Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02594294 2007-07-30
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CA 02594294 2007-07-30
l
DESCRIPTION
PLANT-OPTIMIZED GENES ENCODING
PESTICIDAL TOXINS
10
Backo ound of the Invention
Insects and other pests cost farmers billions of dollars annually in crop
losses and in the
expense of keeping these pests under control. The losses caused by insect
pests in agricultural
production environments include decrease in crop yield, reduced crop quality,
and increased
harvesting costs.
Chemical pesticides have provided an effective method of pest control:
however, the
public has become concerned about the amount of residual chemicals which might
be found in
food, ground water, and the environment. Therefore, synthetic chemical
pesticides are being
inereasingly scrutinized, and correctly so, for their potential toxic
environmental consequences.
Synthetic chemical pesticides can poison the soil and underlying aquifers,
pollute surface waters
as a result oftunoff, and destroy non-target life forms. Synthetic chemical
control agents have
the further disadvantage of presenting public safety hazards when they are
applied in areas
where pets, farm animals, or children may come into contact with them. They
may also provide
health hazards to applicants, especially if the proper application techniques
are not followed.
Regulatory agencies around the world are restricting and/or banning the uses
of many pesticides
and particularly the synthetic chemical pesticides which are persistent in the
environment and
enter the food chain. Examples of widely used synthetic chemical pesticides
include the
organochlorines, e.g., DDT, mirex, kepone, lindane, aldrin, chlordane,
aldicarb. and dieldrin; the
organophosphates, e.g., chlorpyrifos, parathion, malathion, and diazinon; and
carbamates.
Stringent new restrictions on the use of pesticides and the elimination of
some effective
pesticides from the market place could limit economical and effective options
for controlling
costly pests.
Because of the problems associated with the use of synthetic chemical
pesticides, there
exists a clear need to limit the use of these agents and a need to identify
alternative control
CA 02594294 2007-07-30
I
agents. The replacement of synthetic chemical pesticides, or combination of
these agents with
biological pesticides, could reduce the levels of toxic chemicals in the
environment.
A biological pesticidal agent that is enjoying increasing populanty is the
soil microbe
Bacillus thuringiensis (B.t_). The soil microbe Bacillus thuringiensis (B.t. )
is a Gram-positive,
spore-forming bacterium. Most strains of B.t. do not exhibit pesticidal
activity. Some B.I.
strains produce, and can be characterized by, parasporal crystalline protein
inclusions. These
"d-endotoxins," which typically have specific pesticidal activity, are
different from exotoxins,
which have a non-specific host range. These inclusions often appear
microscopically as
distinctively shaped crystals. The proteins can be highlv toxic to pests and
are specific in their
toxic activity.
Preparations of the spores and crystals of B. tlturingiensis subsp. kurstaki
have been used
for many years as commercial insecticides for lepidopteran pests. For example.
B_ t/zuringiensis
var. kurstaki HD- I produces a crystalline 6-endotoxin which is toxic to the
larvae of a number
of lepidopteran insects.
The cloning and expression of a B.r. crystai protein gene in Escizerichia coii
was
described in the pubiished literature more than 15 vears ago (Schnepf, H.E..
H.R. Whiteley
[1981] Proc. ,Vatl..4cad. Sci. USA 78:2893-2897.). U.S. Patent No. 4,448,885
and U.S. Patent
No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coti.
Recombinant DNA-
based B.t. products have been produced and approved for use.
Commercial use of B.I. pesticides was originally restricted to a narrow range
of
lepidopteran (caterpillar) pests. More recently, however, investigators have
discovered B.t.
pesticides with specificities for a much broader range of pests_ For example,
other species of
B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.I. M-7),
have been used
commercially to control insects of the orders Diptera and Coleoptera,
respectivelv (Gaertner,
F.H. [1989) "Cellular Delivery Systems for Insecticidal Proteins: Living and
Non-Living
Microorganisms." in Controlled Delivery of Crop Protection Agents, R.M.
Wilkins, ed., Taylor
and Francis, New York and London, 1990, pp. 245-255).
New subspecies of B.I. have now been identified, and genes responsible for
active 6-
endotoxin proteins have been isolated and sequenced (Hofte, H., H.R. Whiteley
[1989]
Microbiological Reviews 52(2):242-255). Hofte and Whiteley classified B.t.
crvstal protein
genes into four major classes. The classes were crvI (Lepidoptera-specific),
crvll (Lepidoptera-
and Diptera-specific), cryIII (Coleoptera-specific), and crvIV (Diptera-
specific). The discovery
of strains specifically toxic to other pests has been reported (Feitelson,
J.S., J. Pavne, L. Kim
[1992] Bio/Technolo,gy 10:271-275). For example, the designations CryV and
CryVi have been
proposed for two new groups of nematode-active toxins.
