect bioassayed using a standard European corn borer assay
(see below) in which small pieces of leaf of clipped from a lant
and placed in a small petri dish with a number of ECB neonate
larvae. Plants are typically assayed at a height of about 6 inches.
Plants showing 100% mortality to ECB in this assay are
characterized further. ELISA data are shown below. Positive plants
are moved to the greenhouse.
Basta Screening
[0553] Eight of the mature plants from the #170 event were selected
for evaluation of Basta (Hoechst) resistance. On one middle leaf
per plant, an area approximately 1014 cm long.times.the leaf width
was painted with 0, 0.4, 1.0 or 2.0% (10 ml of 200 g/L diluted to
100 ml with deionized water) aqueous Basta containing 2 drops of
Tween 20/100 ml. Two plants were tested per level. Eight wild-type
6N615 plants of the same approximate age were treated as controls.
All plants were observed at 4 and 7 days. All of the control plants
eventually died. Throughout the study, none of the #170 plants
displayed any damage due to the herbicide.
Pollination
[0554] All tassel ears, first ear and, if available, the second ear
on the #170 and #171 plants were pollinated with wild-type 6N615
pollen. At least 90% of the plants were female fertile.
[0555] Pollen from #171 plants was outcrossed to genotypes 6N615,
5N984, 5NA89, 6F010, 5NA56, 2N217AF, 2NDO1 and 3N961. At least 90%
of the plants were shown to be male fertile.
Embryo Rescue
[0556] Embryos from the #171 event have been "rescued." Fourteen to
16 days after pollination, the ear tip with 25-50 kernels was cut
from the ear with a coping saw. Prior to cutting, the husks were
gently peeled away to expose the upper portion of the ear. The cut
end of the ear on the plant was painted with Captan fungicide and
the husks replaced. The seed remaining on the plant was allowed to
dry naturally.
[0557] The excised ear piece was surface sterilized with 20% Clorox
for 20 minutes and rinsed 3 times with sterile water. Individual
embryos were excised and plated scutellum side up on B5 medium
(Gamborg) containing 2% sucrose. B5 vitamins are added to the
medium after autoclaving. Four embryos were plated per GA7
container and the containers incubated in the dark. When
germination occurred, the containers were moved to a light culture
room and incubated at 25.degree. C. using a 16 hour light (50 .mu.E
.m-2. s-1)/8 hour dark photoperiod. The germination frequency is
94%.
[0558] Progeny from 15 plants of the #171 event and 2 of the #176
event were rescued using standard embryo rescue techniques and
evaluated. All plants were evaluated by insect assay. Plants from
the #171 event were also tested in the histochemical GUS assay. In
both the insect assay and the GUS assay, the ratio of segregation
of the transgenes was 1:1, as expected for a single locus insertion
event.
Example 48
Analysis of Transgenic Maize Plants
[0559] ELISA Assay
[0560] Detection of cryIA(b) gene expression in transgenic maize is
monitored using European corn borer (ECB) insect bioassays and
ELISA analysis for a quantitative determination of the level of
cryIA(b) protein obtained.
[0561] Quantitative determination of cryIA(b) IP in the leaves of
transgenic plants was performed using enzyme-linked immunosorbant
assays (ELISA) as disclosed in Clark M F, Lister R M, Bar-Joseph M:
ELISA Techniques. In: Weissbach A, Weissbach H (eds) Methods in
Enzymology 118:742-766, Academic Press, Florida (1986).
Immunoaffinity purified polyclonal rabbit and goat antibodies
specific for the B. thuringiensis subsp. kurstaki IP were used to
determine ng IP per mg soluble protein from crude extracts of leaf
samples. The sensitivity of the double sandwich ELISA is 1-5 ng IP
per mg soluble protein using 50 ug of total protein per ELISA
microtiter dish well.
[0562] Corn extracts were made by grinding leaf tissue in gauze
lined plastic bags using a hand held ball-bearing homogenizer
(AGDIA, Elkart Ind.) in the presence of extraction buffer (50 mM
Na.sub.2CO.sub.3 pH 9.5, 100 mM NaCl, 0.05% Triton, 0.05% Tween, 1
mM PMSF and 1 .mu.M leupeptin). Protein determination was performed
using the Bio-Rad (Richmond, Calif.) protein assay.
[0563] Using the above procedure, the primary maize transformants
described above were analyzed for the presence of cryIA(b) protein
using ELISA. These plants varied in height from 6 inches to about
three feet at the time of analysis. TABLE-US-00038 Plant Bt ng/mg
soluble protein May 27, 1091 176-8 0 0 176-10 700 1585 176-11 760
2195 171-4A 59 171-6 50 171-8 60 171-9 280 171-13 77 171-14A 43
171-14B 60 171-15 55 171-16A 13 171-16B 19 171-18 19 176-30 1160
171-32 980 171-31 166 171-30 370 71-14 #10 leaf 26 1 leaf 17 plant
171-16 #9 leaf 40 #1 leaf 120
[0564] European Corn Borer Assay [0565] 1. One to four 4 cm
sections are cut from an extended leaf of a corn plant. [0566] 2.
Each leaf piece is placed on a moistened filter disc in a
50.times.9 mm petri dish. [0567] 3. Five neonate European corn
borer larvae are placed on each leaf piece. (Making a total of 5-20
larvae per plant.) [0568] 4. The petri dishes are incubated at
29.5.degree. C. [0569] 5. Leaf feeding damage and mortality data
are scored at 24, 48, and 72 hours.
