U.S. patent application number 15/630568 was filed with the patent office on 2017-10-26 for methods for transferring molecular substances into plant cells.
The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to Frank G. Burroughs, Suraj K. Dixit, Jayakumar Pon Samuel, Mark W. Zettler.
Application Number | 20170306339 15/630568 |
Document ID | / |
Family ID | 40239629 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170306339 |
Kind Code |
A1 |
Samuel; Jayakumar Pon ; et
al. |
October 26, 2017 |
METHODS FOR TRANSFERRING MOLECULAR SUBSTANCES INTO PLANT CELLS
Abstract
Provided are methods for introducing a molecule of interest into
a plant cell comprising a cell wall. Methods are provided for
genetically or otherwise modifying plants and for treating or
preventing disease in plant cells comprising a cell wall.
Inventors: |
Samuel; Jayakumar Pon;
(Carmel, IN) ; Burroughs; Frank G.; (Noblesville,
IN) ; Dixit; Suraj K.; (Bloomington, IN) ;
Zettler; Mark W.; (Carmel, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Family ID: |
40239629 |
Appl. No.: |
15/630568 |
Filed: |
June 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15272362 |
Sep 21, 2016 |
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15630568 |
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14275613 |
May 12, 2014 |
9476057 |
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15272362 |
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12245685 |
Oct 3, 2008 |
8722410 |
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14275613 |
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60978059 |
Oct 5, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8206 20130101;
C12N 15/8207 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 15/82 20060101 C12N015/82 |
Claims
1. A method of introducing a molecule of interest into a plant cell
having a cell wall, the method comprising: providing the plant cell
having a cell wall; coating a nanoparticle with a molecule of
interest, wherein the nanoparticle is a gold nanoparticle or a
quantum dot, and wherein the molecule of interest is an herbicide
or a fungicide; placing the cell having a cell wall and the coated
nanoparticle in contact with each other; and allowing uptake of the
nanoparticle and the molecule of interest into the cell comprising
a cell wall.
2. The method according to claim 1, wherein coating a nanoparticle
with a molecule of interest comprises immobilizing the molecule of
interest via noncovalent absorption on the surface of the
nanoparticle.
3. The method according to claim 1, further comprising absorbing
the molecule of interest into the nanoparticle.
4. The method according to claim 1, wherein the plant cell
comprising a cell wall is selected from the group consisting of
tobacco, carrot, maize, canola, rapeseed, cotton, palm, peanut,
soybean, Oryza sp., Arabidopsis sp., Ricinus sp., and sugarcane
cells.
5. The method according to claim 1, wherein the plant cell is from
a tissue selected from the group consisting of embryo,
meristematic, callus, pollen, leaves, anthers, roots, root tips,
flowers, seeds, pods and stems.
6. The method according to claim 1, further comprising derivatizing
the surface of the nanoparticle.
7. The method according to claim 1, further comprising selecting
cells that have stably integrated the molecule of interest.
8. The method according to claim 7, wherein the selected cells are
regenerable cells.
9. The method according to claim 8, further comprising regenerating
a plant from the selected cells.
10. The method according to claim 1, further comprising coating the
nanoparticle with a second molecule of interest.
11. The method according to claim 1, wherein the second molecule of
interest is selected from the group consisting of amino acids,
polypeptides, enzymes, peptide hormones, glycol-peptides, signaling
peptides, and antibodies.
12. The method according to claim 1, wherein the second molecule of
interest is selected from the group consisting of messengers,
second messengers, and cAMP.
13. The method according to claim 1, wherein the second molecule of
interest is selected from the group consisting of sugars, fats,
vitamins, herbicides, fungicides, and antibiotics.
14. A method of introducing a molecule of interest into a plant
cell having a cell wall, the method comprising: providing the plant
cell having a cell wall; coating a nanoparticle with a molecule of
interest, wherein the nanoparticle is a gold nanoparticle or a
quantum dot, and wherein the molecule of interest is selected from
the group consisting of a sugar, a fat, a vitamin, a drug, and an
antibiotic; placing the cell having a cell wall and the coated
nanoparticle in contact with each other; and allowing uptake of the
nanoparticle and the molecule of interest into the cell comprising
a cell wall.
15. The method according to claim 14, wherein coating a
nanoparticle with a molecule of interest comprises immobilizing the
molecule of interest via noncovalent absorption on the surface of
the nanoparticle.
16. The method according to claim 14, further comprising absorbing
the molecule of interest into the nanoparticle.
17. The method according to claim 14, wherein the plant cell
comprising a cell wall is selected from the group consisting of
tobacco, carrot, maize, canola, rapeseed, cotton, palm, peanut,
soybean, Oryza sp., Arabidopsis sp., Ricinus sp., and sugarcane
cells.
18. The method according to claim 14, wherein the plant cell is
from a tissue selected from the group consisting of embryo,
meristematic, callus, pollen, leaves, anthers, roots, root tips,
flowers, seeds, pods and stems.
19. The method according to claim 14, further comprising
derivatizing the surface of the nanoparticle.
20. The method according to claim 14, further comprising selecting
cells that have stably integrated the molecule of interest.
21. The method according to claim 20, wherein the selected cells
are regenerable cells.
22. The method according to claim 21, further comprising
regenerating a plant from the selected cells.
23. The method according to claim 14, further comprising coating
the nanoparticle with a second molecule of interest.
24. The method according to claim 14, wherein the second molecule
of interest is selected from the group consisting of amino acids,
polypeptides, enzymes, peptide hormones, glycol-peptides, signaling
peptides, and antibodies.
25. The method according to claim 14, wherein the second molecule
of interest is selected from the group consisting of messengers,
second messengers, and cAMP.
26. The method according to claim 14, wherein the second molecule
of interest is selected from the group consisting of sugars, fats,
vitamins, herbicides, fungicides, and antibiotics.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/272,362, filed Sep. 21, 2016, which is a
continuation of U.S. patent application Ser. No. 14/275,613, filed
May 12, 2014, which is a continuation of U.S. patent application
Ser. No. 12/245,685, filed Oct. 3, 2008, which issued as U.S. Pat.
No. 8,722,410 on May 13, 2014, which claims the benefit of U.S.
Provisional Application No. 60/978,059, filed Oct. 5, 2007, the
contents of each of which are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] Nanoparticles have unique properties that have been
exploited for use in the delivery of DNA to cells. Among all
nanoparticles investigated gold (Au) nanoparticles tend to be
excellent candidates for delivery because of their low cytotoxicity
and ease of functionalization with various ligands of biological
significance. The commonly used synthesis of Au nanoparticles
yields negatively charged (e.g., citrate coating) surface. Plasmid
DNA, which may be sufficiently flexible to partially uncoil its
bases, can be exposed to gold nanoparticles ("GNPs"). Under these
partially uncoiled conditions, the negative charge on the DNA
backbone may be sufficiently distant so that attractive van der
Waals forces between the bases and the gold nanoparticle are
sufficient to cause plasmid DNA to be attached to the surface of
the gold particle.
[0003] In addition to metal nanoparticles, semi-conductor
nanoparticles (e.g., quantum dots) ("QD") within the size range of
3-5 nm have also been used as carriers to deliver molecules into
cells. DNA and proteins can be linked to the ligand attached to the
QD surface (see, e.g., Patolsky, F., et al., J. Am. Chem. Soc. 125,
13918 (2003)). Carboxylic acid or amine coated QDs can be cross
linked to molecules containing a thiol group see, e.g., Dubertret
B. et al., Science 298, 1759 (2002); Akerman, M. E., W. C. W. Chan,
P. Laakkonen, S. N. Bhatia, E. Ruoslahti, Proc. Natl. Acad. Sci.
U.S.A. 99, 12617 (2002); Mitchell, G. P., C. A. Mirkin, R. L.
Letsinger, J. Am. Chem. Soc. 121, 8122 (1999) or an
N-hydroxysuccinimyl (NHS)ester group by using standard
bioconjugation protocols (see, e.g., Pinaud, F., D. King, H.-P.
Moore, S. Weiss, J. Am. Chem. Soc. 126, 6115 (2004); Bruchez, M.,
M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Science 281, 2013
(1998)). An alternative way is conjugation of streptavidin coated
QDs to biotinylated proteins, oligos or antibodies (see, e.g.,
Dahan M. et al., Science 302, 442 (2003); Pinaud, F., D. King,
H.-P. Moore, S. Weiss, J. Am. Chem. Soc. 126, 6115 (2004); Dahan M.
et al., Science 302, 442 (2003); Wu. X. Y., et al., Nature
Biotechnol. 21, 41 (2003); Jaiswal, J. K., H. Mattoussi, J. M.
Mauro, S. M. Simon, Nature Biotechnol. 21, 47 (2003); and Mansson,
A., et al., Biochem. Biophys. Res. Commun. 314, 529 (2004)).
[0004] Nanoparticles have been used to deliver plasmid DNA to a
variety of animal cells. It has been found that when DNA coated
nanoparticles are incubated with cells not having a cell wall, the
cells take up the nanoparticles and begin expressing any genes
encoded on the DNA. Where nanoparticle delivery to cells normally
having a cell wall is desired, the cells wall is stripped before
the addition of the particles to protoplasts of plant (see, Torney,
F. et al., Nature Nanotechnol. 2, (2007). In plant cells, the cell
wall stands as a barrier for the delivery of exogenously applied
molecules. Many invasive methods, like gene gun (biolistics),
microinjection, electroporation, and Agrobacterium, have been
employed to achieve gene and small molecule delivery into these
walled plant cells, but delivery of proteins have only been
achieved by microinjection. Delivery of small molecules and
proteins in the presence of a cell wall of a plant cell remains
unexplored and would be advantageous in order to develop enabling
technologies to be deployed in intact plant cell/tissue or organ
for in vitro and in vivo manipulations
[0005] The present invention relates to methods using nanoparticles
to non-invasively deliver molecular substances into cells having a
cell wall.
BRIEF SUMMARY OF THE INVENTION
[0006] The following embodiments are described in conjunction with
systems, tools and methods which are meant to be exemplary and
illustrative, and not limiting in scope.
[0007] According to the invention, there are provided methods of
introducing a molecule of interest into a plant cell that includes
a cell wall, the methods comprising: placing the plant cell having
a cell wall in contact with a nanoparticle and a molecule of
interest, and allowing uptake of the nanoparticle and the molecule
of interest into the cell.
[0008] Further provided are methods of introducing a molecule of
interest into a plant cell having a cell wall, the methods
comprising: placing the plant cell having a cell wall in contact
with a nanoparticle and a molecule capable of treating the disease
and allowing uptake of the nanoparticle and the molecule capable of
treating the disease into the cell.
[0009] In addition to the exemplary aspects and embodiments
described above, further aspects and embodiments will become
apparent in view of the following descriptions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 depicts photographs of single cells of BY2 viewed
using a differential Interference Contrast microscope attached to a
confocal imaging system (Panel A). Panel B shows a light
microscopic view of a single cell from a BY2 variant that is
stained with I2KI to highlight the plastid (Amyloplast).
[0011] FIG. 2, Pane A depicts photoautotrophic cells of tobacco
(NT1) maintained in minimal medium and 5% carbon dioxide as seen in
a light microscope, where prominent chloroplasts are visible. FIG.
2, Panel B, shows similar NT1 cells viewed under a fluorescent
microscope with active chloroplasts autofluorescing in red.
[0012] FIG. 3 shows BY2 suspension aggregates treated with SAMSA
fluorescein alone and with SAMSA fluorescein coated GNPs. FIG. 3,
Panel A, shows a DIC image of cells treated with SAMSA fluorescein
alone while FIG. 3, Panel B, shows the fluorescent image of the
same cells. FIG. 3, Panel C, shows a DIC image of cells treated
with SAMSA fluorescein coated GNPs while FIG. 3, Panel D, shows the
fluorescent image of the SAMSA fluorescein coated GNPs-treated
cells. Positions of Nucleus (Nu) and Cell Wall (CW) are as
indicated.
[0013] FIG. 4 shows SAMSA fluorescein coated GNP-treated single
cells under high magnification. Panel B shows the presence of large
number of GNPs in the nucleolus. Panel A shows a bright-field view
of the same nucleolus shown in Panel B under a different plane of
focus.
[0014] FIG. 5 shows photoautotrophic cells treated with SAMSA
fluorescein coated GNP. Panel A shows hyaline cells in 3-4 cell
clusters with large chloroplasts lining the inner side of the cell
wall. Panel B shows accumulation of nanoparticles in the
chloroplast. Panels C and D show higher power magnification of a
single chloroplast using a fluorescent microscope. Nanoparticles
are visible in the membrane lamellations of the chloroplast and
interspersed among the red autofluorescing chlorophyll
pigments.
[0015] FIG. 6 shows reflectance and fluorescent microscopic images
of cells containing nanoparticles. Panel A of FIG. 6 shows a
reflectance image where the GNPs are preferentially seen. Panel B
shows fluorescing particles within the background of red
autofluorescing chloroplast. A merged reflectance and fluorescent
image is shown in Panel C, wherein the yellow fluorescing particles
are within the boundary of the chloroplast.
[0016] FIG. 7 shows a graphical representation of one possible
transformation scheme according to an embodiment of the present
invention.
[0017] FIG. 8 shows cellular internalization of GFP as visualized
through reflectance microscopy after two hours of treatment. Panels
A and A1 show untreated control cells under DIC scope (Panel A),
and GFP tethered Au-NP treated cells as seen under DIC scope (Panel
A1); Panels B and B1 show control cells under reflectance scope
(Panel B), and GFP tethered Au-NP treated cells as seen under
reflectance scope (Panel B1), showing particle internalization from
the reflected Au-NPs; Panels C and C1 show control cells
superimposed images of DIC and reflectance scope (Panel C), and
treated cells superimposed images of DIC and reflectance scope
(Panel C1); Panels D and D1 show control cells reflectance inverted
image to show no particle in the background (Panel D), and treated
cells reflectance inverted image to show very clearly particle
internalization (Panel D1).