CA 02594294 2007-07-30
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Many Bacillus thuringiensis 6-endotoxin crystal protein molecules are composed
of two
functional segments. For these proteins, the protease-resistant core toxin is
the first segment and
corresponds to about the first half of the protein molecule. The three-
dimensional structure of
a core segment of a CryllIA B.t. S-endotoxin is known, and it was proposed
that all related
toxins have that same overall structure (Li, J., J. Carroll, D.J. Ellar [19911
Nature 353:81 5-821).
The second half of the molecule is often referred to as the "protoxin
segment." The protoxin
segment is believed to participate in toxin crystal formation (Arvidson, H.,
P.E. Dunn, S. Strand,
A.I. Aronson [1989] iVfolecularMicrodiology 3:1533-1534; Choma, C.T., W.K.
Surewicz, P.R.
Carey, M. Pozsgay, T. Raynor, H. Kaplan [1990] Eur. J. Biochem. 189:523-527).
The :ull 130
kDa toxin molecule is typically processed to the resistant core segment by
proteases in the insect
gut. The protoxin segment may thus convey a partial insect specificity for the
toxin by limiting
the accessibility of the core to the insect bv reducing the protease
processing of the toxin
molecule (Haider, M.Z., B.H. Knowles. D.J. Ellar [1986] Eur. J. Biochem.
156:531-540) or by
reducing toxin solubility (Aronson, A.L. E.S. Han. W. McGaughey, D. Johnson [
1991 ].-lppl.
Environ. Microbiol. 57:981-986).
The 1989 nomenclature and classification scheme of Hofte and Whiteiey was
based on
both the deduced amino acid sequence and the host range of the toxin. That
system was adapted
to cover 14 different types of toxin genes which were divided into five major
classes. The
number of sequenced Bacillus thuringiensis crystal protein genes currently
stands at more than
50. A revised nomenclature scheme has been proposed which is based solely on
amino acid
identity (Criclanore et al. [ 19961 Society for lnvertebrate Pathology, 29th
Annual Meeting, IIIrd
International Colloquium on Bacillus thuringiensis, University of Cordoba.
Cordoba. Spain,
September 1-6, 1996, abstract). The mnemonic "cry" has been retained for all
of the toxin genes
except cvtA and cytB, which remain a separate class. Roman numerals have been
exchanged
for Arabic numerals in the primary rank, and the parentheses in the tertiary
rank have been
removed. Many of the original names have been retained, although a number have
been
reclassified.
With the use of genetic engineering techniques, new approaches for delivering
B.t.
toxins to agricultural environments are under development, including the use
of plants
genetically engineered with B.t. toxin genes for insect resistance and the use
of stabilized,
microbial cells as delivery vehicles of B.t. toxins (Gaertner, F.H., L. Kim
[1988] TIBTECH6:S4-
S7). Thus, isolated B_t. endotoxin genes are becoming commercially valuable.
Vanous improvements have been achieved by modifying B.I. toxins and/or their
genes.
For example, U.S. Patent Nos. 5,380,831 and 5.567,862 relate to the production
of synthetic
insecticidal crystal protein genes having improved expression in plants.
CA 02594294 2007-07-30
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Obstacles to the successful agricultural use of B.t. toxins include the
deveioprnent of
resistance to B.t. toxins by insects. In addition, certain insects can be
refractory to the effects
of B.t. The latter includes insects such as boll weevil and black cutworm as
well as adult insects
of most species which heretofore have demonstrated no apparent significant
sensitivity to B.I.
S-endotoxins.
Thus, resistance management strategies in B.t. plant technology have become of
great
interest, and there remains a great need for new toxin genes. As a result of
extensive research
and resource investment, other patents have issued for new B.t. isolates,
toxins, and genes, and
for new uses of B_t. isolates. See Feitelson el al., supra, for a review.
Additional examples
include the following:
B.t. Isolate, Toxin, and/or Exemplified Pesticidal U.S. Patent Lo. (unless
Gene Activitv of Toxin otherwise indicated)
PS81I, 81IA, 8l IB2 lepidopteran 5,126,133; 5,188.960
CrylAc lepidopteran Adang et al.. GENBANK Acc.
No. M 11068
IC / IA(b) chimeric toxin lepidopteran 5.593,881
IF / IA(b) chimeric toxin lepidopteran 5,527,883
PS 158C, 158C2c lepidopteran 5,268,172; 5,723,758
PS31 G 1, 31 G 1 a lepidopteran WO 98/00546 (published PCT
application)
However, the discovery of new B.t. isolates and new uses of known B.t.
isolates remains an
empirical, unpredictable art.