Example 49
Expression of Bt Endotoxin in Progeny of Transformed Maize
Plants
[0570] The transformed maize plants were fully fertile and were
crossed with several genotypes of maize. Progeny from these crosses
were analyzed for their ability to kill European corn borer (ECB)
in a standard ECB bioassay (described immediately above) as well as
for the presence of the cryIA(b) protein using ELISA as described
above. The ability to kill ECB and the production of cryIA(b)
protein correlated. These traits segregated to the progeny with a
1:1 ratio, indicating a single site of insertion for the active
copy of the synthetic gene. This 1:1 ratio was true for both the
constitutive promoter/synthetic-cryIA(b) plants and the tissue
specific promoter/synthetic-cryIA(b) plants (data not shown).
[0571] FIG. 23A is a table containing a small subset of the total
number of progeny analyzed. This table is representative of a
number of different crosses.
[0572] Insect assays were done with Diatrea saccharalis and
Ostrinia nubilalis using leaf material (as described above) of
transgenic progeny containing a maize optimized CryIA(b) gene. The
results of these assays are shown in FIG. 23B. They demonstrate
that the maize optimized CryIA(b) gene functions in transformed
maize to provide resistance to Sugarcane borer and Ostrinia
nubilalis.
Example 50
Expression of the CryIA(b) Gene in Maize Pollen
[0573] Progeny of the transformed maize plants containing the
chimeric pollen promoter/synthetic cryIA(b) gene derived from
pCIB4431 were grown in the field to maturity. Pollen was collected
and analyzed for the presence of the cryIA(b) protein using
standard ELISA techniquesd as described elsewhere. High levels of
cryIA(b) protein were detected in the pollen. Progeny from the 35S
promoter/synthetic cryIA(b) transformed plant were grown in the
greenhouse. Pollen from these plants was analyzed using ELISA, and
cryIA(b) protein was detected. Results are shown below in FIG.
23C.
[0574] It is recognized that factors including selection of plant
lines, plant genotypes, synthetic sequences and the like, may also
affect expression.
Example 51
Expression of the CryIA(b) Gene Fused to a Pith-Preferred
Promoter
[0575] pCIB4433 (FIG. 36) is a plasmid containing the maize
optimized CryIA(b) gene fused with the pith-preferred promoter
isolated from maize. This plasmid was constructed using a three-way
ligation consisting of:
[0576] 1) pCIB4418, cut with BstEII and BamHI; 1.8 Kb fragment
[0577] 2) pBtin1, cut with NsiI and BstEII; 5.9 Kb fragment; pBtin1
is described elsewhere in this application
[0578] 3) PCR fragment VI-151 was generated in a PCR reaction using
standard conditions as described elsewhere in this application.
[0579] PCR Primers Utilized were: TABLE-US-00039 KE150A28: (SEQ ID
NO:93) 5'-ATT CGC ATG CAT GTT TCA TTA TC-3' KE151A28: (SEQ ID
NO:94) 5'-GCT GGT ACC ACG GAT CCG TCG CTT CTG TGC AAC AAC C-3'
[0580] After the PCR reaction, the DNA was checked on an agarose
gel to make sure the reaction had proceeded properly. DNA was
recovered from the PCR reaction using standard conditions described
elsewhere and subsequently cut with the restriction enzymes NsiI
and BamHI using standard condition. After cutting, the fragment was
run on a 2% NuSieve gel and the desired band recovered as described
elsewhere. The DNA was used in the ligation described above.
[0581] After ligation (under standard condition), the DNA was
transformed into competent E. coli cell.
[0582] Transformation was carried out using microprojectile
bombardment essentially as described elsewhere in this application.
Embryos were transferred to medium containing 102 .mu.g/ml PPT 24
hours after microprojectile bombardment. Resulting callus was
transferred to medium containing 40 .mu.g/ml PPT after four weeks.
Plants were regenerated without selection.
[0583] A small sample of plants (3-5) was assayed by PCR for each
event. Further codes were added to indicate different positions and
distances of embryos with respect to the microprojectile
bombardment device. Plants were sent to the greenhouse having the
following codes: TABLE-US-00040 JS21A TOP Plants B.t. PCR Positive
JS21A MID Plants B.t. PCR Positive JS21C BOT Plants B.t. PCR
Positive JS22D MID Plants B.t. PCR Positive JS23B MID Plants B.t.
PCR Negative (for control)
[0584] Leaf samples from the regenerated plants were bioassayed for
insecticidal activity against European corn borer as described in
Example 48 with the results shown in FIG. 23D.
[0585] ELISA analysis of leaf samples to quantify the level of
CryIA(b) protein expressed in the leaves was carried out as
described in Example 48 with the results shown in FIG. 23E.
Deposits
[0586] The following plasmids have been deposited with the
Agricultural Research Culture Collection (NRRL) (1818 N. University
St., Peoria, Ill. 61604) under the provisions of the Budapest
Treaty: pCIB4418, pCIB4420, pCIB4429, pCIB4431, pCIB4433, pCIB5601,
pCIB3166 and pCIB3171.
[0587] The present invention has been described with reference to
specific embodiments thereof; however it will be appreciated that
numerous variations, modifications, and embodiments are possible.
Accordingly, all such variations, modifications and embodiments are
to be regarded as being within the spirit and scope of the present
invention.
Sequence CWU 1
1
* * * * *