Note: Descriptions are shown in the official language in which they were submitted.
~ -
215~gS76
WO94/16565 ~ PCT~S94100630
~ , ~ .
Aprotinin and Synergistic Combinations Thereof
with Lectins as Larvicides Against Insect
ests of Agronomic Crops, Harvested Material Thereof, and
Products Obtained from the ~arvested Material
Technical Field
This invention relates to materials and methods for
killing insect larvae which are harmful to plants, and
materials and methods for imparting insect resistance to
plants, material harvested from the plants, and products
derived from the harvested material.
Background of the Invention
Numerous insects are serious pests of common agricul-
tural crops. One method of controlling insects has been to
apply insecticidal organic or semiorganic chemicals to
crops. This method has numerous, art-recognized problems.
A more recent method of control of insect pests has been the
use of biological control organisms which are typically
natural predators of the troublesome insects. These include
other insects, fungi (milky-spore) and bacteria (Bacillus
thuringiensis cv., commonly referred to as "Bt"). However,
it is difficult to apply biological control organisms to
large areas, and even more difficult to have those living
organisms remain and survive in the treated area for an
extended period. Still more recently, techniques in
recombinant DNA have provided the opportunity to insert into
plant cells cloned genes which express insecticidal toxins
derived from biological control organisms such as Bt. This
technology has given rise to additional concerns about
eventual insect resistance to well-known, naturally
occurring insect toxins, particularly in the face of heavy
selection pressure, which may occur in some areas. Thus, a
continuing need exists to identify naturally occurring
WO94/16565 PCT~S94/00~0
;i 16 2 -
insecticidal toxins which can be formed by plant cells
directly by translation of a single strudtural gene.
More recently, certain specific plant lectins have come
to be recognized as larvicidal and insecticidal agents of
some merit, particularly since they are naturally produced
by plant gene expression systems. However, some lectins are
active at relatively high levels which limit flexibility in
that they require maximal expression systems for most
effective larval/insect control. Thus, a continuing need
also exists for substances or combinations of substances
which are effective in controlling larvae at lower
concentrations. It would be particularly desirable to
identify compounds or compositions which could be used to
potentiate the larvicidal action of existing agents such as
lectins. These and other objectives of this invention will
be evident from the following disclosure.
Disclosure of the Invention
It has now been determined that the serine-specific
proteinase inhibitor aprotinin has potent larvicidal
activity when administered enterally to the larvae of
insects such as European corn borer and corn rootworm.
Thus, this invention provides a method for killing
susceptible insect larvae, including larvae of European corn
borer and corn rootworm comprising administering enterally
to the larvae a larvicidal amount of aprotinin or a serine
proteinase inhibitor which is at least 90% homologous to
aprotinin by amino acid sequence.
The terms "protease inhibitor" and "proteinase
inhibitor" are considered equivalent. The terms "insect"
and "larva", although not equivalent when used specifically,
should be understood to include both adult and larval forms
of a species when used generically. Thus, the term "insect
resistance" should be understood to include resistance to
larval forms as well as adults, and "larvicidal" materials
should be considered insecticidal, particularly since
killing larvae produces a corresponding absence of adults.
2151576
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The proteinase inhibitor can be effectively applied to
plants, harvested materials or products consumed by the
insects by spray; dust or other formulation common to the
insecticidal arts. By "harvested plant material" herein is
meant any material harvested from an agricultural or
horticultural crop, including without limitation grain,
fruit, leaves, fibers, seeds, or other plant parts.
Products derived or obtained from such harvested material
include flour, meal, and flakes derived from grain; and
products in which such materials are admixed, such as, for
example, cake, cookie, muffin, pancake and biscuit mixes.
Alternatively, the larvicidal proteinase inhibitor can be
incorporated into the tissues of a susceptible plant so that
in the course of infesting and consuming the plant, its
harvested material, or a product derived from harvested
plant material, the larvae consume larvicidal amounts of the
proteinase inhibitor. One method of doing this is to
incorporate the proteinase inhibitor in a non-phytotoxic
vehicle which is adapted for systemic administration to the
susceptible plants. This method is commonly employed with
insecticidal materials which are designed to attack chewing
insects and is well within the purview of one of ordinary
skill in the art of insecticide and larvicide formulation,
but is a method which may not be as suitable for active
enzyme blockers such as proteinase inhibitors.