[0018] FIG. 9 shows SAMSA stain coated GNP internalization in
Single cells. Panel A shows fluorescein stained single cells, with
the cell wall and the medium showing fluorescence, but no
internalization of stain; Panel B shows single cells under DIC
scope; Panel C shows phase contrast imaging to show the
nanoparticle (GNP 150 nm) internalization into the cytosol and the
nucleus, with the fluorescein internalized only with the particle
and the plasmalyzed cells under prolonged exposure up to 1 hr in
the UV light.
[0019] FIG. 10 shows Au-GFP conjugate with fluorescing GFP
molecules, prior to mixing the single cells. Panel A (FITC), B
(Brightfield), C (Reflectance), D (Panels A+B+C):GFP fluorescing
Au-GNPs as observed through fluorescence microscopy, 2 hrs after
incubation, but prior to mixing cells. Similar fluorescing
particles could be seen on the particle showing reflectance in the
nucleus (see FIG. 8).
[0020] FIG. 11 shows nanoparticle (GNP 90 nm) mediated cellular
internalization of GFP into BY2-E cell lines. Panel A shows
dividing control cells with active cytoplasmic strands (Phase
contrast image); Panel B shows the same cells as in Panel A when
examined through FITC filter, where the autofluorescene from the
nongreen plastids in the cytoplasm form the periphery and also from
those plastids associated with the dividing nucleus; and where the
cytoplasmic strands and the cytoplasm near the periphery of the
cells do not show autofluorescence; Panel C shows control BY2 cells
treated with GFP that are not attached to GNPs (FITC), where the
cells do not show GFP uptake, but the GFP are surrounding the
cells, but are not internalized; Panel D shows GNP mediated GFP
internalization as observed through FITC filter, with the
peripheral cytoplasm, cytoplasmic strands and the nucleus showing
internalization of GFP as compared to the control in Panel B.
[0021] FIG. 12 shows BY2-E single cell lines showing GNP mediated
YFP internalization 2 hrs after incubation of cells. Panel A
(FITC), B (Rhodamine), C (DIC), D (Panels A+B), E (Panels A+B+C):F
(Reflectance image inverted):YFP internalization as observed
through fluorescence microscopy. Arrows in yellow show the
internalization in a live single cell with YFP in the cytosol
(diffused and concentrated in the nucleus). Arrows in orange show
internalization in a plasmalyzed cells where the shrunk protoplast
mass within the cell shows intense fluorescence indicating the YFP
internalization in the cell. This cell is found in the same focal
plane of the live cell that is placed adjacent, but below other
live cells. The cells that accumulate a high level of particle and
YFP fluorescence show cell death on prolonged examination under
fluorescent scope.
[0022] FIGS. 13 and 14 show gel images of PAT and YFP amplified
gene products were amplified.
[0023] FIG. 15 shows gel electrophoresis carried out on QD-peptide
conjugates to confirm the attachment of peptides to QDs.
[0024] FIG. 16 shows Plasmid pDAB3831.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In the description and tables which follow, a number of
terms are used. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided:
[0026] Backcrossing. Backcrossing may be a process in which a
breeder repeatedly crosses hybrid progeny back to one of the
parents, for example, a first generation hybrid F.sub.1 with one of
the parental genotypes of the F.sub.1 hybrid.
[0027] Embryo. The embryo may be the small plant contained within a
mature seed.
[0028] Nanoparticle. A microscopic particle with at least one
nanoscale dimension, usually less than 100 nm. Nanoparticles
suitable for use in the present invention may have a size of 1
nm-0.4 um. A quantum dot may have a median diameter of 1 nm-10 nm,
preferably 2-4 nm. The nanoparticle may be selected from: gold
nanoparticles, gold coated nanoparticles, porous nanoparticles,
mesoporous nanoparticles, silica nanoparticles, polymer
nanoparticles, tungsten nanoparticles, gelatin nanoparticles,
nanoshells, nanocores, nanospheres, nanorods, magnetic
nanoparticles, and combinations thereof.
[0029] Quantum dot. A quantum dot is a semiconductor nanostructure
that confines the motion of conduction band electrons, valence band
holes, or excitons (bound pairs of conduction band electrons and
valence band holes) in all three spatial directions. The
confinement can be due to electrostatic potentials (generated by
external electrodes, doping, strain, impurities), the presence of
an interface between different semiconductor materials (e.g., in
core-shell nanocrystal systems), the presence of the semiconductor
surface (e.g., semiconductor nanocrystal), or a combination of
these. A quantum dot can have a discrete quantized energy spectrum.
The corresponding wave functions are spatially localized within the
quantum dot, but extend over many periods of the crystal lattice. A
quantum dot contains a small finite number (of the order of 1-100)
of conduction band electrons, valence band holes, or excitons
(i.e., a finite number of elementary electric charges).
[0030] Resistant to Glyphosate. Resistance to a dosage of
glyphosate refers to the ability of a plant to survive (i.e., the
plant may be not killed) by that dosage of glyphosate. In some
cases, tolerant plants may temporarily yellow or otherwise exhibit
some glyphosate-induced injury (e.g., excessive tillering and/or
growth inhibition), but recover.
[0031] Stabilized. Stabilized refers to characteristics of a plant
that are reproducibly passed from one generation to the next
generation of inbred plants of the same variety.
[0032] Uptake. Uptake refers to the translocation of a particle,
such as a nanoparticle, for example, gold or quantum dots, across a
cell wall or a cellular membrane, wherein the translocation does
not occur solely as a result of momentum imparted to the particle
by something other than the cell into which the particle is being
uptaken. Non-limiting examples of devices or methods which cause
translocation of a particle across a cell wall or a cell membrane
solely as a result of momentum imparted to the particle are
biolistic, gene gun, microinjection, and/or impalefection
technologies.
[0033] According to embodiments the invention, there may be
provided a method of introducing a molecule of interest into a
plant cell comprising a cell wall, the method comprising placing a
nanoparticle containing, and a molecule of interest in contact
with, the plant cell and allowing uptake of the nanoparticle across
the plant cell wall. In particular aspects of invention, the
nanoparticle may be any nanoparticle and may reversibly or
irreversibly contain, be coated with, or otherwise be bound to
and/or carry a molecule of interest. In certain embodiments, a
molecule of interest may be introduced to the nanoparticles before
contact with a plant cell having a cell wall or concurrently with
the introduction of the nanoparticle to a plant cell having a cell
wall. Examples of nanoparticles that can be used in embodiments of
the present invention include, but are not limited to, gold,
quantum dots, gold coated nanoparticles, porous nanoparticles,
mesoporous nanoparticles, silica nanoparticles, polymer
nanoparticles, tungsten nanoparticles, gelatin nanoparticles,
nanoshells, nanocores, nanospheres, nanorods, magnetic
nanoparticles, and/or combinations thereof.
[0034] According to embodiments of the present invention, a plant
cell having a cell wall may be any plant cell comprising an intact
and whole cell wall. Examples of cells having a cell wall include,
but are not limited to, algal, tobacco, carrot, maize, canola,
rapeseed, cotton, palm, peanut, soybean, sugarcane, Oryza sp.,
Arabidopsis sp., and Ricinus sp., preferably tobacco, carrots
maize, cotton, canola, soybean and sugarcane; more preferably
tobacco and carrots. Embodiments of the invention may include cells
comprising a cell wall from any tissue or wherever they are found,
including but not limited to, in embryos, meristematic cells,
callus, pollen, leaves, anthers, roots, root tips, flowers, seeds,
pods, stems, and tissue culture.
[0035] In embodiments of the invention, a molecule of interest may
be any molecule that can be delivered to a plant cell according to
the present invention. Molecules of interest, or components of
molecules of interest, may comprise, but are not limited to,
nucleic acids, DNA, RNA, RNAi molecules, genes, plasmids, cosmids,
YACs, BACs, polypeptides, enzymes, hormones, glyco-peptides,
sugars, fats, signaling peptides, antibodies, vitamins, messengers,
second messengers, amino acids, cAMP, drugs, herbicides,
fungicides, antibiotics, and/or combinations thereof.
[0036] Embodiments of the invention include methods for the
prevention or treatment of disease. Non-limiting example
embodiments include the delivery of fungicides, antibiotics, and/or
other drugs to cells in need thereof using methods of the present
invention.
[0037] In particular embodiments of the invention, the surface of
the nanoparticle may be functionalized, which may, for example,
allow for targeted uptake or allow for reversible or irreversible
binding of other substances to the surface of the nanoparticle. By
way of non-limiting example, the surface of a nanoparticle (e.g.,
gold nanoparticle or quantum dots) might be functionalized with a
self-assembled monolayer of, for example, alkanethiolates, which
can be further functionalized or derivatized. In a further
non-limiting example, the surface of a nanoparticle may be
derivatized with linkers which themselves may be further
functionalized or derivatized. In one embodiment, a nanoparticle
may be PEGylated. In other embodiments, the nanoparticle may
comprise, or may be multifunctionalized with, one or more of a core
(active or inactive), a steric coat (active or inert), a cleavable
linkage, and/or a targeting molecule or ligand.
[0038] In aspects of the invention, the nanoparticle may be uptaken
into various parts of cells. Examples of locations that a
nanoparticle may be uptaken into include, but are not limited to,
cytosol, nucleus, tonoplasts, plastids, etioplasts, chromoplasts,
leucoplasts, elaioplasts, proteinoplasts, amyloplasts,
chloroplasts, and the lumen of a double membrane. In other
embodiments of the invention, nanoparticle uptake into a cell
comprising a cell wall may occur via the symplastic or apoplastic
pathway.
[0039] Additional embodiments of the invention include genetically
modified plant cells and methods for generating them, wherein the
plant cells have one or more nucleic acids introduced therein via
methods of the present invention. In one example of an embodiment,
a plasmid comprising a gene of interest and a selectable marker may
be in introduced into a plant cell having a cell well via a
nanoparticle according to the present invention. In further
embodiments, stable transformants may be selected that have stably
integrated the gene of interest and/or the selectable marker. In
alternative embodiments, a plant cell now comprising the gene of
interest may be propagated to produce other cells comprising a
molecule of interest. In other embodiments, plant cells now
comprising a molecule of interest may be a regenerable cell that
may be used to regenerate a whole plant including the molecule of
interest.
[0040] In another aspect, the present invention provides methods of
creating regenerable plant cells comprising a molecule of interest
for use in tissue culture. The tissue culture will preferably be
capable of regenerating plants having substantially the same
genotype as the regenerable cells. The regenerable cells in such
tissue cultures can be embryos, protoplasts, meristematic cells,
callus, pollen, leaves, anthers, roots, root tips, flowers, seeds,
pods or stems. Still further, an embodiment of the invention
provides plants regenerated from the tissue cultures of the
invention.
[0041] Alternatively, the present invention provides a method of
introducing a desired trait into a plant cell having a cell wall,
wherein the method comprises: placing a nanoparticle and a molecule
of interest capable of providing the desired trait to the plant
cell in contact with the cell and allowing uptake of the
nanoparticle across the cell wall. Examples of desired traits
include, but are not limited to, traits selected from male
sterility, herbicide resistance, insect resistance, and resistance
to bacterial disease, fungal disease, and/or viral disease.
[0042] Further aspects of the invention provide for the methods of
generating of stabilized plant lines comprising a desired trait or
molecule of interest, wherein the desired trait or molecule of
interest may be first introduced by uptake of a nanoparticle across
a plant cell wall. Methods of generating stabilized plant lines are
well known to one of ordinary skill in the art and may include
techniques such as, but not limited to, selfing, backcrosses,
hybrid production, crosses to populations, and the like. All plants
and plant cells comprising a desired trait or molecule of interest
first introduced into the plant cell (or its predecessors) by
uptake of a nanoparticle across a cell wall are within the scope of
this invention. Advantageously, the plant cells comprising a
desired trait or molecule of interest first introduced into the
plant or cell (or its predecessors) by uptake of a nanoparticle
across a cell wall can be used in crosses with other, different,
plant cells to produce first generation (F.sub.1) hybrid cells,
seeds, and/or plants with superior characteristics.
[0043] In embodiments wherein the molecule of interest comprises
one or more gene(s), the gene(s) may be a dominant or recessive
allele. By way of example, the gene(s) will confer such traits as
herbicide resistance, insect resistance, resistance for bacterial
resistance, fungal resistance, viral disease resistance, male
fertility, male sterility, enhanced nutritional quality, and
industrial usage.
[0044] With the advent of molecular biological techniques that have
allowed the isolation and characterization of genes that encode
specific protein or RNA products (e.g., RNAi), scientists in the
field of plant biology developed a strong interest in engineering
the genome of cells to contain and express foreign genes, or
additional or modified versions of native or endogenous genes
(perhaps driven by different promoters) in order to alter the
traits of a cell in a specific manner. Such foreign additional
and/or modified genes are referred to herein collectively as
"transgenes." Over the last fifteen to twenty years, several
methods for producing transgenic cells have been developed and, in
particular embodiments, the present invention relates to
transformed versions of cells and methods of producing them via
introducing into a cell having a cell wall a transgene via uptake
of a nanoparticle across a cell wall. In embodiments of the
invention, the transgene may be contained in an expression
vector.
[0045] Cell transformation may involve the construction of an
expression vector which will function in a particular cell. Such a
vector may comprise DNA that includes a gene under control of, or
operatively linked to, a regulatory element (for example, a
promoter). The expression vector may contain one or more such
operably linked gene/regulatory element combinations. The vector(s)
may be in the form of a plasmid and can be used alone or in
combination with other plasmids to provide transformed cells using
transformation methods as described herein to incorporate
transgene(s) into the genetic material of a plant cell comprising a
cell wall.
Expression Vectors for Uptake Via Nanoparticle: Marker Genes
[0046] Expression vectors may include at least one genetic marker,
operably linked to a regulatory element (a promoter, for example)
that allows transformed cells containing the marker to be either
recovered by negative selection (i.e., inhibiting growth of cells
that do not contain the selectable marker gene) or by positive
selection (i.e., screening for the product encoded by the genetic
marker). Many selectable marker genes for transformation are well
known in the transformation arts and include, for example, genes
that code for enzymes that metabolically detoxify a selective
chemical agent which may be an antibiotic or an herbicide, or genes
that encode an altered target which may be insensitive to the
inhibitor. A few positive selection methods are also known in the
art.