There remains a great need for new toxin genes that can be successfully
expressed at
adequate levels in plants in a manner that will result in the effective
control of insects and other
pests.
Brief Summary of the Invention
The subject invention concerns materials and methods useful in the control of
pests and,
particularly, plant pests. More specifically, the subject invention provides
plant-optimized
poiynucleotide sequences that encode pesticidal toxins (full-length and
truncated). Truncated
polynucleotide sequences can be used to produce truncated toxins or for the
production of fusion
(or chimeric) genes and proteins. The polynucleotide sequences of the subject
invention have
certain modifications, compared to wild-type sequences, that make them
particularly well-suited
CA 02594294 2007-07-30
for optimized expression in plants. Using techniques known to those skilled in
the art, the
polynucleotide sequences described herein can be used to transform plants in
order to confer pest
resistance upon said plants.
In one preferred embodiment, the subject invention provides plant-optimized
genes that
5 encode other proteins that are toxic to pests. Preferred embodiments are
referred to herein as
1 AC 1 AB-N-PO, 1 AC 1 AB-PO, 1 AC 1 AB-B-PO, 1 AC-T-PO, 1 AC-TB-PO, 1 AC-TBX-
PO,
1 C-T-PO, l C I AB-PO, 15 8C2c-PO, 15 8C2c-T-PO, and 31 Gla-PO.
The subject invention also provides other plant-optimized polynucleotide
sequences which
encode Cry 1 F toxins that are active against lepidopteran insects. These
polynucleotide sequences
include plant-optimized genes designated 1 F 1 AB-PO, 1 F-T-PO, 1 F-7G-PO, and
1 F-7Z-PO.
The subject invention further provides plant-optimized polynucleotide
sequences that
encode C-terminal, protoxin portions that can be used with genes encoding
truncated, core toxins
to produce full-length toxins. Preferred embodiments of plant-optimized
protoxins are designated
PT-1 AB-PO and PT-1 AB-2-PO.
In addition, the subject invention provides unique amino acids sequences for
pesticidal
toxins. These toxins are encoded by the genes designated 1F1AB-PO; 1F-T-PO, 1F-
7G-PO, and
1 F-7Z-PO; 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO; 1 C 1 AB-PO; 15
8C2c-PO;
158C2c-T-PO; and 31 G 1 a-T-PO. Furthermore, the subject invention provides
unique, C-terminal
amino acid sequences for protoxin portions (of full-length Bacillus
thuringiensis toxins) encoded
by the polynucleotide sequences designated PT-IAB-PO and PT-lAB-2-PO.
Brief Description of the Sequences
SEQ ID NO. 1 is a polynucleotide sequence for a full-length, plant-optimized
crylFlcrylA(b) hybrid gene designated 1 F 1 AB-PO.
SEQ ID NO. 2 is an amino acid sequence for a full-length, plant-optimized
CryIF/CryIA(b) chimeric toxin. The 1F1AB-PO gene encodes this toxin.
SEQ ID NO. 3 is a polynucleotide sequence for a truncated, plant-optimized
crylF gene
designated IF-T-PO.
SEQ ID NO. 4 is an amino acid sequence for a truncated, plant-optimized CryIF
toxin.
The genes designated 1F-T-PO, 1F-7G-PO, and 1F-7Z-PO encode this toxin.
SEQ ID NO. 5 is the native polynucleotide sequence of the wild-type, full
length B.t.
toxin gene designated 81 IA (crylF).
SEQ ID NO. 6 is the amino acid sequence of the full length, wild-type B.t.
toxin
designated 81 IA (Cry1F).
CA 02594294 2007-07-30
6
SEQ ID NO. 7 is a polynucleotide sequence for a gene designated 1 F-7G-PO,
which
is optimized for expression in cotton.
SEQ ID NO. 8 is a polynucleotide sequence for a gene designated 1 F-7Z-PO,
which is
optimized for expression in maize.
SEQ ID NO. 9 is a polynucleotide sequence designated PT-1 AB-PO, wiuch is
optimized
for expression in plants. This gene, which encodes a Cry 1Ab protoxin portion,
can be used in
conjunction with truncated genes (genes encoding truncated, core toxins) to
make full-length
toxins. Unless otherwise indicated, the chimeric genes exemplified herein are
shown with this
polynucleotide sequence (PT-1 AB-PO).