Alternatively, a dietary bait containing one or more of the
selected proteinase inhibitors can be employed, with,
optionally, an added pheromonal or other larval attractant
material. However, the genes which code for these peptides
can be isolated and cloned. Alternatively, they can be
synthesized directly using a DNA sequence obtained by
working backwards from the known amino acid sequence for
aprotinin or a related proteinase inhibitor, preferably
using plant-preferred codons. The resulting sequence can be
35 inserted into an appropriate expression cassette and
introduced into cells of a susceptible plant species, so
that an especially preferred embodiment of this method
involves inserting into the genome of the plant a DNA
~ s ~ j
WO94/1656~ PCT~S94/00630
2 ~5 45~ 6 - 4 -
sequence coding for one or more insecticidal proteinase
inhibitors selected from aprotinin and serine proteinase
inhibitors having at least 90% homology to aprotinin by
amino acid sequence, in proper reading frame relative to
transcription initiator and promoter sequences active in the
plant. Transcription and translation of the DNA sequence
under control of the plant-active regulatory sequences
causes expression of the larvicidal gene product at levels
which provide an insecticidal amount of the proteinase
inhibitor in the tissues of the plant which are normally
infested by the larvae.
This method offers particular advantages when the
potential for insects becoming resistant to these materials
is considered. Insecticide-resistant insects become a
problem as a result of application of strong selection
pressure which highly favors naturally resistant individuals
and any resistant mutants which occur. As a result, over
the course of a few generations the resistant insects become
the predominant type.
Heavy application of insecticidal materials generally
to a field or a geographical area by dust or spray or by
soil incorporation tends to impose strong selection
pressures of the kind described, since insects have no "safe
havens" where non-resistant individuals can survive.
However, many insect pests of crop plants also attack non-
crop species. Limiting the insecticidal materials to the
crop plants in the region by expressing the insecticidal
materials only in those plants permits continued survival of
non-resistant insects in associated weed plants which
provide not only "safe havens" from the toxic compound but
food for the insects. This reduces selection pressure
significantly and thus slows development and spread of
resistant insects.
This method also offers advantages from the standpoint
of soil and groundwater contamination, since no application
vehicle is required. The insecticidal components themselves
are of natural origin and break down naturally when the
plant is digested or decomposes. The method offers further
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._ .;,
- 5 - ~
advantages from the standpoint of cost, since no applicàt~ion
expense is involved and the cost of the insecticidal
materials is factored into the price of the seed or other
reproductive material which the grower purchases.
The plant should be a plant which is susceptible to
infestation and damage, or whose harvested material or
products are susceptible to infestation and damage by the
larvae of European corn borer and corn rootworm. These
include corn (Zea mays), wheat (Triticum aestivum) and
sorghum (Sorghum bicolor). However, this short list is not
to be construed as limiting, inasmuch as these species are
among the most difficult commercial crops to reliably
transform and regenerate, and these insects (under other
common names) also infest other crops. Thus the methods of
this invention are readily applicable via conventional
techniques to numerous plant species, if they are found to
be susceptible to the plant pests listed hereinabove,
including, without limitation, species from the genera
Fragaria, Lotus, Medicago, Onobrychis, Trifolium,
Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus,
Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum,
Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum,
Petunia, Digitalis, Majorana, Cichorium, Helianthus,
Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis,
Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus,
Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium,
Triticum, and Datura.
Preferred plants that are to be transformed according
to the methods of this invention are cereal crops, including
maize, rye, barley, wheat, sorghum, oats, millet, rice,
triticale, sunflower, alfalfa, rapeseed and soybean, fiber
crops, such as cotton, fruit crops, such as melons, and
vegetable crops, including onion, pepper, tomato, cucumber,
squash, carrot, crucifer (cabbage, broccoli, cauliflower),
eggplant, spinach, potato and lettuce.