[0047] One commonly used selectable marker gene suitable for plant
transformation may include the neomycin phosphotransferase II
(nptII) gene under the control of plant regulatory signals, which
confers resistance to kanamycin. See, e.g., Fraley et al., Proc.
Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used
selectable marker gene may be the hygromycin phosphotransferase
gene, which confers resistance to the antibiotic hygromycin. See,
e.g., Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).
[0048] Additional selectable marker genes of bacterial origin that
confer resistance to antibiotics include gentamycin acetyl
transferase, streptomycin phosphotransferase,
aminoglycoside-3'-adenyl transferase, and the bleomycin resistance
determinant. See Hayford et al., Plant Physiol. 86:1216 (1988),
Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab et al., Plant
Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol. 7:171
(1986). Other selectable marker genes confer resistance to
herbicides such as glyphosate, glufosinate or bromoxynil. See Comai
et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell
2:603-618 (1990) and Stalker et al., Science 242:419-423
(1988).
[0049] Other selectable marker genes suitable for plant
transformation are not of bacterial origin. These genes include,
for example, mouse dihydrofolate reductase, plant
5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate
synthase. See Eichholtz et al., Somatic Cell Mol. Genet. 13:67
(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant
Cell Rep. 8:643 (1990).
[0050] Another class of marker genes suitable for plant
transformation requires screening of presumptively transformed
plant cells rather than direct genetic selection of transformed
cells for resistance to a toxic substance, such as an antibiotic.
These genes are particularly useful to quantify or visualize the
spatial pattern of expression of a gene in specific tissues and are
frequently referred to as reporter genes because they can be fused
to a gene or gene regulatory sequence for the investigation of gene
expression. Commonly used genes for screening transformed cells
include .beta.-glucuronidase (GUS), .beta.-galactosidase,
luciferase and chloramphenicol acetyltransferase. See Jefferson, R.
A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343
(1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987),
DeBlock et al., EMBO J. 3:1681 (1984).
[0051] Recently, in vivo methods for visualizing GUS activity that
do not require destruction of plant tissue have been made
available. Molecular Probes publication 2908, Imagene Green. T M.,
p. 1-4(1993) and Naleway et al., J. Cell Biol. 115:151a (1991).
However, these in vivo methods for visualizing GUS activity have
not proven useful for recovery of transformed cells because of low
sensitivity, high fluorescent backgrounds, and limitations
associated with the use of luciferase genes as selectable
markers.
[0052] More recently, genes encoding Fluorescent Proteins (e.g.,
GFP, EGFP, EBFP, ECFP, and YFP) have been utilized as markers for
gene expression in prokaryotic and eukaryotic cells. See Chalfie et
al., Science 263:802 (1994). Fluorescent proteins and mutations of
fluorescent proteins may be used as screenable markers.
Expression Vectors for Uptake Via Nanoparticle: Promoters
[0053] Genes included in expression vectors must be driven by a
nucleotide sequence comprising a regulatory element, for example, a
promoter. Several types of promoters are now well known in the
transformation arts, as are other regulatory elements that can be
used alone or in combination with promoters.
[0054] As used herein, "promoter" includes reference to a region of
DNA that may be upstream from the start of transcription and that
may be involved in recognition and binding of RNA polymerase and
other proteins to initiate transcription. A "plant promoter" may be
a promoter capable of initiating transcription in plant cells.
Examples of promoters under developmental control include promoters
that preferentially initiate transcription in certain tissues, such
as leaves, roots, seeds, fibers, xylem vessels, tracheids, or
sclerenchyma. Such promoters are referred to as "tissue-preferred."
Promoters which initiate transcription only in certain tissues are
referred to as "tissue-specific." A "cell type" specific promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" promoter may be a promoter which may be under
environmental control. Examples of environmental conditions that
may effect transcription by inducible promoters include anaerobic
conditions or the presence of light. Tissue-specific,
tissue-preferred, cell type specific and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter may be a promoter which may be active under
most environmental conditions.
[0055] A. Inducible Promoters
[0056] An inducible promoter may be operably linked to a gene for
expression in a cell. Optionally, the inducible promoter may be
operably linked to a nucleotide sequence encoding a signal sequence
which may be operably linked to a gene for expression in a cell.
With an inducible promoter, the rate of transcription increases in
response to an inducing agent.
[0057] Any inducible promoter can be used in the instant invention.
See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary
inducible promoters include, but are not limited to: those from the
ACEI system that responds to copper (Mett et al., PNAS 90:4567-4571
(1993)); In2 gene from maize that responds to benzenesulfonamide
herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237
(1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)); and
Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics
227:229-237 (1991)). A particularly useful inducible promoter may
be a promoter that responds to an inducing agent to which plants do
not normally respond. An exemplary inducible promoter may be the
inducible promoter from a steroid hormone gene, the transcriptional
activity of which may be induced by a glucocorticosteroid hormone.
Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).
[0058] B. Constitutive Promoters
[0059] A constitutive promoter may be operably linked to a gene for
expression in a cell or the constitutive promoter may be operably
linked to a nucleotide sequence encoding a signal sequence which
may be operably linked to a gene for expression in a cell.
[0060] Different constitutive promoters can be utilized in the
instant invention. Exemplary constitutive promoters include, but
are not limited to: promoters from plant viruses, such as the 35S
promoter from CaMV (Odell et al., Nature 313:810-812 (1985));
promoters from rice actin genes (McElroy et al., Plant Cell
2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol.
12:619-632 (1989) and Christensen et al., Plant Mol. Biol.
18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet.
81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730
(1984)); and maize H3 histone (Lepetit et al., Mol. Gen. Genetics
231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):
291-300 (1992)). The ALS promoter, Xbal/NcoI fragment 5' to the
Brassica napus ALS3 structural gene (or a nucleotide sequence
similarity to said Xbal/NcoI fragment), represents a particularly
useful constitutive promoter. See PCT application WO 96/30530.
[0061] C. Tissue-Specific or Tissue-Preferred Promoters
[0062] A tissue-specific promoter may be operably linked to a gene
for expression in a cell. Optionally, the tissue-specific promoter
may be operably linked to a nucleotide sequence encoding a signal
sequence which may be operably linked to a gene for expression in a
cell. Plants transformed with a gene of interest operably linked to
a tissue-specific promoter can produce the protein product of the
transgene exclusively, or preferentially, in a specific tissue.
[0063] Any tissue-specific or tissue-preferred promoter can be
utilized in the instant invention. Exemplary tissue-specific or
tissue-preferred promoters include, but are not limited to, a
root-preferred promoter--such as that from the phaseolin gene
(Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et
al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a
leaf-specific and light-induced promoter such as that from cab or
rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko
et al., Nature 318:579-582 (1985)); an anther-specific promoter
such as that from LAT52 (Twell et al., Mol. Gen. Genetics
217:240-245 (1989)); a pollen-specific promoter such as that from
Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a
microspore-preferred promoter such as that from apg (Twell et al.,
Sex. Plant Reprod. 6:217-224 (1993)).
[0064] Transport of protein produced by transgenes to a subcellular
compartment, such as the chloroplast, vacuole, peroxisome,
glyoxysome, cell wall or mitochondrion or for secretion into the
apoplast, can be accomplished by means of operably linking the
nucleotide sequence encoding a signal sequence to the 5' and/or 3'
region of a gene encoding the protein of interest. Targeting
sequences at the 5' and/or 3' end of the structural gene may
determine, during protein synthesis and processing, where the
encoded protein may be ultimately compartmentalized. Alternatively
such subcellular compartment targeting proteins can be directly
linked to a nanoparticle to direct the nanoparticle coated with the
molecule of interest to the desired subcellular compartment.
[0065] The presence of a signal sequence directs a polypeptide to
either an intracellular organelle or subcellular compartment, or
for secretion to the apoplast. Many signal sequences are known in
the art. See, e.g., Becker et al., Plant Mol. Biol. 20:49 (1992),
Close, P. S., Master's Thesis, Iowa State University (1993), Knox,
C., et al., "Structure and Organization of Two Divergent
Alpha-Amylase Genes from Barley", Plant Mol. Biol. 9:3-17 (1987),
Lerner et al., Plant Physiol. 91:124-129 (1989), Fontes et al.,
Plant Cell 3:483-496 (1991), Matsuoka et al., Proc. Natl. Acad.
Sci. 88:834 (1991), Gould et al., J. Cell. Biol. 108:1657 (1989),
Creissen et al., Plant J. 2:129 (1991), Kalderon, et al., A short
amino acid sequence able to specify nuclear location, Cell
39:499-509 (1984), Steifel, et al., Expression of a maize cell wall
hydroxyproline-rich glycoprotein gene in early leaf and root
vascular differentiation, Plant Cell 2:785-793 (1990).
Foreign Protein Genes and Agronomic Genes
[0066] With transgenic plants according to the present invention, a
foreign protein can be produced in commercial quantities. Thus,
techniques for the selection and propagation of transformed plants,
which are well understood in the art, yield a plurality of
transgenic plants which are harvested in a conventional manner, and
a foreign protein then can be extracted from a tissue of interest
or from total biomass. Protein extraction from plant biomass can be
accomplished by known methods which are discussed, for example, by
Heney and Orr, Anal. Biochem. 114:92-6 (1981).
[0067] In aspects of the invention, the transgenic plant provided
for commercial production of foreign protein may be a cell or a
plant. In other aspects, the biomass of interest may be seed. For
the relatively small number of transgenic plants that show higher
levels of expression, a genetic map can be generated primarily via
conventional RFLP, PCR and SSR analysis, which identifies the
approximate chromosomal location of the integrated DNA molecule.
For exemplary methodologies in this regard, see Glick and Thompson,
Methods in Plant Molecular Biology and Biotechnology CRC Press,
Boca Raton 269:284 (1993). Map information concerning chromosomal
location may be useful for proprietary protection of a subject
transgenic plant. If unauthorized propagation may be undertaken and
crosses made with other germplasm, the map of the integration
region can be compared to similar maps for suspect plants to
determine if the latter have a common parentage with the subject
plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR
and sequencing, all of which are conventional techniques.
[0068] Likewise, agronomic genes can be expressed in transformed
cells or their progeny. More particularly, plants can be
genetically engineered via the methods of the invention to express
various phenotypes of agronomic interest. Exemplary genes that may
be used in this regard include, but are not limited to, those
categorized below.
1. Genes that Confer Resistance to Pests or Disease and that
Encode:
[0069] A) Plant disease resistance genes. Plant defenses are often
activated by specific interaction between the product of a disease
resistance gene (R) in the plant and the product of a corresponding
avirulence (Avr) gene in the pathogen. A plant variety can be
transformed with cloned resistance genes to engineer plants that
are resistant to specific pathogen strains. See, e.g., Jones et
al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for
resistance to Cladosporium fulvum); Martin et al., Science 262:1432
(1993) (tomato Pto gene for resistance to Pseudomonas syringae pv.
tomato encodes a protein kinase); Mindrinos et al., Cell 78:1089
(1994) (Arabidopsmay be RSP2 gene for resistance to Pseudomonas
syringae).
[0070] B) A gene conferring resistance to a pest, such as soybean
cyst nematode. See, e.g., PCT Application WO 96/30517; PCT
Application WO 93/19181.
[0071] C) A Bacillus thuringiensis protein, a derivative thereof or
a synthetic polypeptide modeled thereon. See, e.g., Geiser et al.,
Gene 48:109 (1986), which discloses the cloning and nucleotide
sequence of a Bt .delta.-endotoxin gene. Moreover, DNA molecules
encoding .delta.-endotoxin genes can be purchased from American
Type Culture Collection, Manassas, Va., for example, under ATCC
Accession Nos. 40098, 67136, 31995 and 31998.
[0072] D) A lectin. See, for example, the disclosure by Van Damme
et al., Plant Molec. Biol. 24:25 (1994), who disclose the
nucleotide sequences of several Clivia miniata mannose-binding
lectin genes.
[0073] E) A vitamin-binding protein, such as avidin. See PCT
application US93/06487. The application teaches the use of avidin
and avidin homologues as larvicides against insect pests.
[0074] F) An enzyme inhibitor, for example, a protease or
proteinase inhibitor or an amylase inhibitor. See, e.g., Abe et
al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice
cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol.
21:985 (1993) (nucleotide sequence of cDNA encoding tobacco
proteinase inhibitor I), Sumitani et al., Biosci. Biotech. Biochem.
57:1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus
.alpha.-amylase inhibitor) and U.S. Pat. No. 5,494,813 (Hepher and
Atkinson, issued Feb. 27, 1996).
[0075] G) An insect-specific hormone or pheromone such as an
ecdysteroid or juvenile hormone, a variant thereof, a mimetic based
thereon, or an antagonist or agonist thereof. See, for example, the
disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus
expression of cloned juvenile hormone esterase, an inactivator of
juvenile hormone.
[0076] H) An insect-specific peptide or neuropeptide which, upon
expression, disrupts the physiology of the affected pest. For
example, see the disclosures of Regan, J. Biol. Chem. 269:9 (1994)
(expression cloning yields DNA coding for insect diuretic hormone
receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243
(1989) (an allostatin may be identified in Diploptera puntata). See
also U.S. Pat. No. 5,266,317 to Tomalski et al., which discloses
genes encoding insect-specific, paralytic neurotoxins.
[0077] I) An insect-specific venom produced in nature by a snake, a
wasp, or any other organism. For example, see Pang et al., Gene
116:165 (1992), for disclosure of heterologous expression in plants
of a gene coding for a scorpion insectotoxic peptide.
[0078] J) An enzyme responsible for a hyperaccumulation of a
monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a
phenylpropanoid derivative or another non-protein molecule with
insecticidal activity.