SEQ ID NO. 10 is a polynucleotide sequence designated PT-IAB-2-PO, which is
optimized for expression in cotton. This polvnucleotide sequence is an
alternative to PT-1 AB-
PO (and also encodes a CrylAb protoxin portion) and can also be used in
conjunction with
uvncated genes (genes encoding truncated, core toxins) to make full-length
toxins. PT- i AB-?-
PO is preferred for use in a host that is transformed with more than one twe
of endotoxin
transgene.
SEQ ID NO. 11 is an amino acid sequence of a protoxin portion encoded by the
genes
designated PT-IAB-PO and PT-lAB-2-PO.
SEQ ID NO. 12 is a polynucleotide sequence for a gene designated 1 AC 1.-a.B-N-
PO,
which is optimized for expression in plants. This gene encodes a chimenc Cry 1
Ac ( N-terminal)
: Cry 1 Ab (protoxin) toxin.
SEQ ID NO. 13 is a polynucleotide sequence for a gene designated I AC I AB-PO,
which
is optimized for expression in plants_ This gene encodes a chimenc CrvlAc (N-
terminal) %
Cry 1 Ab (protoxin) toxin.
SEQ ID NO. 14 is a polynucleotide sequence for a gene designated 1 AC 1 AB-B-
PO,
which is optimized for expression in plants. This gene encodes a chimeric Cry
I Ac (N-terminal)
! Cry I Ab (protoxin) toxin.
SEQ ID NO. 15 is an amino acid sequence of a toxin encoded by the genes
designated
I AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO.
SEQ ID NO. 16 is a polynucleotide sequence for a gene designated 1 AC-T-PO,
which
is optimized for expression in plants. This plant-optimized gene encodes a
core toxin, the amino
acid sequence of which is the same as that of the truncated form of a Cry lAc
toxin described by
Adang et al. in GENBANK (Acc. No. M 11068).
SEQ ID NO. 17 is a polynucleotide sequence for a gene designated I AC-TB-PO,
which
is optimized for expression in plants. This piant-optimized gene encodes a
core toxin, the amino
CA 02594294 2007-07-30
7
acid sequence of which is the same as that of the truncated form of a Cry I Ac
toxin descnbed by
Adang et al, in GENBAINK (Acc. No. M 11068)_
SEQ ID NO. 18 is an alternative polynucleotide sequence for a gene designated
I AC-TBX-PO, which is optimized for expression in plants. This plant-optimized
gene encodes
a core toxin, the amino acid sequence of which is the same as that of the
truncated form of a
Cry 1 Ac toxin described by Adang et al. in GENBANK (Acc. No. M 11068).
SEQ ID NO. 19 is a polynucleotide sequence, optimized for expression in
dicots, for
a gene designated 1 C-T-PO, which encodes the truncated form of a Cry1 C toxin
designated
811B2 in U.S. Patent No. 5,246,852.
SEQ ID NO. 20 is a polynucleotide sequence for a gene designated 1 C 1 AB-PO,
which
is optimized for expression in plants. This gene encodes a chimeric Crv1C (N-
terminal) i
Cry 1Ab (protoxin) toxin.
SEQ ID NO. 21 is an amino acid sequence of a toxin encoded by the gene
designated
1 C 1 AB-PO.
SEQ LD NO. 22 is a polynucleotide sequence for a gene designated 158C2c-PO.
SEQ ID'1O. 23 is an amino acid sequence for a full-length toxin encoded bv the
;ene
desimated 158C2c-PO.
SEQ ID NO. 24 is a polynucleotide sequence for a gene designated 158C2c-T'-PO.
SEQ LD NO. 25 is an amino acid sequence for a truncated toxin encoded by the
gene
designated 158C2c-T-PO.
SEQ ID NO. 26 is a poiynucleotide sequence for a gene designated 31 G I a-T-
PO, which
is optimized for expression in maize.
SEQ m NO. 27 is an amino acid sequence for a truncated toxin encoded by the
gene
desienated 31 G 1 a-T-PO.
Detailed Disclosure of the Invention
The subject invention concerns materials and methods useful in the control of
pests and,
particularly, plant pests. More specifically, the subject invention provides
plant-optimized
polynucleotide sequences that encode pesticidal toxins (full-length and
truncated). Truncated
polynucleotide sequences can be used to produce truncated toxins or for the
production of fusion
(or chimeric) genes and proteins. The polynucleotide sequences of the subject
invention have
certain modifications, compared to wild-type sequences, that make them
particularly well-suited
for optimized expression in plants. Using techniques known to those skilled in
the art, the
polynucleotide sequences described herein can be used to transform ptants in
order to confer pest
resistance upon said plants.