The DNA sequence which when expressed imparts insecti-
cidal activity is a structural gene which codes for
aprotinin, or a proteinase inhibitor having at least 90%
W094/l6565 ~16 ~ PCT~594/00~0
homology to aprotinin. It has been found that these
proteinase inhibitors have sufficient insecticidal
(larvicidal) activity to be operative in a plant cell
expression system. That is, while certain other proteinase
inhibitors such as cowpea trypsin inhibitors have some
larvicidal activity at high concentrations in pure form,
plant cell expression at such high concentrations is either
not possible in a living plant cell system, or is not
feasible if the commercially useful characteristics of the
plant are to be preserved in terms of production of oils,
starches, fibers, or other materials. A tissue-specific
promoter can be used in any instance where it may be
desirable to localize production of the proteinase inhibitor
to an infested tissue or to a tissue which is efficient in
production of the proteinase inhibitor.
In carrying out this invention, it will be appreciated
that numerous plant expression cassettes and vectors are
well known in the art. By the term "expression cassette" is
meant a complete set of control sequences including
initiation, promoter and termination sequences which
function in a plant cell when they flank a structural gene
in the proper reading frame. Expression cassettes
frequently and preferably contain an assortment of restric-
tion sites suitable for cleavage and insertion of any
desired structural gene. It is important that the cloned
gene have a start codon in the correct reading frame for the
structural sequence. In addition, the plant expression
cassette preferably includes a strong constitutive promoter
sequence at one end to cause the gene to be transcribed at ~
high frequency, and a poly-A recognition sequence at the
other end for proper processing and transport of the
messenger RNA. An example of such a preferred (empty)
expression cassette into which the DNA sequence of the
present invention can be inserted is the pPHI414 plasmid
developed by Beach et al. of Pioneer Hi-Bred International,
Inc., Johnston, IA and disclosed in U.S. Patent application
No. 07/785,648, filed October 31, 1991. Highly preferred
plant expression cassettes will be designed to include one
21;54576 ~ t~ ~YJS '3,
WO 94/16565 ~ 4/00630
_, . .
or more selectable marker genes, such as kanamycin
resistance or herbicide tolerance genes.
By the term "vector" herein is meant a DNA sequence
which is able to replicate and express a foreign gene in a
host cell. Typically, the vector has one or more endo-
nuclease recognition sites which may be cut in a predictable
fashion by use of the appropriate enzyme. Such vectors are
preferably constructed to include additional structural gene
sequences imparting antibiotic or herbicide resistance,
which then serve as selectable markers to identify and
separate transformed cells. Preferred selection agents
include kanamycin, chlorosulfuron, phosphonothricin,
glyphosate, hygromycin and methotrexate, and preferred
markers are genes conferring resistance to these agents. A
cell in which the foreign genetic material in a vector is
functionally expressed has been "transformed" by the vector
and is referred to as a "transformant".
A particularly preferred vector is a plasmid, by which
is meant a circular double-stranded DNA molecule that is not
a part of the chromosomes of the cell.
As mentioned above, genomic, synthetic and cDNA
encoding the gene of interest may be used in this invention.
The vector of interest may also be constructed partially
from a cDNA clone, partially from a synthetic sequence and
partially from a genomic clone. When the gene sequence of
interest is in hand, genetic constructs are made which
contain the necessary regulatory sequences to provide for
efficient expression of the gene -in the host cell.
According to this invention, the genetic construct will
contain (a) a first genetic sequence coding for the
proteinase inhibitor of interest and (b) one or more
regulatory sequences operably linked on either side of the
structural gene of interest. Typically, the regulatory
sequences will be selected from the group comprising of
promoters and terminators. The regulatory sequences may be
from autologous or heterologous sources.
WO94/16565 PCT~S94/00630
5~6 8 -
Promoters that may be used in the genetic sequence
include nos, ocs, phaseolin, CaMV, FMV and other promoters
isolated from plants or plant pests.
An efficient plant promoter that may be used is an
overproducing plant promoter. Overproducing plant promoters
that may be used in this invention include the promoter of
the small sub-unit (ss) of the ribulose-1,5-biphosphate
carboxylase from soybean (Berry-Lowe et al, J. Molecular and
App. Gen., 1:483-498 (1982)), and the promoter of the
cholorophyll a-b binding protein. These two promoters are
known to be light-induced, in eukaryotic plant cells (see,
for example, Genetic Engineering of Plants, An Agricultural
Perspective, A. Cashmore, Pelham, New York, 1983, pp. 29-38,
G. Coruzzi et al., J. Biol. Chem., 258:1399 (1983), and P.
Dunsmuir, et al., J. Molecular and App. Gen., 2:285 (1983)).