[0079] K) An enzyme involved in the modification, including the
post-translational modification, of a biologically active molecule;
for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic
enzyme, a nuclease, a cyclase, a transaminase, an esterase, a
hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase,
an elastase, a chitinase and a glucanase, whether natural or
synthetic. See PCT application WO 93/02197 in the name of Scott et
al., which discloses the nucleotide sequence of a callase gene. DNA
molecules which contain chitinase-encoding sequences can be
obtained, for example, from the ATCC under Accession Nos. 39637 and
67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23:691
(1993), who teach the nucleotide sequence of a cDNA encoding
tobacco hornworm chitinase, and Kawalleck et al., Plant Molec.
Biol. 21:673 (1993), who provide the nucleotide sequence of the
parsley ubi4-2 polyubiquitin gene.
[0080] L) A molecule that stimulates signal transduction. For
example, see the disclosure by Botella et al., Plant Molec. Biol.
24:757 (1994), of nucleotide sequences for mung bean calmodulin
cDNA clones, and Griess et al., Plant Physiol. 104:1467 (1994), who
provide the nucleotide sequence of a maize calmodulin cDNA
clone.
[0081] M) A hydrophobic moment peptide. See PCT application WO
95/16776 (disclosure of peptide derivatives of Tachyplesin which
inhibit fungal plant pathogens) and PCT application WO 95/18855
(teaches synthetic antimicrobial peptides that confer disease
resistance).
[0082] N) A membrane permease, a channel former or a channel
blocker. For example, see the disclosure of Jaynes et al., Plant
Sci 89:43 (1993), of heterologous expression of a cecropin-.beta.
lytic peptide analog to render transgenic tobacco plants resistant
to Pseudomonas solanacearum.
[0083] O) A viral-invasive protein or a complex toxin derived
therefrom. For example, the accumulation of viral coat proteins in
transformed plant cells imparts resistance to viral infection
and/or disease development effected by the virus from which the
coat protein gene may be derived, as well as by related viruses.
See Beachy et al., Ann. rev. Phytopathol. 28:451 (1990). Coat
protein-mediated resistance has been conferred upon transformed
plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco
streak virus, potato virus X, potato virus Y, tobacco etch virus,
tobacco rattle virus and tobacco mosaic virus. Id.
[0084] P) An insect-specific antibody or an immunotoxin derived
therefrom. Thus, an antibody targeted to a critical metabolic
function in the insect gut would inactivate an affected enzyme,
killing the insect. Cf. Taylor et al., Abstract #497, Seventh Int'l
Symposium on Molecular Plant-Microbe Interactions (Edinburgh,
Scotland) (1994) (enzymatic inactivation in transgenic tobacco via
production of single-chain antibody fragments).
[0085] Q) A virus-specific antibody. See, for example, Tavladoraki
et al., Nature 366:469 (1993), who show that transgenic plants
expressing recombinant antibody genes are protected from virus
attack.
[0086] R) A developmental-arrestive protein produced in nature by a
pathogen or a parasite. For example, fungal endo
.alpha.-1,4-D-polygalacturonases facilitate fungal colonization and
plant nutrient release by solubilizing plant cell wall
homo-.alpha.-1,4-D-galacturonase. See Lamb et al., Bio/Technology
10:1436 (1992). The cloning and characterization of a gene which
encodes a bean endopolygalacturonase-inhibiting protein may be
described by Toubart et al., Plant J. 2:367 (1992).
[0087] S) A developmental-arrestive protein produced in nature by a
plant. For example, Logemann et al., Bio/Technology 10:305 (1992),
have shown that transgenic plants expressing the barley
ribosome-inactivating gene have an increased resistance to fungal
disease.
2. Genes that Confer Resistance to an Herbicide:
[0088] A) An herbicide that inhibits the growing point or meristem,
such as an imidazolinone or a sulfonylurea. Exemplary genes in this
category code for mutant ALS and AHAS enzyme as described, for
example, by Lee et al., EMBO J. 7:1241 (1988), and Mild et al.,
Theor. Appl. Genet. 80:449 (1990), respectively.
[0089] B) Glyphosate (resistance conferred by, e.g., mutant
5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) genes (via the
introduction of recombinant nucleic acids and/or various forms of
in vivo mutagenesis of native EPSPs genes), aroA genes and
glyphosate acetyl transferase (GAT) genes, respectively), other
phosphono compounds such as glufosinate (phosphinothricin acetyl
transferase (PAT) genes from Streptomyces species, including
Streptomyces hygroscopicus and Streptomyces viridichromogenes), and
pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase
inhibitor-encoding genes), See, for example, U.S. Pat. No.
4,940,835 to Shah, et al. and U.S. Pat. No. 6,248,876 to Barry et.
al., which disclose nucleotide sequences of forms of EPSPs which
can confer glyphosate resistance to a plant. A DNA molecule
encoding a mutant aroA gene can be obtained under ATCC accession
number 39256, and the nucleotide sequence of the mutant gene may be
disclosed in U.S. Pat. No. 4,769,061 to Comai. European patent
application No. 0 333 033 to Kumada et al., and U.S. Pat. No.
4,975,374 to Goodman et al., disclose nucleotide sequences of
glutamine synthetase genes which confer resistance to herbicides
such as L-phosphinothricin. The nucleotide sequence of a PAT gene
may be provided in European application No. 0 242 246 to Leemans et
al., DeGreef et al., Bio/Technology 7:61 (1989), describe the
production of transgenic plants that express chimeric bar genes
coding for PAT activity. Exemplary of genes conferring resistance
to phenoxy proprionic acids and cyclohexones, such as sethoxydim
and haloxyfop include the Accl-S1, Accl-S2 and Accl-S3 genes
described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).
GAT genes capable of conferring glyphosate resistance are described
in WO 2005012515 to Castle et. al. Genes conferring resistance to
2,4-D, fop and pyridyloxy auxin herbicides are described in WO
2005107437 assigned to Dow AgroSciences LLC.
[0090] C) An herbicide that inhibits photosynthesis, such as a
triazine (psbA and gs+ genes) or a benzonitrile (nitrilase gene).
Przibila et al., Plant Cell 3:169 (1991), describe the
transformation of Chlamydomonas with plasmids encoding mutant psbA
genes. Nucleotide sequences for nitrilase genes are disclosed in
U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing
these genes are available under ATCC Accession Nos. 53435, 67441,
and 67442. Cloning and expression of DNA coding for a glutathione
S-transferase may be described by Hayes et al., Biochem. J. 285:173
(1992).
3. Genes that Confer or Contribute to a Value-Added Trait, Such
as:
[0091] A) Modified fatty acid metabolism, for example, by
transforming a plant with an antisense gene of stearyl-ACP
desaturase to increase stearic acid content of the plant. See
Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992).
[0092] B) Decreased phytate content--1) Introduction of a
phytase-encoding gene would enhance breakdown of phytate, adding
more free phosphate to the transformed plant. For example, see Van
Hartingsveldt et al., Gene 127:87 (1993), for a disclosure of the
nucleotide sequence of an Aspergillus niger phytase gene. 2) A gene
could be introduced that reduced phytate content. In maize, for
example, this could be accomplished by cloning and then
reintroducing DNA associated with the single allele which may be
responsible for maize mutants characterized by low levels of phytic
acid. See Raboy et al., Maydica 35:383 (1990).
[0093] C) Modified carbohydrate composition effected, for example,
by transforming plants with a gene coding for an enzyme that alters
the branching pattern of starch. See Shiroza et al., J. Bacteol.
170:810 (1988) (nucleotide sequence of Streptococcus mutants
fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet.
20:220 (1985) (nucleotide sequence of Bacillus subtil may be
levansucrase gene), Pen et al., Bio/Technology 10:292 (1992)
(production of transgenic plants that express Bacillus lichenifonn
may be .alpha.-amylase), Elliot et al., Plant Molec. Biol. 21:515
(1993) (nucleotide sequences of tomato invertase genes), Sogaard et
al., J. Biol. Chem. 268:22480 (1993) (site-directed mutagenes may
be of barley .alpha.-amylase gene), and Fisher et al., Plant
Physiol. 102:1045 (1993) (maize endosperm starch branching enzyme
II).
EXAMPLES
[0094] The present invention is further described in the following
examples, which are offered by way of illustration and are not
intended to limit the invention in any manner.
Example 1
Preparation of Single Cell Plant Material
[0095] Both BY2 cells and NT1 cells were used. BY2 cells are a
non-green, fast growing tobacco cell line. NT1 cells are
photoautotrophic cells isolated from tobacco. Three to four days
prior to transformation, a one-week-old suspension culture was
subcultured to fresh medium by transfer of 2 ml of NT1 or BY2
culture into 40 ml NT1B or LSBY2 media containing 50 nM DAS-PMTI-1
(a microtubule inhibitor) and 0.5-0.1% (v/v) DMSO in a 250-mL
flask. Single cells were collected either at four days or seven
days after the microtubule inhibitor treatment. The BY2 single
cells used were processed through a Beckman Flow cytometer to count
the viable cells. There were 658250 viable cells/ml with a mean
diameter of 10.43 um and a volume of 593.8 .mu.m.sup.3. As visible
in FIG. 1, all the cells were single cells (the pair in FIG. 1 has
overlapping edges). The cells were examined using a Differential
Interference Contrast (DIC) microscope attached to a confocal
imaging system (Panel A). Panel B shows a light microscopic view of
single cell from BY2 cells (EP12% medium habituated and maintained
cultures) that were stained with I2KI to highlight the plastid
(Amyloplast). As is visible therein, single cells of BY2 cells
comprise large numbers of plastids (amyloplasts) distributed
throughout the cytoplasm of the cell.
[0096] FIG. 2, Panel A, depicts light microscope photoautotrophic
cells of tobacco (NT1) having prominent chloroplasts, which were
maintained in minimal medium and 5% carbon dioxide. These cells
were sub cultured once in every 14 days by transferring 1 ml of
suspension at 3.0OD.sup.600. FIG. 2, Panel B, shows similar NT1
cells as viewed under a fluorescent microscope in which the active
chloroplasts can be seen to be autofluorescing in red.
[0097] The cell types described above were used as target cells for
transformation. The green cells (NT1 cells) are an optimum cell
type to track a nanoparticle into the chloroplast as they have few
cells in a given cluster and are hyaline. In addition, the cells
have very prominent chloroplasts that autofluoresce red (as visible
in FIG. 2, Panel B).
Example 2
Nanoparticle Preparation and Treatment of Cells
[0098] To determine if cells took up fluorescent dye in culture,
single cells and multicellular standard aggregate suspension
culture of BY2 cells was used. The cell suspension cultures were
exposed to SAMSA fluorescein (5-((2-(and-3)-S-(acetylmercapto)
succinoyl)amino) fluorescein) from Molecular Probes in the absence
of nanoparticles for 20 minutes and then were briefly washed before
being observed under a fluorescent microscope.
[0099] Gold nanoparticles (GNP) were coated with SAMSA fluorescein
as per the product technical guidelines (available on the world
wide web at probes.invitrogen.com/media/pis/mp00685.pdf).
Gold-fluorescein conjugate was prepared by using a method described
hereafter. 1 mg of SAMSA fluorescein was dissolved in 100 .mu.l of
0.1 M NaOH and vortexed for 15 minutes to remove the acetyl group
protecting the thiol. This activated SAMSA was then mixed with 100
.mu.l of 150 nm gold colloids (.about.109 particles/ml). The
resulting solution was then incubated for 1 hour to ensure the
completion of the reaction. Then 50 .mu.L of 1M HCl was added to
neutralize the solution. It was centrifuged at 3000 RPM for 30
minutes and supernatant was removed. The yellow pellet obtained was
re-suspended in 200 .mu.L of 0.1 M PBS, resulting in an orange
colored solution. This purification step was repeated 2 times to
ensure removal of free SAMSA fluorescein. The mode of attachment of
SAMSA to gold is mainly via thiol bonding. Due to the significant
electrostatic repulsion (SAMSA is dianionic at pH>7), SAMSA is
thought to lie perpendicular to the gold colloidal surface. The
particles showed clear fluorescence without any background when
observed under a DIC and multiphoton confocal microscope. 20 and 40
.mu.l of coated gold nanoparticle were transferred to 500 .mu.l of
BY2/NT1 tobacco suspensions or Photoautotrophic tobacco cells and
incubated for 20 minutes in dark.
[0100] After incubation, 50 .mu.l aliquots of cell suspensions were
mounted on microscopic perfusion slides and observed under the
microscope to track the particles. In addition, aliquots of samples
were prepared for microscopic observation at 2-20 hrs after the 20
minute incubation.
Example 3
Fluorescein Coated Nanoparticle Delivery and Accumulation in
BY2/NT1 Cell Aggregates and in Nucleus and Plastids of Single
Photoautotrophic Tobacco Cells
[0101] The BY2/NT1 suspension aggregates treated with SAMSA
fluorescein alone and with SAMSA fluorescein coated GNPs were
examined under low and high magnification using DIC, bright-field,
and fluorescent scopes. FIG. 3, Panel A, shows a DIC image of cells
treated with SAMSA fluorescein alone, while FIG. 3, Panel B, shows
a fluorescent image of the same cells. FIG. 3, Panel C, shows a DIC
image of cells treated with SAMSA fluorescein coated GNPs, while
FIG. 3, Panel D, shows the fluorescent image of the SAMSA
fluorescein coated GNPs-treated cells. As is clearly visible in
FIG. 3, Panel B, only the cell walls of the cells treated with
SAMSA fluorescein alone stained with the fluorescein and very
little other background fluorescence was visible. This indicates
that the cells did not uptake the SAMSA fluorescein in the absence
of nanoparticles.