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8
In one preferred embodiment, the subject invention provides plant-optimized
genes that
encode other proteins that are toxic to pests. Preferred embodiments are
referred to herein as
1 AC 1 AB-N-PO, 1 AC I AB-PO, 1 AC 1 AB-B-PO, 1 AC-T-PO, IAC-TB-PO, IAC-TBX-
PO,
IC-T-PO, 1 C 1 AB-PO, 158C2c-PO, 158C2c-T-PO, and 31 Gla-PO.
The subject invention also provides other plant-optimized polynucleotide
sequences which
encode Cryl F toxins that are active against lepidopteran insects. These
polynucleotide sequences
include plant-optimized genes designated 1 F 1 AB-PO, 1 F-T-PO, 1 F-7G-PO, and
1 F-7Z-PO.
The subject invention further provides plant-optimized polynucleotide
sequences that
encode C-terminal, protoxin portions that can be used with genes encoding
truncated, core toxins
to produce full-length toxins. Preferred embodiments of plant-optimized
protoxins are designated
PT-1 AB-PO and PT-1 AB-2-PO.
In addition, the subject invention provides unique amino acids sequences for
pesticidal
toxins. These toxins are encoded by the genes designated 1 F 1 AB-PO; 1 F-T-
PO, 1 F-7G-PO, and
1 F-7Z-PO; 1 AC 1 AB-N-PO, 1 AC 1 AB-PO, and 1 AC 1 AB-B-PO; 1 C 1 AB-PO;
158C2c-PO;
158C2c-T-PO; and 31GIa-T-PO. Furthermore, the subject invention provides
unique, C-terminal
amino acid sequences for protoxin portions (of full-length Bacillus
thuringiensis toxins) encoded
by the polynucleotide sequences designated PT-IAB-PO and PT-1AB-2-PO.
In one embodiment the subject invention provides genes which express a CryIF
toxin that
is truncated compared to the full length CryIF toxin. The truncated toxins of
the subject invention
are typically missing all or a portion of the protoxin segment. Also, the
truncated genes of the
subject invention can be used for the production of fusion (or chimeric) genes
and proteins. One
example is the plant-optimized gene comprising a crylF portion and a cryIA(b)
portion, wherein
the hybrid gene encodes a chimeric toxin. In a preferred embodiment, the CryIF
portion of the
chimeric toxin is itself pesticidal.
More specifically, one example of a chimeric DNA molecule of the subject
invention is
shown in SEQ IF NO. 1, which as a crylF 5' portion and a 3' crylA(b) portion
of the DNA
molecule. The chimeric toxin encoded by SEQ IF NO. I is shown in SEQ ID NO. 2.
The chimeric
toxin encoded by SEQ ID NO. 1 comprises a Cryl F core toxin comprising
approximately the first
605 amino acids encoded by the nucleotides from approximately 1 to
approximately 1815. This
chimeric gene also comprises a crylAb protoxin portion, which encodes amino
acids from
approximately 606 to approximately 1148. The CrylAb protoxin portion is
encoded by the
nucleotides from approximately 1816 to approximately 3444.
The sequence of a preferred, truncated crylF gene of the subject invention
(1815
nucleotides) is shown in SEQ ID NO. 3. This truncated gene corresponds to
nucleotides 1-1815
CA 02594294 2007-07-30
9
of the chimenc gene of SEQ ID NO. 1. A stop codon, such as TAA or TAG, can be
added to
this sequence at positions 1816-1818. for example, if the use of a truncated
toxin, without a
protoxin portion, is desired. Other polvnucleotide sequences and genes of the
subject invention
can be similarly modified, as would be recognized by one skilled in the art.
The synthetic,
truncated Cry iF toxin (encoded by SEQ ID NO. 3) is shown in SEQ ID NO. 4.
As can be seen by comparing, for example. SEQ ID NOS. l and 2 with SEQ ID NOS.
3 and 4, and .vith SEQ ID NOS. 9 and 10, there can be some overlap between the
sequences for
the "truncated genes" and the sequences for the "protoxin portions"
exemplified herein.
PT-IAB-PO can be used in preferred embodiments in combination with other
truncated
oenes of the subject invention, such as the 1 C-T-PO gene, in order to form
other hybrid genes
that encode full-length toxins. PT-1 AB-2-PO (an altemative polynucleotide
sequence that
encodes a protoxin portion) can also be used with truncated genes (which are
smailer than full-
length toxin genes, so long as the protein encoded bv the truncated gene
retains pesticidal
activity) to encode chimeric or hybrid toxins. Preferred uses of PT-lAB-2-PO
are described
above in the section entitled "Description of the Sequences."