[0102] In contrast, the SAMSA fluorescein-coated GNPs were tracked
into the cells and the nucleus (Nu), as seen in FIG. 3, Panel D. It
was clear from the DIC observations that the SAMSA
fluorescein-coated GNPs were found in all compartments of the
cells, except vacuoles. The cytoplasmic strands lining the vacuoles
also had SAMSA fluorescein-coated GNPs in addition to the nuclear
compartment. The nanoparticles do not appear to have been hindered
in their transport across the cell walls. Thus, the accumulation of
SAMSA fluorescein coated GNPs seems to be in the symplastic, as
opposed to the apoplastic, continuum. Further examination of the
SAMSA fluorescein-coated GNP treated single cells under high
magnifications showed the presence of a large number of GNPs in
nucleolus and it appeared that the GNPs are preferentially
accumulated in these organelles (FIG. 4: Panel B). Panel A of FIG.
4 shows a bright-field image of the same nucleus as Panel B under a
different plane of focus.
[0103] FIG. 5. shows photoautotrophic cells treated with SAMSA
fluorescein coated GNP. Panel A shows very hyaline cells in 3-4
cell clusters with large chloroplasts lining the inner side of the
cell wall. Panel B shows accumulation of nanoparticles in the
chloroplast. Panels C and D show higher power magnification of a
single chloroplast using a fluorescent microscope. Nanoparticles
are visible in the membrane lamellations of the chloroplast and
interspersed among the red autofluorescing chlorophyll
pigments.
[0104] Thus, the live photoautotrophic cell tracking with the
bright-field and fluorescent microscopes in the real time
demonstrated that the nanoparticles were accumulating in both
membrane and chloroplast matrix. The particles could be also
tracked in the lumen of the double membrane of the chloroplast.
[0105] While the experiments done to track the particles within the
chloroplast revealed that the particles appear to accumulate in the
plastids, it was difficult to visually identify the presence of
particles within the chloroplast envelope by using light
microscopes, due to insufficient resolution. Thus, the particles
were additionally tracked using reflectance and fluorescent
microscopes, and the images were merged to clearly locate the
particle, as seen in FIG. 6. Panel A of FIG. 6 shows a reflectance
image where the GNPs are preferentially seen. This picture not only
indicates the presence of nanoparticles in the chloroplast, but
also shows heavy accumulation of the nanoparticles within
chloroplast, indicating active uptake. Panel B shows fluorescing
particles within the background of red autofluorescing chloroplast.
A merged reflectance and fluorescent image is shown in Panel C,
wherein the yellow fluorescing particles are within the boundary of
the chloroplast, confirming the presence of the particle in the
plastids.
Example 4
DNA Attached GNP Delivery for Nuclear Transformation
[0106] DNA coated GNPs were synthesized via 2 pathways, i.e.,
non-specific interaction and specific interaction (using PEG as a
platform) and incubated with BY2/NT1 cells. For non-specific
interaction, 9 mL of 3% mannitol was added to 1 mL of cell
suspension and then centrifuged for 5 minutes at 1000 rpm. The
supernatant was then decanted and the cells resuspended in 300
.mu.l of 3% mannitol. 50 .mu.l of 150 nm diameter gold
nanoparticles (available from BBI International (EM. GC150)) and 50
.mu.g of plasmid DNA (pDAB3831) (FIG. 16) (SEQ. ID. NOS. 1 and 2)
encoding YFP were added to the resuspended cells, and the mixture
was allowed to incubate for 20 minutes. After incubation, 20 mL of
3% mannitol were added to the solution and the resulting mixture
centrifuged for 5 minutes at 1000 rpms. The supernatant was then
decanted and the cells resuspended in 3 mL growth media. The
resuspended cells were then transferred to microwells for at least
48 hours before transfer to selection plates. For specific
interaction (PEG pathway), a large excess equivalent of thiol
ligand was used: 100 monolayers/particle, estimated by assuming
that the occupied surface area by a single thiol molecule is ca.
0.20 nm. Using this calculation, 2 mg of SH-PEG(3)-OCH3 was added
into the citrate GNPs solution. The mixture was rapidly stirred at
room temperature for 20 h during which the color of the solution
became slightly darker. Then, 3 volumes of THF were added to the
reaction mixture and the resulting solution was centrifuged at 13 K
rpm at 4.degree. C. for 30 min. The supernatant was removed, the
pellet was re-dissolved into 10 mL of ultra-pure water (18
M.OMEGA.cm), 30 mL of THF was added, and a second centrifugation in
the same conditions was carried out. The pellet was then dissolved
into ultra-pure water (18 M.OMEGA.cm) and kept at room temperature.
To coat plasmid DNA onto H.sub.3CO-PEG-SH-GNPs for transformation
experiments, 1 mg of purified plasmid DNA was incubated with 10 mg
gold particles in 50 ml water for 2 h at 23.degree.. (see, Torney,
F. et al., Nature Nanotechnol. 2, (2007)).
[0107] A graphical representation of one possible transformation
scheme is outlined in FIG. 7. For the transformation, a plasmid
DNA, pDAB3831 comprising a YFP reporter gene was used. BY2 cells
were treated as described supra and suspensions were incubated for
48 to 72 hrs with slow agitation in micro-well plates. A 50 .mu.l
aliquot of suspension was taken from the total 0.5-1 ml mixture and
examined under a fluorescent microscope to observe for any
expression of the reporter gene. BY2 cells transformed with the
plasmid containing the YFP reporter gene showed transient
expression of the YFP.
Example 5
DNA Attached PEGylated Quantum Dot Delivery for Nuclear
Transformation
[0108] PEG functionalization of the QD for the cell entry
evaluation studies: This protocol was adopted from Dubertret B, et
al., Science 298, 1759 (2002)). 2 mg of TOPO (tri-octyl phosphine
oxide)-coated CdSe/ZnS QDs (Ocean nanotechnology, Cat #
QSO0630-0010) were suspended with 0.015 g (5.5 .mu.mol) of PEG-PE
(1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-poly(ethylene
glycol)]) (Avanti lipids, Cat #880160) in chloroform followed by
evaporation of the solvent and solubilization with water. PEG
conjugation was done to make sure that there is complete protection
from cytotoxicity
[0109] QD conjugation to plasmid DNA: 2 mg of TOPO (tri-octyl
phosphine oxide)-coated QDs (Ocean nanotechnology, Cat #
QSO0630-0010) were suspended with 4 mg of HS-PEG-OCH.sub.3
(Prochimia, Cat #TH 014-01) overnight at .about.60-70.degree. C.
The solvent was removed in a vacuum oven. The residue was then
suspended in 1 mL of water (18 M). The last step is accompanied by
a change of the red residue to an orange, optically clear,
transparent solution. To coat plasmid DNA onto H.sub.3CO-PEG-SH-QDs
for transformation experiments, 0.02 mg of purified plasmid DNA
(pDAB 3831) was incubated with resultant QD conjugate in 2 ml of
water for 2 h at 23.degree. in dark. (Torney, F. et al., Nature
Nanotechnol. 2, (2007)).
[0110] Incubation of QDs with tobacco intact cells: Experiments
with cell lines were performed using Bright Yellow (BY2) tobacco
single cell lines, maintained at 25.degree. C. in LSBY2 medium.
These single cell lines are produced by the same methodology
outlined in IDM#64901. A concentration of 1-3 .mu.L/mL was added to
500 .mu.l of cells in a 24-well micro titer plate, and rotated on a
shaker gently for 20 min before analyzing the cells.
Example 6
Nanoparticle Mediated Transduction and Cellular Internalization of
Fluorescent Proteins into Intact Plant Cells and Potential
Applications
[0111] Materials to test nanoparticle mediation transduction and
cellular internalization of proteins into plant cells include gold
colloids of 150 nm diameter in size (BBI International, GC150),
5-((2-(and-3)-S(acetylmercapto)succinoyl)amino)fluorescein (SAMSA
fluorescein: Invitrogen, A-685), nanoparticles of size 80 and 90 nm
carboxylic acid coated gold Colloids (TedPella, 32019), Sulfo-NHS
(N-hydroxysulfosuccinimide),
EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride),
(Pierce Bitoechnology, 24510, 22980,), IVIES
(2-[N-morpholino]ethane sulfonic acid)(Fisher Scientific,
AC32776-1000), Phosphate buffered saline buffer packets (Sigma,
P5368-10PAK), Histdine tagged GFP (Evrogen, Excitation max--482 nm,
Emission max--502 nm, FP611), turbo YFP (Evrogen, Excitation
max--525 nm, Emission max--538 nm, FP611), and Propidium iodide
(Sigma-P4864), Fluorescein diacetate (Sigma, F7378).
[0112] Cell cultures (BY2-E tobacco single cells): Both BY2 cells
and NT1 cells were used. BY2/NT1 cells are a non-green, fast
growing tobacco cell line. Three to four days prior to the
experiments, a one-week-old suspension culture was sub-cultured to
fresh medium by transfer of 2 ml of NT1 or BY2 culture into 40 ml
NT1B or LSBY2 media containing 1 .mu.l DAS-proprietary MTI-1
(PMTI-1) (a microtubule inhibitor), 1-3% Glycerol, and 0.05-0.1%
(v/v) DMSO in a 250-mL flask. Single cells were collected either at
3.5 days or 7 days after the microtubule inhibitor treatment. The
BY2 single cells used were processed through a Beckman Flow
cytometer to count the viable cells. There were 658250 viable
cells/ml with a mean diameter of 10.43 um (volume of 593.8
.mu.m.sup.3)-50.42 um (volume of 67079.05 .mu.m.sup.3). Live cells
in these cultures, after PMTI-1 treatment, were all single cells.
The cells were examined using a Differential Interference Contrast
(DIC) microscope attached to a confocal fluorescence imaging
system.
[0113] Nanoparticle conjugates: gold-fluorescein conjugate,
gold-histidine tagged GFP conjugate, and gold-YFP conjugate were
synthesized.
[0114] Synthesis of Gold-fluorescein conjugate: Gold-fluorescein
conjugate was prepared by using a method described previously
(Cannone, F., G. Chirico, et al. (2006), Quenching and Blinking of
Fluorescence of a Single Dye Molecule Bound to Gold Nanoparticles.
J. Phys. Chem. B 110(33): 16491-16498.). 1 mg of SAMSA fluorescein
was dissolved in 100 .mu.l of 0.1 M NaOH and vortexed for 15
minutes to remove the acetyl group protecting the thiol. This
activated SAMSA was mixed with 100 .mu.l of 150 nm gold colloids
(.about.10.sup.9 particles/ml). This solution was then incubated
for 1 hour to ensure the completion of the reaction. After
incubation, 50 .mu.L of 1M HCl was added to neutralize the
solution. It was centrifuged at 3000 RPM for 30 minutes and
supernatant was removed. The yellow pellet obtained was
re-suspended in 200 .mu.L of 0.1 M PBS, resulting in an orange
colored solution. This purification step was repeated 2 times to
ensure removal of free SAMSA fluorescein. The mode of attachment of
SAMSA to gold is mainly via thiol bonding. Due to the significant
electrostatic repulsion (SAMSA is dianionic at pH>7), SAMSA is
believed to lie perpendicular to the gold colloidal surface
(Cannone et. al. 2006).
[0115] Synthesis of Gold-histidine tagged GFP and Gold-YFP
conjugate: Gold-protein conjugates were synthesized using a slight
modification of protocol described by Grabarek (Grabarek, Z. and J.
Gergely (1990), Zero-length cross linking procedure with the use of
active esters. Analytical Biochemistry 185(1): 131-135)), which was
illustrated for sequential coupling of two proteins. 0.25 ml of 90
nm carboxyl acid coated gold colloidal solution (.about.10.sup.9
particles/ml) was centrifuged at 3000 RPM for 10 minutes. After
discarding the supernatant, the red pellet was suspended in 1 ml of
activation buffer (0.1 M MES, 0.5 M NaCl, pH 6.0). 0.4 mg EDC and
1.1 mg of sulfo-NHS was added to this solution and vortexed for 15
minutes at room temperature. Then, 9 .mu.l of protein (histidine
tagged GFP or turbo YFP) was added and the resulting solution was
incubated for 2 hours in the dark at room temperature in order for
the protein and gold to react completely. The ratio of gold
colloids and protein used in this reaction was determined by
finding the number of carboxylic acids present on gold colloids.
First, number of carboxylic groups present on one gold colloid was
calculated by dividing the surface area of 1 gold particle (sphere
assumption) by surface occupied by one carboxylic group (0.20
nm.sup.2 Kimura, K.; Takashima, S.; Ohshima, H. Journal of Physical
Chemistry B 2002, 106, 7260-7266). This result was multiplied by
total number of gold colloids present to obtain the total number of
carboxylic groups present in entire gold colloidal solution. This
was equated with the number of amino groups present in a given
amount of protein. These gold colloids attach to protein via the
formation of amide bond between carboxylic acid present on gold and
amino group present on protein (Grabarek, Z. and J. Gergely (1990).
Zero-length cross linking procedure with the use of active esters.
Analytical Biochemistry 185(1): 131-135). There are roughly 127285
protein molecules tethered to one gold nanoparticle.
[0116] Cell treatments--Three separate samples were prepared for
testing, as follows:
[0117] Time Course of Gold Uptake and Cell Viability--
[0118] The following samples were prepared in a 24 well sterile
plates:
[0119] i) 500 .mu.l of single BY2-E cells (control);
[0120] ii) 500 .mu.l of single BY2-E cells+20 .mu.l of GNP+25 .mu.l
of Fluorescein di-acetate (FDA)+25 .mu.l of Propidium iodide;
and
[0121] iii) Other treatments include 40, 60, 80 .mu.l of GNP with
the cells and cell viability stains as mentioned above. Treated
samples (Ref) were examined under fluorescence microscope at 5, 20,
120 min and finally after 18-20 hrs.
[0122] Gold-SAMSA Fluorescein Treatments--
[0123] The following samples were prepared in a 24 well sterile
plates:
[0124] i) 500 .mu.l of single BY2-E cells (control);
[0125] ii) 500 .mu.l of single BY2-E cells+20 .mu.l of
SAMSA-fluorescein (control); and
[0126] iii) 500 .mu.l of single BY2-E cells+20 .mu.l of
Au-SAMSA-fluorescein.
[0127] The treated cells were incubated for 20 minutes in dark at
room temperature before conducting microscopy studies.
[0128] Gold-Histidine Tagged GFP Treatments--
[0129] The following samples were prepared in a 24 well sterile
plates:
[0130] i) 500 .mu.l of single BY2-E cells (control).
[0131] ii) 500 .mu.l of single BY2-E cells+10 .mu.l of histidine
tagged GFP (control).
[0132] iii) 500 .mu.l of single BY2-E cells+20 .mu.l of
Au-histidine tagged GFP.
[0133] The treated cells were incubated for 2 hours in dark at room
temperature before conducting microscopy studies.
[0134] Microscopy: Phase contrast and Fluorescence microscopy of
the single cell experiments with Au-SAMSA fluorescein and
Au-histidine tagged GFP was carried out using Leica inverted
fluorescence microscope (DAS). All the experiments were carried out
at 20.times. magnification. FITC (fluorescein isothiocyanate) and
GFP filter was used for SAMSA fluorescein and GFP single cell
treatments respectively.
[0135] Differential Image Contrast (DIC), Confocal and Reflectance
Microscopy:
[0136] These studies were carried out at UIUC (University of
Illinois at Urbana Champaign) microscopy center on a Zeiss inverted
microscope. For all these methods, the magnification was kept at
63.times.. For confocal, FITC, GFP and YFP filters were used for
different cell treatments. For reflectance studies, dichroic mirror
was replaced by a transparent glass slide and emission filter was
removed.
[0137] Image Acquisition:
[0138] Suspension cultured tobacco cells were imaged using a Zeiss
Axiovert M 200 microscope equipped with apotome optical sectioning
system coupled with X-Cite 120 illumination system (Carl Zeiss
microimaging, Obercohen, Germany). The gold particles were imaged
under a reflectance imaging setup using the mercury illumination
through 635/20 excitation filter and imaged using a IGS polarizing
filter set (available from 33001, Chroma Technology Corp.,
Rockingham, Vt.) consisting of GG420 glass to block the UV, KG5 (IR
blocker), 50/50 beam splitter and an excitation and emission
parallel polarizers. Simultaneously, DIC/transmitted light images
were acquired using standard DIC optics and the GFP in GFP-DNA
coated gold particles were (psuedocolored green) acquired with a
band pass FITC filter (HQ480/40 excitation filter, Q505LP dichroic
mirror and HQ535/50 emission). Cells were aliquoted in a chambered
cover glass setup having a thickness of 500 microns (Grace
Bio-labs, Bend, Oreg.) for high resolution imaging. Most of the
images were acquired with a 63.times.1.4 NA Planapochromat
objective or with a 40.times.1.4 NA Planapochromat objective,
depending on the cell size (available from Carl Zeiss Microimaging,
Obercohen, Germany). Exposure times were set for each channel
(i.e., DIC, Reflectance and/or FITC) and exposed sequentially using
the Axiovision 4.6 program coupled with a high resolution Axiocam
MRm monochrome camera (available from Carl Zeiss, Obercohen,
Germany) with the dimension of 1388.times.1034 pixels. When needed,
the resolution is set at 1024.times.1024 and a time lapse sequence
of images obtained at the highest possible speed to resolve the
particle dynamics over a period of 2-5 min consisting around
150-250 frames. The images were prepared either in the Axiovision
4.6 gallery module or Adobe Photoshop (Adobe Systems, San Jose,
Calif.).
[0139] Time Course and GNP Internalization Studies:
[0140] To evaluate the impact of particle uptake and concentration
of GNPs on cell intactness and viability, time course experiments
were performed on BY2-E single cell lines incubated with citrate
functionalized GNP (90 nm diameter). Various concentrations of GNP
(20, 40, 60, 80 .mu.l) were used in this experiment. The particles
were internalized within 5 minutes after mixing with cells, while
particle accumulation took up to 2 hrs to show increased levels in
the cytosol and nucleus of the cells. Among the concentration
tested, a higher level of cell viability and cell vigor was
observed with 20 .mu.l treatment as studied by FDA and PI
(live/dead cell staining) protocol. In all the treated samples, the
average viability of the cells was close to 98%, but with the
highest concentration tested, no FDA stained nucleus was seen in 80
.mu.l treatment. However, these unstained nuclei did not respond to
PI, thus indicating no cell death. This result indicates the
highest concentration of particle could lead to internal
disturbances to an extent that the cell may be quiescent but still
alive after 20 hrs after treatment.
[0141] Reflectance Microscope Tracking Studies:
[0142] Reflectance studies on single BY2/NT1 tobacco cells treated
with gold Protein (GFP/YFP) conjugates show the presence of gold
nanoparticles inside the cells. This was compared to untreated
control single BY2 cells, which appeared to be dark under similar
conditions, as shown in FIG. 8. Single gold nanoparticles emitting
bright reflectance were observed, as shown in FIG. 8. This is a
clear indication of uptake of gold nanoparticles by these walled
BY2 cells.
[0143] Gold-SAMSA Fluorescein Experiments:
[0144] Phase contrast experiments conducted at DAS revealed a
bright yellow staining of the intracellular space and nucleus for
the treated cells as compared to silver contrast observed for
control single cells. Also, in many cells, under conditions of
plasmalysis, the plasma membrane withdrew itself from the cell
wall, leaving a space in between indicating partial or complete
plasmolysis of the cell, as shown in FIG. 9. Such cells, when
observed under confocal fluorescence experiments of single cells
treated with SAMSA fluorescein alone, showed fluorescence in the
cytoplasm and the nucleus while it appeared to be dark in the
untreated control cells. Also, the cells treated with SAMSA
fluorescein stain alone showed some wall fluorescence, but not
inside the cells. This means that SAMSA fluorescein is not
internalized by the cells on its own and that the gold nanoparticle
is acting like a carrier for its uptake.
[0145] Gold-Histidine Tagged GFP Experiments:
[0146] In order to establish the protein delivery to the intact
cells via GNPs, we confirmed the GFP attachment to GNP using
fluorescence microscopy, as shown in FIG. 10. Fluorescence images
of BY2 cells treated with histidine tagged GFP show extracellular
fluorescence with dark cells no fluorescence in the center. This
indicates that in control treatments where histidine tagged GFP is
added to the cells without Au particles do not internalize the
particles. The evidence which support the intake of protein inside
the cells were: i) increased fluorescence intensity of fluorescence
in treated as cells internally, ii) fluorescing cytoplasmic strands
in treated cells as compared to dark strands in control cells (see
FIG. 11). Similar observations were made with YFP tethered GNPs,
indicating the clear internalization of these fluorescent protein
into the plant cells with intact cells (see FIG. 12).
[0147] There is a certain level of background reflectance and
auto-fluorescence in the single cells that are inherent in
plasmalyzing/dying or cells showing program cell death (PCD)-like
cytological characteristics. In order to delineate the cells that
have internalized the protein from such background problems and to
unequivocally prove with the direct evidence for protein
internalization, extensive reflectance scope investigation was
carried out to focus and track individual particle or particle
aggregate levels. The results of this study clearly showed
internalization of particles with protein inside the cells and
nucleus. However, the cells that accumulated increased number of
particles with the fluorescent protein had a tendency to plasmalyze
when observed under the microscope. It is likely that the increased
concentration of protein due to the accumulation of high GNPs
tethered to either GFP or YFP reaches toxic levels or the prolonged
observation under the scope induces ROS which in turn has
deleterious effect in such cells.
Example 7
Molecular Analysis and Proof for the Genomic Integration of
Transgenes in the T1 Progeny of Arabidopsis thaliana cv
Columbia
[0148] Genomic DNA from Arabidopsis transgenic plants was extracted
from total leaf material of 6-week-old using DNeasy Plant Mini kit
according to the manufacturer's instructions (Qiagen Inc). The
following YFP and PAT PCR primers were used to in the PCR reactions
using the template genomic DNA from the T1 seedlings that tolerated
4-5.times. field level spray of Finale herbicide.
TABLE-US-00001 YFP (SEQ ID NO: 3) Forward Primer:
5'-TGTTCCACGGCAAGATCCCCTACG-3' (SEQ ID NO: 4) Reverse Primer:
5'-TATTCATCTGGGTGTGATCGGCCA-3' PAT (SEQ ID NO: 5) Forward Primer:
5'-GGAGAGGAGACCAGTTGAGATTAG-3' (SEQ ID NO: 6) Reverse Primer:
5'-AGATCTGGGTAACTGGCCTAACTG-3'
[0149] The PCR for PAT and YFP (Yellow Florescent tag, Evrogen)
gene products were amplified in total reaction volume 50 .mu.L of
the mixture containing 100 ng genomic template DNA, 1.times.ExTaq
reaction buffer (TaKaRa Bio), 0.2 mM dNTP, 10 pmol each primer, and
0.025 units/.mu.L ExTaq. The following PCR conditions were used: 1
cycle at 96.degree. C. for 5 min and 31 cycles of the following PCR
program: 94.degree. C., 15 s; 65.degree. C., 30 s; 72.degree. C., 1
min. and final extension was performed at 72.degree. C. for 7 min
to complete product synthesis. The gel images were obtained using
Bio Rad Gel imagining System. (FIGS. 13 and 14). The amplified
fragments were gel-purified using a gel purification kit (Qiagen
Inc) according to the manufacturer's instructions
[0150] The PCR fragments were sequenced using PAT forward primer
and YFP forward at using advanced Sanger sequencing technology (MWG
Biotechnologies, Inc) and the sequence was analyzed using
Sequencher software.
[0151] The results show that the PAT and YFP sequences were
delivered through the nanoparticle and Quantum dot mediated DNA
delivery, thus providing clear evidence of stable genomic
integration of transgenes in the genomic DNA of the T1 plants.
Example 8
Facilitated Delivery of QD Across the JTNT1 Tobacco Single Cell
Wall
[0152] Several peptides were surface functionalized based on the
procedure discussed in Example 7 to test the noninvasive delivery
of the QDs across the cell wall. Cell Penetrating Peptide
(CPP)/Protein Transduction unit (PTD) attachment determination was
carried out via gel electrophoresis as described below.
[0153] Gel electrophoresis was carried out on QD-peptide conjugates
to confirm the attachment of peptides to QDs. The samples used were
QD-Amine (control), QD-Amine-R9, QD-Amine-Zein, QD-Amine-Pep1 and
QD-Amine-MPG. R9, Zein, Pep1 and MPG are peptides. A 2% (w/v)
agarose gel was run at 120 V in TBE (1.times., pH 8) buffer for 1
hour. The QD-Amine-peptides migrated towards the negative end of
the electrode showing the attachment of strongly positive character
of the peptides attached to QDs while QD-Amine remained static
showing weak positive charge of the amine group due to the
neutralizing effect of gel buffer at a basic Ph, as shown in FIG.
15 (Lane 1: QD-Amine; 2: QD Amine-R9; 3: QD-Amine-Y-Zein; 4: QD
Amine-Pep1; 5: QD-Amine-MPG).
[0154] The peptides were tested for internalization into the cells
and the emission of QD inside the cell was used as a measure to
track the level of internalization of the particles inside the cell
and compartments. JTNT1 single cells with intact walls were used as
target cells in these experiments. Table 1 shows the treatments of
the samples. The cells were tracked under the scope.
[0155] Microscopy was carried out within 1 minute after the
preparation of the sample on Spinning Disk Confocal microscope
(Andor Technology Revolution System). The excitation filter was set
at 488 nm while the emission filter was set beyond 650 nm.
[0156] As shown in Table 1, the control protoplasts and JTNT1
showed no auto fluorescence at the emission wavelength used for
QDs. Significant debris (broken cell parts) was observed in each of
these samples. Samples 4, 5, 6, and 7 did not show internalization
of QDs inside single cells or protoplasts. Sample 8 and 9 indicated
a clear presence of QDs surrounding the nucleus in both walled
single cells and protoplasts. This was due to the presence of the
cell penetrating peptide on the QDs which has the nuclear
localization signal (NLS). However, sample #6 and 7 had y-Zein
tethered to the QDs showed no QD internalization inside the cell.
This indicates that the QDs were taken into the cells due to the
Cell Penetrating Peptide (CPP) or the Protein Transduction unit
(PTD), y-Zein as the QDs that were functionalized only with amine
and not CPP/PTD did not get internalized.
TABLE-US-00002 TABLE 1 Single Cell type QD used (100 Functionalized
Autofluorescence localization in Functionalization type ul) QD
volume 480-650 nm the cell 1 QD-PEG-Amine-Control-1 NA 20 ul No NA
2 Protoplast-Control-2 Tobacco 0 ul No NA JTNT1 protoplast 3 Single
cells (Control-3) Tobacco 0 ul No NA JTNT1 walled single cells 4
QD-PEG-Amine Tobacco 20 ul No No JTNT1 protoplast 5 QD-PEG-Amine
Tobacco 20 ul No No JTNT1 walled single cells 6 QD-PEG-Amine-Zein
Tobacco 20 ul No No JTNT1 protoplast 7 QD-PEG-Amine-Zein Tobacco 20
ul No No JTNT1 walled single cells 8 QD-Amine-y-Zein Tobacco 20 ul
No Yes JTNT1 protoplast 9 QD-Amine-y-Zein Tobacco 20 ul No Yes
JTNT1 walled single cells
[0157] This data demonstrates evidence of cell internalization of
Quantum dots tethered to CPP/PTD with the nuclear localization
signal (NLS) taking the QDs across the cell wall of the intact
functional cell via Spinning Disk Confocal microscope (Andor
Technology Revolution System). The nuclear localization of the QDs
is possible across the cell wall in sample 9 and in the absence of
the cell wall as seen the protoplast based cell internalization
that is relieved of a cell wall through enzyme treatment. Thus the
mere presence of the cell wall does not hinder the internalization
of Quantum dots, evidencing particle entry is demonstrated
non-invasively with a CPP/PTD in plant cells with intact cell
wall.