Using techniques such as computer- or software-assisted sequence alinments,
differences can be noted in the nucleotide sequence of the subject plant-
optimized "enes as
compared to the wild-type genes or to previously lmown genes. For example, SEQ
ID NO. I
or SEQ ID NO 3 can be compared to SEQ ID NO. 5, which is the 3522-basepair,
wild-type crvlF
gene. Similarly, differences in the unique amino acid sequences of the subject
invention can be
noted as compared to wild-type toxins or to previously known toxins.
It should be apparent to a person skilled in this art that, given the
sequences of the genes
as set forth herein, the genes of the subject invention can be obtained
through several means.
In preferred embodiments, the subject genes may be constructed synthetically
by using a gene
synthesizer, for example. The specific genes exemplified herein can also be
obtained by
modifying, according to the teachings of the subject invention, certain wild-
type genes (for
example, by point-mutation techniques) from certain isolates deposited at a
culture depository
as discussed below. For example, a wild-type crvIF gene can be obtained from
B.t. isolate
PS81I. Likewise, the crylA(b) portions of the hybrid genes of the subject
invention can be
produced synthetically or can be derived by modifying wild-type genes.
CryIA(b) toxins and
genes have been described in, for example, H6fte et al. (1986) Eur. J.
Biochem. 161:273; Geiser
et al. (1986) Gene 48:109; and Haider et al. (1988) Nucleic,4cids Res_
16:10927. Clones and
additional wild-type isolates are discussed in more detail, above, in the
section entitled
"Background of the Invention" and in the list, below.
CA 02594294 2007-07-30
Cultures discussed in this application have becn deposited in the Agricultural
IZcsearch
Service Patent Culture Collection (NRRL), Nortllern Regional Research Center,
1815 North
University Street, Peona, Illinois 61604, USA. "I'he deposited strains listed
below are disclosed
in the patent references as discussed above in the section entitled
"Background of the lnventron."
Subculture Accession Number Deposit Date
B. PS81I NRRL B-18484 April 19. 1989
E. coli (NM522) (pMYC1603) (81IA) NRRI. B-185 17 June 30, 1989
10 E. coli (NM522) (pMYC394) (81IB22) NRRL B-18500 iMav 17. 1989
B.t. PS158C2 NRRL B-18872 Aug. 27, I991
E. coli (NM522) (pMYC2383) (158C2c) NRRI. B-21428 April 11, 1995
B_t. PS31G1 NRRL B-21560 April 18, 1996
E. coli (N'Vf522) (pMYC2454) c.:lGlal NRRL B-21796N Sept. 30, 1997
It should be understood that the availabilitv of a deposit does not constitute
a license to practice
the subject invention in derogation of patent rights granted by govesnmental
action.
Genes and toxins. The polvnucleotides of the subject invention can be used to
form
complete "genes" to encode proteins or peptides in a desired host cell. For
example, as the
skilled artisan would readily recognize, the polynucleotides of the subject
invention are shown
without stop codons. Also, the subject polynucleotides can be appropriately
placed under the
control of a promoter in a host of interest. as is readily lmown in the art.
As the skilled artisan would readily recognize, DNA can exist in a double-
stranded form.
In this arrangement, one strand is complementary to the other strand and vice
versa. "I tle
"coding strand" is often used in the art to refer to the strand having a
series of codons (a codon
is three nucleotides that can be read three-at-a-time to yield a particular
amino acid) that can be
read as an open reading frame (ORF) to form a protein or peptide of interest.
In order to express
a protein in vivo, a strand of DNA is typically translated into a
complementary strand of RNA
which is used as the template for the protein. As DNA is replicated in a plant
(for example)
additional, complementary strands of DNA are produced. Thus, the subject
invention includes
the use of either the exemplified polynucleotides shown in the attached
sequence listing or the
complementary strands. RNA and PNA (peptide nucleic acids) that are
functionally equivalent
to the exeniplified DNA are included in the subject invention.
Certain DNA sequences of the subject rnverltion have been specifically
exerriplified
iierein_ These sequences are exemplary of the subject inventron. It should be
readily apparent
CA 02594294 2007-07-30
11
that the subject invention includes not only the genes and sequences
specifically exemplified
herein but also equivalents and variants thereof(such as mutants, fusions,
chimenes, truncations,
fragments, and smaller genes) that exhibit the same or similar charactenstics
relating to
expressing toxins in plants, as compared to those specifically disclosed
herein. As used herein,
"variants" and "equivalents" refer to sequences which have nucleotide (or
amino acid)
substitutions, deletions (internal and/or terminal), additions, or insertions
which do not
materially affect the expression of the subject genes, and the resultant
pesticidal activity, ;n
plants. Fragments retaining pesticidal activity are also included in this
deftnitiori. Thus,
polynucleotides that are smaller than those specifically exemplified are
included in the subject
invention, so long as the polvnucleotide encodes a pesticidal toxin.
Genes can be modified, and variations of genes may be readilv constructed,
usin"
standard techniques. For example, techniques for malcing point mutations are
well known in the
art. In addition, commercially available exonucleases or endonucleases can be
used accordinc,
to standard procedures, and enzymes such as Ba131 or site-directed mutagenesis
can be used to
systematically cut off nucleotides from the ends of these genes. Useful genes
can also be
obtained using a variety of restriction enzymes.
It shouid be noted that equivalent genes will encode toxins that have high
amino acid
identity or homology with the toxins encoded by the subject genes. The amino
acid homology
will be highest in critical regions of the toxin which account for biological
activity or are
involved in the determination of three-dimensional configuration which
ultimately is responsible
for the biological activity. In this regard, certain substitutions are
acceptable and can be
expected if these substitutions are in regions which are not critical to
activity or are conservative
amino acid substitutions which do not affect the three-dimensional
configuration of the
molecule. For example, amino acids may be placed in the following classes: non-
polar,
uncharged polar, basic, and acidic. Conservative substitutions whereby an
amino acid of one
class is replaced with another amino acid of the same type fall within the
scope of the subject
invention so long as the substitution does not materially alter the biological
activity of the
compound. Table I provides a listing of examples of amino acids belonging to
each class.
CA 02594294 2007-07-30
12
Table 1.
Class of Amino Acid Examples of Amino Acids
Nonpolar Ala, Vat, Leu. Ile, Pro, Met. Phe, Trp
Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin
Acidic Asp, Glu
Basic Lys, Arg, His
In some instances, non-conservative substitutions can also be made. The
critical factor
is that these substitutions must not significantly detract from the ability of
plants to express the
subject DNA sequences or from the biological activity of the toxin.
As used herein, reference to "isolated" polynucleotides and / or "puritied"
toxins refers
to these molecuies when they are not associated with the other molecules with
which they would
be found in nature and would include their use in plants. Thus, reference to
"isolated and
purified" signifies the involvement of the "hand of man" as described herein.
Recombinant hosts. The toxin-encoding genes of the subject invention can be
introduced into a wide variety of microbial or plant hosts. In some
embodiments of the subject
invention, transformed microbial hosts can be used in preiiminary steps for
preparing precursors,
for example, that will eventually be used to transform. in preferred
embodiments, plant cells and
plants so that they express the toxins encoded by the genes of the subject
invention. Microbes
transformed and used in this manner are within the scope of the subject
invennon. Recombinant
microbes may be, for exampie, B.t., E. coli, or Pseudomonas. Transformations
can be made by
those skilled in the art using standard techniques_ Materials necessary for
these transformations
are disclosed herein or are otherwise readily available to the slcilled
artisan.
Thus, in preferred embodiments, expression of the toxin gene results, directly
or
indirectiy, in the intracellular production and maintenance of the pesticide.
When transformed
plants are ingested by the pest, the pests will ingest the toxin. The result
is a control of the pest.
The B.t. toxin gene can be introduced via a suitable vector into a host,
preferably a plant
host. There are many crops of interest, such as corn, wheat, rice, cotton,
soybeans, and
sunflowers. The genes of the subject invention are particulariv well suited
for providing stable
maintenance and expression, in the transfocmed plant, of the gene expressing
the polypeptide
pesticide, and, desirably, provide for improved protection of the pesticide
from environmental
degradation and inactivation.
CA 02594294 2007-07-30
13
While the subject invention provides specific embodiments of synthetic genes,
other
genes that are functionally equivalent to the genes exemplified herein can
also be used to
transform hosts, preferably plant hosts. Additional guidance for the
production of synthetic
genes can be found in, for example, U.S. Patent No. 5,380,83 1.
Following is an example which illustrates procedures for practicing the
invention. This
example should not be construed as limiting.
Example l- Insertion of Toxin Genes Into Plants
One aspect of the subject invention is the transformation of plants with the
subject
polynucleotide sequences encoding insecticidal toxins. The transformed plants
are resistant to
attack by the target pest. The genes of the subject invention are optimized
for use in plants.
Obviously, a promoter region capable of expressing the gene in a plant is
needed. Thus,
for in planta expression, the DNA of the subject invention is under the
control of an appropriate
promoter region. Techniques for obtaining in planta expression by using such
constructs is
known in the art.