[0158] While this invention has been described in certain
embodiments, the present invention can be further modified within
the spirit and scope of this disclosure. This application is
therefore intended to cover any variations, uses, or adaptations of
the invention using its general principles. Further, this
application is intended to cover such departures from the present
disclosure as come within known or customary practice in the art to
which this invention pertains and which fall within the limits of
the appended claims and their equivalents.
Sequence CWU 1
1
618448DNAArtificial SequencePlasmid pDAB3831 1ggccgctaaa cccagaaggt
aattatccaa gatgtagcat caagaatcca atgtttacgg 60gaaaaactat ggaagtatta
tgtaagctca gcaagaagca gatcaatatg cggcacatat 120gcaacctatg
ttcaaaaatg aagaatgtac agatacaaga tcctatactg ccagaatacg
180aagaagaata cgtagaaatt gaaaaagaag aaccaggcga agaaaagaat
cttgaagacg 240taagcactga cgacaacaat gaaaagaaga agataaggtc
ggtgattgtg aaagagacat 300agaggacaca tgtaaggtgg aaaatgtaag
ggcggaaagt aaccttatca caaaggaatc 360ttatccccca ctacttatcc
ttttatattt ttccgtgtca tttttgccct tgagttttcc 420tatataagga
accaagttcg gcatttgtga aaacaagaaa aaatttggtg taagctattt
480tctttgaagt actgaggata caacttcaga gaaatttgta agtttgtaga
tctccatggg 540ctccagcggc gccctgctgt tccacggcaa gatcccctac
gtggtggaga tggagggcaa 600tgtggatggc cacaccttca gcatccgcgg
caagggctac ggcgatgcca gcgtgggcaa 660ggtggatgcc cagttcatct
gcaccaccgg cgatgtgccc gtgccctgga gcaccctggt 720gaccaccctg
acctacggcg cccagtgctt cgccaagtac ggccccgagc tgaaggattt
780ctacaagagc tgcatgcccg atggctacgt gcaggagcgc accatcacct
tcgagggcga 840tggcaatttc aagacccgcg ccgaggtgac cttcgagaat
ggcagcgtgt acaatcgcgt 900gaagctgaat ggccagggct tcaagaagga
tggccacgtg ctgggcaaga atctggagtt 960caatttcacc ccccactgcc
tgtacatctg gggcgatcag gccaatcacg gcctgaagag 1020cgccttcaag
atctgccacg agatcaccgg cagcaagggc gatttcatcg tggccgatca
1080cacccagatg aataccccca tcggcggcgg ccccgtgcac gtgcccgagt
accaccacat 1140gagctaccac gtgaagctga gcaaggatgt gaccgatcac
cgcgataata tgagcctgaa 1200ggagaccgtg cgcgccgtgg attgccgcaa
gacctacctg tgagagctcg catgcggtca 1260ccaaaccttg gactcccatg
ttggcaaagg caaccaaaca aacaatgaat gatccgctcc 1320tgcatatggg
gcggtttgag tatttcaact gccatttggg ctgaattgaa gacatgctcc
1380tgtcagaaat tccgtgatct tactcaatat tcagtaatct cggccaatat
cctaaatgtg 1440cgtggcttta tctgtctttg tattgtttca tcaattcatg
taacgtttgc ttttcatatg 1500aattttcaaa taaattatcg cgatagtact
acgaatattt cgtatcgctg atcttctcaa 1560tcacaatgat gcgtagtgac
ccgacaaata atttaagcgt ccttaatacc aatcctaaaa 1620taattgaggc
aaataaaatt tttttgtaat ttttatgata gcagatcgat tctccagcaa
1680gcctgcaaca aaatattgtg tatttctaaa tagattttga tattaaaatc
ccgagaaagc 1740aaaattgcat ttaacaaaac agtaatttag tacattaata
aaaattatgc tcggccggcc 1800gcggccgctt aattaaattt aaatgtttaa
accccgcctg caggtcaacg gatcaggata 1860ttcttgttta agatgttgaa
ctctatggag gtttgtatga actgatgatc taggaccgga 1920taagttccct
tcttcatagc gaacttattc aaagaatgtt ttgtgtatca ttcttgttac
1980attgttatta atgaaaaaat attattggtc attggactga acacgagtgt
taaatatgga 2040ccaggcccca aataagatcc attgatatat gaattaaata
acaagaataa atcgagtcac 2100caaaccactt gcctttttta acgagacttg
ttcaccaact tgatacaaaa gtcattatcc 2160tatgcaaatc aataatcata
caaaaatatc caataacact aaaaaattaa aagaaatgga 2220taatttcaca
atatgttata cgataaagaa gttacttttc caagaaattc actgatttta
2280taagcccact tgcattagat aaatggcaaa aaaaaacaaa aaggaaaaga
aataaagcac 2340gaagaattct agaaaatacg aaatacgctt caatgcagtg
ggacccacgg ttcaattatt 2400gccaattttc agctccaccg tatatttaaa
aaataaaacg ataatgctaa aaaaatataa 2460atcgtaacga tcgttaaatc
tcaacggctg gatcttatga cgaccgttag aaattgtggt 2520tgtcgacgag
tcagtaataa acggcgtcaa agtggttgca gccggcacac acgagtcgtg
2580tttatcaact caaagcacaa atacttttcc tcaacctaaa aataaggcaa
ttagccaaaa 2640acaactttgc gtgtaaacaa cgctcaatac acgtgtcatt
ttattattag ctattgcttc 2700accgccttag ctttctcgtg acctagtcgt
cctcgtcttt tcttcttctt cttctataaa 2760acaataccca aagcttcttc
ttcacaattc agatttcaat ttctcaaaat cttaaaaact 2820ttctctcaat
tctctctacc gtgatcaagg taaatttctg tgttccttat tctctcaaaa
2880tcttcgattt tgttttcgtt cgatcccaat ttcgtatatg ttctttggtt
tagattctgt 2940taatcttaga tcgaagacga ttttctgggt ttgatcgtta
gatatcatct taattctcga 3000ttagggtttc ataaatatca tccgatttgt
tcaaataatt tgagttttgt cgaataatta 3060ctcttcgatt tgtgatttct
atctagatct ggtgttagtt tctagtttgt gcgatcgaat 3120ttgtcgatta
atctgagttt ttctgattaa caggtaagga tccaaccatg gcttctccgg
3180agaggagacc agttgagatt aggccagcta cagcagctga tatggccgcg
gtttgtgata 3240tcgttaacca ttacattgag acgtctacag tgaactttag
gacagagcca caaacaccac 3300aagagtggat tgatgatcta gagaggttgc
aagatagata cccttggttg gttgctgagg 3360ttgagggtgt tgtggctggt
attgcttacg ctgggccctg gaaggctagg aacgcttacg 3420attggacagt
tgagagtact gtttacgtgt cacataggca tcaaaggttg ggcctaggat
3480ccacattgta cacacatttg cttaagtcta tggaggcgca aggttttaag
tctgtggttg 3540ctgttatagg ccttccaaac gatccatctg ttaggttgca
tgaggctttg ggatacacag 3600cccggggtac attgcgcgca gctggataca
agcatggtgg atggcatgat gttggttttt 3660ggcaaaggga ttttgagttg
ccagctcctc caaggccagt taggccagtt acccagatct 3720gaggtaccct
gagcttgagc ttatgagctt atgagcttag agctcggatc cactagtaac
3780ggccgccagt gtgctggaat tcgcccttga ctagataggc gcccagatcg
gcggcaatag 3840cttcttagcg ccatcccggg ttgatcctat ctgtgttgaa
atagttgcgg tgggcaaggc 3900tctctttcag aaagacaggc ggccaaagga
acccaaggtg aggtgggcta tggctctcag 3960ttccttgtgg aagcgcttgg
tctaaggtgc agaggtgtta gcgggatgaa gcaaaagtgt 4020ccgattgtaa
caagatatgt tgatcctacg taaggatatt aaagtatgta ttcatcacta
4080atataatcag tgtattccaa tatgtactac gatttccaat gtctttattg
tcgccgtatg 4140taatcggcgt cacaaaataa tccccggtga ctttctttta
atccaggatg aaataatatg 4200ttattataat ttttgcgatt tggtccgtta
taggaattga agtgtgcttg cggtcgccac 4260cactcccatt tcataatttt
acatgtattt gaaaaataaa aatttatggt attcaattta 4320aacacgtata
cttgtaaaga atgatatctt gaaagaaata tagtttaaat atttattgat
4380aaaataacaa gtcaggtatt atagtccaag caaaaacata aatttattga
tgcaagttta 4440aattcagaaa tatttcaata actgattata tcagctggta
cattgccgta gatgaaagac 4500tgagtgcgat attatggtgt aatacatagc
ggccgggttt ctagtcaccg gtgtagcttg 4560gcgtaatcat ggtcatagct
gtttcctgtg tgaaattgtt atccgctcac aattccacac 4620aacatacgag
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
4680acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
gtgccagctg 4740cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
gtattgggcg ctcttccgct 4800gcgcacgctg cgcacgctgc gcacgcttcc
tcgctcactg actcgctgcg ctcggtcgtt 4860cggctgcggc gagcggtatc
agctcactca aaggcggtaa tacggttatc cacagaatca 4920ggggataacg
caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
4980aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
tcacaaaaat 5040cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
aaagatacca ggcgtttccc 5100cctggaagct ccctcgtgcg ctctcctgtt
ccgaccctgc cgcttaccgg atacctgtcc 5160gcctttctcc cttcgggaag
cgtggcgctt tctcatagct cacgctgtag gtatctcagt 5220tcggtgtagg
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
5280cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
cgacttatcg 5340ccactggcag cagccactgg taacaggatt agcagagcga
ggtatgtagg cggtgctaca 5400gagttcttga agtggtggcc taactacggc
tacactagaa ggacagtatt tggtatctgc 5460gctctgctga agccagttac
cttcggaaaa agagttggta gctcttgatc cggcaaacaa 5520accaccgctg
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
5580ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
gaacgaaaac 5640tcacgttaag ggattttggt catgagatta tcaaaaagga
tcttcaccta gatcctttta 5700aattaaaaat gaagttttaa atcaatctaa
agtatatatg agtaaacttg gtctgacagt 5760taccaatgct taatcagtga
ggcacctatc tcagcgatct gtctatttcg ttcatccata 5820gttgcctgac
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
5880agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
agcaataaac 5940cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
ctttatccgc ctccatccag 6000tctattaatt gttgccggga agctagagta
agtagttcgc cagttaatag tttgcgcaac 6060gttgttgcca ttgctacagg
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6120agctccggtt
cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
6180gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
gttatcactc 6240atggttatgg cagcactgca taattctctt actgtcatgc
catccgtaag atgcttttct 6300gtgactggtg agtactcaac caagtcattc
tgagaatagt gtatgcggcg accgagttgc 6360tcttgcccgg cgtcaatacg
ggataatacc gcgccacata gcagaacttt aaaagtgctc 6420atcattggaa
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
6480agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
tttcaccagc 6540gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
aaaagggaat aagggcgaca 6600cggaaatgtt gaatactcat actcttcctt
tttcaatatt attgaagcat ttatcagggt 6660tattgtctca tgagcggata
catatttgaa tgtatttaga aaaataaaca aataggggtt 6720ccgcgcacat
ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
6780ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
cggtgatgac 6840ggtgaaaacc tctgacacat gcagctcccg gagacggtca
cagcttgtct gtaagcggat 6900gccgggagca gacaagcccg tcagggcgcg
tcagcgggtg ttggcgggtg tcggggctgg 6960cttaactatg cggcatcaga
gcagattgta ctgagagtgc accatatgcg gtgtgaaata 7020ccgcacagat
gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
7080aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
ggcgaaaggg 7140ggatgtgctg caaggcgatt aagttgggta acgccagggt
tttcccagtc acgacgttgt 7200aaaacgacgg ccagtgaatt acaccggtgt
gatcatgggc cgcgattaaa aatctcaatt 7260atatttggtc taatttagtt
tggtattgag taaaacaaat tcgaaccaaa ccaaaatata 7320aatatatagt
ttttatatat atgcctttaa gactttttat agaattttct ttaaaaaata
7380tctagaaata tttgcgactc ttctggcatg taatatttcg ttaaatatga
agtgctccat 7440ttttattaac tttaaataat tggttgtacg atcactttct
tatcaagtgt tactaaaatg 7500cgtcaatctc tttgttcttc catattcata
tgtcaaaacc tatcaaaatt cttatatatc 7560tttttcgaat ttgaagtgaa
atttcgataa tttaaaatta aatagaacat atcattattt 7620aggtatcata
ttgattttta tacttaatta ctaaatttgg ttaactttga aagtgtacat
7680caacgaaaaa ttagtcaaac gactaaaata aataaatatc atgtgttatt
aagaaaattc 7740tcctataaga atattttaat agatcatatg tttgtaaaaa
aaattaattt ttactaacac 7800atatatttac ttatcaaaaa tttgacaaag
taagattaaa ataatattca tctaacaaaa 7860aaaaaaccag aaaatgctga
aaacccggca aaaccgaacc aatccaaacc gatatagttg 7920gtttggtttg
attttgatat aaaccgaacc aactcggtcc atttgcaccc ctaatcataa
7980tagctttaat atttcaagat attattaagt taacgttgtc aatatcctgg
aaattttgca 8040aaatgaatca agcctatatg gctgtaatat gaatttaaaa
gcagctcgat gtggtggtaa 8100tatgtaattt acttgattct aaaaaaatat
cccaagtatt aataatttct gctaggaaga 8160aggttagcta cgatttacag
caaagccaga atacaatgaa ccataaagtg attgaagctc 8220gaaatatacg
aaggaacaaa tatttttaaa aaaatacgca atgacttgga acaaaagaaa
8280gtgatatatt ttttgttctt aaacaagcat cccctctaaa gaatggcagt
tttcctttgc 8340atgtaactat tatgctccct tcgttacaaa aattttggac