Genes encoding pesticidal toxins, as disclosed herein, can be inserted into
plant cells
using a variety of techniques which are well 4atown in the art. For example, a
large number of
cloning vectors compnsing a replication system in E. coli and a marker that
permits selection
of the transformed cells are available for preparation for the insertion of
foreign genes into
higher plants. The vectors comprise, for example, pBR322, pUC series, M l 3mp
senes.
pACYC184, etc. Accordingly, the sequence encoding the B.t. toxin can be
inserted into the
vector at a suitable restriction site. The resulting plasmid is used for
transformation into E. coli.
The E. coli cells are cultivated in a suitable nutrient medium, then harvested
and lysed. The
plasmid is recovered. Sequence analysis, restriction analysis,
electrophoresis. and other
biochemical-molecular biological methods are generally carried out as methods
of analysis.
After each manipulation, the DNA sequence used can be cleaved and joined to
the next DNA
sequence. Each plasmid sequence can be cloned in the same or other plasmids.
Depending on the method of inserting desired genes into the plant, other DNA
sequences
may be necessary. If, for example, the Ti or Ri plasmid is used for the
transfot7nation of the
plant cell, then at least the right border, but often the right and the left
border of the Ti or Ri
plasmid T-DNA, has to be joined as the flanking region of the genes to be
inserted. The use of
T-DNA for the transformation of plant cells has been intensiveiy researched
and sufficiently
described in EP 120 516; Hoekema (1985) In: The Binary Planr Vector Sysieni,
Offset-durkkenj
CA 02594294 2007-07-30
14
Kanters B.V., Aiblasserdam, Chapter 5; Fraley et al., (1986) Crit. Rev. Plant
Sci. 4:1-46; and
An et al. (1985) FAIBO J. 4:277-287.
Once the inserted DNA has been integrated in the genome, it is relatively
stable there
and, as a rule, does not come out again. It normally contains a selection
marker that confers on
the transformed plant cells resistance to a biocide or an antibiotic, such as
kanamvcin, G 418,
bleomycin, hygromycin, or chloramphenicol, inter alia. The individuallv
emploved marker
should accordingly permit the selection of transformed cells rather than cells
that do not contain
the inserted DNA.
A large number of techniques are available for inserting DNA into a plant host
cell.
Those techniques include transformation with T-DNA using 4grobacteriuni
tunzefaciens or
Agrobacterium rhizogenes as transformation agent, fusion, injection,
biolistics (microparticle
bombardment), or electroporation as well as other possible methods. If
Agrobacteria are used
for the transformation, the DNA to be inserted has to be cioned into special
piasmids, namely
either into an intermediate vector or into a binary vector. The intermediate
vectors can be
integrated into the Ti or Ri plasmid by homologous recombination owing to
sequences that are
homologous to sequences in the T-DNA_ The Ti or Ri plasmid also comprises the
vir region
necessary for the transfer of the T-DNA. Intermediate vectors cartnot
repiicate themselves in
Agrobacteria. The intermediate vector can be transferred into Agrobacterium
tinnefaciens by
means of a helper plasmid (conjugation). Binary vectors can replicate
themselves both in E. coli
and in Agrobacteria. They comprise a selection marker gene and a linker or
polvlinker which
are framed by the right and left T-DNA border regions. They can be transformed
directly into
Agrobacteria (Holsters et al. [ 1978] rLlol. Gen. Genet. 163:181-187). The
Agrobacteriunl used
as host cell is to comprise a plasmid carrying a vir region. The vir region is
necessary for the
transfer of the T-DNA into the plant cell. Additional T-DNA may be contained.
The bacterium
so transformed is used for the transformation of plant cells. Plant explants
can advantageously
be cultivated with Agrobacterium rumefaciens or Agrobacterium rhizogenes for
the transfer of
the DNA into the plant ceil. Whole plants can then be regenerated from the
infected plant
material (for example, pieces of leaf, segments of stalk, roots, but also
protoplasts or suspension-
cultivated cells) in a suitable medium, which may contain antibiotics or
biocides for selection.
The plants so obtained can then be tested for the presence of the inserted
DNA. No special
demands are made of the plasmids in the case of injection and electroporation.
lt is possible to
use ordinary plasmids, such as, for example. pUC derivatives.
The transformed cells grow inside the plants in the usual manner. They can
form germ
cells and transmit the transformed trait(s) to progeny plants. Such plants can
be grown in the
normal manner and crossed with plants that have the same transformed
hereditary factors or
CA 02594294 2007-07-30
other hereditary factors. The resulting hybrid individuals have the
corresponding phenotypic
properties.
It should be understood that the exampies and embodiments described herein are
for
~ illustrative purposes only and that various modifications or changes in
light thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of this
application and the scope of the appended claims.
CA 02594294 2007-07-30
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