tactattggg aacttcttct 8400gaaaatagtg gccaccgctt aattaaggcg
cgccatgccc gggcaagc 844828448DNAArtificial sequencereverse
complement of SEQ ID NO 1 2gcttgcccgg gcatggcgcg ccttaattaa
gcggtggcca ctattttcag aagaagttcc 60caatagtagt ccaaaatttt tgtaacgaag
ggagcataat agttacatgc aaaggaaaac 120tgccattctt tagaggggat
gcttgtttaa gaacaaaaaa tatatcactt tcttttgttc 180caagtcattg
cgtatttttt taaaaatatt tgttccttcg tatatttcga gcttcaatca
240ctttatggtt cattgtattc tggctttgct gtaaatcgta gctaaccttc
ttcctagcag 300aaattattaa tacttgggat atttttttag aatcaagtaa
attacatatt accaccacat 360cgagctgctt ttaaattcat attacagcca
tataggcttg attcattttg caaaatttcc 420aggatattga caacgttaac
ttaataatat cttgaaatat taaagctatt atgattaggg 480gtgcaaatgg
accgagttgg ttcggtttat atcaaaatca aaccaaacca actatatcgg
540tttggattgg ttcggttttg ccgggttttc agcattttct ggtttttttt
ttgttagatg 600aatattattt taatcttact ttgtcaaatt tttgataagt
aaatatatgt gttagtaaaa 660attaattttt tttacaaaca tatgatctat
taaaatattc ttataggaga attttcttaa 720taacacatga tatttattta
ttttagtcgt ttgactaatt tttcgttgat gtacactttc 780aaagttaacc
aaatttagta attaagtata aaaatcaata tgatacctaa ataatgatat
840gttctattta attttaaatt atcgaaattt cacttcaaat tcgaaaaaga
tatataagaa 900ttttgatagg ttttgacata tgaatatgga agaacaaaga
gattgacgca ttttagtaac 960acttgataag aaagtgatcg tacaaccaat
tatttaaagt taataaaaat ggagcacttc 1020atatttaacg aaatattaca
tgccagaaga gtcgcaaata tttctagata ttttttaaag 1080aaaattctat
aaaaagtctt aaaggcatat atataaaaac tatatattta tattttggtt
1140tggttcgaat ttgttttact caataccaaa ctaaattaga ccaaatataa
ttgagatttt 1200taatcgcggc ccatgatcac accggtgtaa ttcactggcc
gtcgttttac aacgtcgtga 1260ctgggaaaac cctggcgtta cccaacttaa
tcgccttgca gcacatcccc ctttcgccag 1320ctggcgtaat agcgaagagg
cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 1380tggcgaatgg
cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
1440catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
agccccgaca 1500cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
ctcccggcat ccgcttacag 1560acaagctgtg accgtctccg ggagctgcat
gtgtcagagg ttttcaccgt catcaccgaa 1620acgcgcgaga cgaaagggcc
tcgtgatacg cctattttta taggttaatg tcatgataat 1680aatggtttct
tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
1740tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
cctgataaat 1800gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
catttccgtg tcgcccttat 1860tccctttttt gcggcatttt gccttcctgt
ttttgctcac ccagaaacgc tggtgaaagt 1920aaaagatgct gaagatcagt
tgggtgcacg agtgggttac atcgaactgg atctcaacag 1980cggtaagatc
cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
2040agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
aactcggtcg 2100ccgcatacac tattctcaga atgacttggt tgagtactca
ccagtcacag aaaagcatct 2160tacggatggc atgacagtaa gagaattatg
cagtgctgcc ataaccatga gtgataacac 2220tgcggccaac ttacttctga
caacgatcgg aggaccgaag gagctaaccg cttttttgca 2280caacatgggg
gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
2340accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
tgcgcaaact 2400attaactggc gaactactta ctctagcttc ccggcaacaa
ttaatagact ggatggaggc 2460ggataaagtt gcaggaccac ttctgcgctc
ggcccttccg gctggctggt ttattgctga 2520taaatctgga gccggtgagc
gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 2580taagccctcc
cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
2640aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
tgtcagacca 2700agtttactca tatatacttt agattgattt aaaacttcat
ttttaattta aaaggatcta 2760ggtgaagatc ctttttgata atctcatgac
caaaatccct taacgtgagt tttcgttcca 2820ctgagcgtca gaccccgtag
aaaagatcaa aggatcttct tgagatcctt tttttctgcg 2880cgtaatctgc
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
2940tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
agataccaaa 3000tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
aagaactctg tagcaccgcc 3060tacatacctc gctctgctaa tcctgttacc
agtggctgct gccagtggcg ataagtcgtg 3120tcttaccggg ttggactcaa
gacgatagtt accggataag gcgcagcggt cgggctgaac 3180ggggggttcg
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
3240acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
acaggtatcc 3300ggtaagcggc agggtcggaa caggagagcg cacgagggag
cttccagggg gaaacgcctg 3360gtatctttat agtcctgtcg ggtttcgcca
cctctgactt gagcgtcgat ttttgtgatg 3420ctcgtcaggg gggcggagcc
tatggaaaaa cgccagcaac gcggcctttt tacggttcct 3480ggccttttgc
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
3540taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
cgaccgagcg 3600cagcgagtca gtgagcgagg aagcgtgcgc agcgtgcgca
gcgtgcgcag cggaagagcg 3660cccaatacgc aaaccgcctc tccccgcgcg
ttggccgatt cattaatgca gctggcacga 3720caggtttccc gactggaaag
cgggcagtga gcgcaacgca attaatgtga gttagctcac 3780tcattaggca
ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
3840gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
agctacaccg 3900gtgactagaa acccggccgc tatgtattac accataatat
cgcactcagt ctttcatcta 3960cggcaatgta ccagctgata taatcagtta
ttgaaatatt tctgaattta aacttgcatc 4020aataaattta tgtttttgct
tggactataa tacctgactt gttattttat caataaatat 4080ttaaactata
tttctttcaa gatatcattc tttacaagta tacgtgttta aattgaatac
4140cataaatttt tatttttcaa atacatgtaa aattatgaaa tgggagtggt
ggcgaccgca 4200agcacacttc aattcctata acggaccaaa tcgcaaaaat
tataataaca tattatttca 4260tcctggatta aaagaaagtc accggggatt
attttgtgac gccgattaca tacggcgaca 4320ataaagacat tggaaatcgt
agtacatatt ggaatacact gattatatta gtgatgaata 4380catactttaa
tatccttacg taggatcaac atatcttgtt acaatcggac acttttgctt
4440catcccgcta acacctctgc accttagacc aagcgcttcc acaaggaact
gagagccata 4500gcccacctca ccttgggttc ctttggccgc ctgtctttct
gaaagagagc cttgcccacc 4560gcaactattt caacacagat aggatcaacc
cgggatggcg ctaagaagct attgccgccg 4620atctgggcgc ctatctagtc
aagggcgaat tccagcacac tggcggccgt tactagtgga 4680tccgagctct
aagctcataa gctcataagc tcaagctcag ggtacctcag atctgggtaa
4740ctggcctaac tggccttgga ggagctggca actcaaaatc cctttgccaa
aaaccaacat 4800catgccatcc accatgcttg tatccagctg cgcgcaatgt
accccgggct gtgtatccca 4860aagcctcatg caacctaaca gatggatcgt
ttggaaggcc tataacagca accacagact 4920taaaaccttg cgcctccata
gacttaagca aatgtgtgta caatgtggat cctaggccca 4980acctttgatg
cctatgtgac acgtaaacag tactctcaac tgtccaatcg taagcgttcc
5040tagccttcca gggcccagcg taagcaatac cagccacaac accctcaacc
tcagcaacca 5100accaagggta tctatcttgc aacctctcta gatcatcaat
ccactcttgt ggtgtttgtg 5160gctctgtcct aaagttcact gtagacgtct
caatgtaatg gttaacgata tcacaaaccg 5220cggccatatc agctgctgta
gctggcctaa tctcaactgg tctcctctcc ggagaagcca 5280tggttggatc
cttacctgtt aatcagaaaa actcagatta atcgacaaat tcgatcgcac
5340aaactagaaa ctaacaccag atctagatag aaatcacaaa tcgaagagta
attattcgac 5400aaaactcaaa ttatttgaac aaatcggatg atatttatga
aaccctaatc gagaattaag 5460atgatatcta acgatcaaac ccagaaaatc
gtcttcgatc taagattaac agaatctaaa 5520ccaaagaaca tatacgaaat
tgggatcgaa cgaaaacaaa atcgaagatt ttgagagaat 5580aaggaacaca
gaaatttacc ttgatcacgg tagagagaat tgagagaaag tttttaagat
5640tttgagaaat tgaaatctga attgtgaaga agaagctttg ggtattgttt
tatagaagaa 5700gaagaagaaa agacgaggac gactaggtca cgagaaagct
aaggcggtga agcaatagct 5760aataataaaa tgacacgtgt attgagcgtt
gtttacacgc aaagttgttt ttggctaatt 5820gccttatttt taggttgagg
aaaagtattt gtgctttgag ttgataaaca cgactcgtgt 5880gtgccggctg
caaccacttt gacgccgttt attactgact cgtcgacaac cacaatttct
5940aacggtcgtc ataagatcca gccgttgaga tttaacgatc gttacgattt
atattttttt 6000agcattatcg ttttattttt taaatatacg gtggagctga
aaattggcaa taattgaacc 6060gtgggtccca ctgcattgaa gcgtatttcg
tattttctag aattcttcgt gctttatttc 6120ttttcctttt tgtttttttt
tgccatttat ctaatgcaag tgggcttata aaatcagtga 6180atttcttgga
aaagtaactt ctttatcgta taacatattg tgaaattatc catttctttt
6240aattttttag tgttattgga tatttttgta tgattattga tttgcatagg
ataatgactt 6300ttgtatcaag ttggtgaaca agtctcgtta aaaaaggcaa
gtggtttggt gactcgattt 6360attcttgtta tttaattcat atatcaatgg
atcttatttg gggcctggtc catatttaac 6420actcgtgttc agtccaatga
ccaataatat tttttcatta ataacaatgt aacaagaatg 6480atacacaaaa
cattctttga
ataagttcgc tatgaagaag ggaacttatc cggtcctaga 6540tcatcagttc
atacaaacct ccatagagtt caacatctta aacaagaata tcctgatccg
6600ttgacctgca ggcggggttt aaacatttaa atttaattaa gcggccgcgg
ccggccgagc 6660ataattttta ttaatgtact aaattactgt tttgttaaat
gcaattttgc tttctcggga 6720ttttaatatc aaaatctatt tagaaataca
caatattttg ttgcaggctt gctggagaat 6780cgatctgcta tcataaaaat
tacaaaaaaa ttttatttgc ctcaattatt ttaggattgg 6840tattaaggac
gcttaaatta tttgtcgggt cactacgcat cattgtgatt gagaagatca
6900gcgatacgaa atattcgtag tactatcgcg ataatttatt tgaaaattca
tatgaaaagc 6960aaacgttaca tgaattgatg aaacaataca aagacagata
aagccacgca catttaggat 7020attggccgag attactgaat attgagtaag
atcacggaat ttctgacagg agcatgtctt 7080caattcagcc caaatggcag
ttgaaatact caaaccgccc catatgcagg agcggatcat 7140tcattgtttg
tttggttgcc tttgccaaca tgggagtcca aggtttggtg accgcatgcg
7200agctctcaca ggtaggtctt gcggcaatcc acggcgcgca cggtctcctt
caggctcata 7260ttatcgcggt gatcggtcac atccttgctc agcttcacgt
ggtagctcat gtggtggtac 7320tcgggcacgt gcacggggcc gccgccgatg
ggggtattca tctgggtgtg atcggccacg 7380atgaaatcgc ccttgctgcc
ggtgatctcg tggcagatct tgaaggcgct cttcaggccg 7440tgattggcct
gatcgcccca gatgtacagg cagtgggggg tgaaattgaa ctccagattc
7500ttgcccagca cgtggccatc cttcttgaag ccctggccat tcagcttcac
gcgattgtac 7560acgctgccat tctcgaaggt cacctcggcg cgggtcttga
aattgccatc gccctcgaag 7620gtgatggtgc gctcctgcac gtagccatcg
ggcatgcagc tcttgtagaa atccttcagc 7680tcggggccgt acttggcgaa
gcactgggcg ccgtaggtca gggtggtcac cagggtgctc 7740cagggcacgg
gcacatcgcc ggtggtgcag atgaactggg catccacctt gcccacgctg
7800gcatcgccgt agcccttgcc gcggatgctg aaggtgtggc catccacatt
gccctccatc 7860tccaccacgt aggggatctt gccgtggaac agcagggcgc
cgctggagcc catggagatc 7920tacaaactta caaatttctc tgaagttgta
tcctcagtac ttcaaagaaa atagcttaca 7980ccaaattttt tcttgttttc
acaaatgccg aacttggttc cttatatagg aaaactcaag 8040ggcaaaaatg
acacggaaaa atataaaagg ataagtagtg ggggataaga ttcctttgtg
8100ataaggttac tttccgccct tacattttcc accttacatg tgtcctctat
gtctctttca 8160caatcaccga ccttatcttc ttcttttcat tgttgtcgtc
agtgcttacg tcttcaagat 8220tcttttcttc gcctggttct tctttttcaa
tttctacgta ttcttcttcg tattctggca 8280gtataggatc ttgtatctgt
acattcttca tttttgaaca taggttgcat atgtgccgca 8340tattgatctg
cttcttgctg agcttacata atacttccat agtttttccc gtaaacattg
8400gattcttgat gctacatctt ggataattac cttctgggtt tagcggcc
8448324DNAArtificial sequenceForward primer for YFP 3tgttccacgg
caagatcccc tacg 24424DNAArtificial sequenceReverse primer for YFP
4tattcatctg ggtgtgatcg gcca 24524DNAArtificial sequenceForward
primer for PAT 5ggagaggaga ccagttgaga ttag 24624DNAArtificial
sequenceReverse primer for PAT 6agatctgggt aactggccta actg 24
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