The expression cassette comprising the structural gene
for the proteinase inhibitor of interest operably linked to
the desired control sequences can be ligated into a suitable
cloning vector. In general, plasmid or viral (bacterio-
phage) vectors containing replication and control sequencesderived from species compatible with the host cell are used.
The cloning vector will typically carry a replication
origin, as well as specific genes that are capable of
providing phenotypic selection markers in transformed host
cells. Typically, genes conferring resistance to anti-
biotics or selected herbicides are used. After the genetic
material is introduced into the target cells, successfully
transformed cells and/or colonies of cells can be isolated
by selection on the basis of these markers.
Typically, an intermediate host cell will be used in
the practice of this invention to increase the copy number
of the cloning vector. With an increased copy number, the
vector containing the gene of interest can be isolated in
significant quantities for introduction into the desired
plant cells. Host cells that can be used in the practice of
this invention include prokaryotes, including bacterial
hosts such as E. coli, S. typhimurium, and S. marcescens.
2159576 ~
WO94/16~65 -- PCT~S94/00630
_ g _ ~ ~
Eukaryotic hosts such as yeast or filamentous fungi may also
be used in this invention.
The isolated cloning vector will then be introduced
into the plant cell using any convenient technique, includ-
ing electroporation (in protoplasts), retroviruses,microparticle bombardment, and microinjection, into cells
from monocotyledonous or dicotyledonous plants, in cell or
tissue culture, to provide transformed plant cells
containing as foreign DNA at least one copy of the DNA
sequence of the plant expression cassette. Preferably, the
monocotyledonous species will be selected from maize,
sorghum, wheat and rice, and the dicotyledonous species will
be selected from soybean, sunflower, cotton, rapeseed
(either edible or industrial), alfalfa, tobacco, and
Solanaceae such as potato and tomato. Using known
techniques, protoplasts can be regenerated and cell or
tissue culture can be regenerated to form whole fertile
plants which carry and express the desired gene for the
selected protein. Accordingly, a highly preferred
embodiment of the present invention is a transformed maize
plant, the cells of which contain as foreign DNA at least
one copy of the DNA sequence of an expression cassette of
this invention.
This invention also provides methods of imparting
resistance to European corn borer and corn rootworm to
plants of a susceptible taxon, comprising the steps of:
a) culturing cells or tissues from at least one plant
from the taxon,
b) introducing into the cells of the cell or tiss~le
culture at least one copy of an expression cassette compris-
ing a structural gene coding for a proteinase inhibitor
selected from aprotinin and serine proteinase inhibitors
having at least 90% homology thereto by amino acid sequence,
or a combination of such proteinase inhibitors, operably
linked to plant regulatory sequences which cause the
expression of the protein structural gene in the cells, and
c) regenerating insect-resistant whole plants from the
cell or tissue culture. Once whole plants have been
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WO 94/16565 PCT/US94/00630
21S45'~ 5
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obtained in this manner, they can be sexually or clonally
reproduced in any manner such that at least one copy of the
sequence provided by the expression cassette is present in
the cells of progeny of the reproduction.
Alternatively, once a single transformed plant has been
obtained by the foregoing recombinant DNA method, conven-
tional plant breeding methods can be used to transfer the
protein structural gene and associated regulatory sequences
via crossing and backcrossing. Such intermediate methods
will comprise the further steps of
a) sexually crossing the insect-resistant plant with a
plant from the insect-susceptible taxon;
b) recovering reproductive material from insect-
resistant progeny of the cross; and
c) growing insect-resistant plants from the
reproductive material. Where desirable or necessary, the
agronomic characteristics of the susceptible taxon can be
substantially preserved by expanding this method to include
the further steps of repetitively:
a) backcrossing the insect-resistant progeny with
insect-susceptible plants from the susceptible taxon; and
b) selecting for expression of insect resistance (or
an associated marker gene) among the progeny of the back-
cross, until the desired percentage of the characteristics
of the susceptible taxon are present in the progeny along
with the gene imparting insect resistance. This will be
important, for example, where the taxon is a substantially
homozygous plant variety, such as an inbred line of maize or
a variety of a self-pollinated crop such as soybeans. sy
"substantially homozygous" is meant homozygous within the
limits commonly accepted in the commercial production of
certified seed of the species. For example, an inbred line
of maize used in commercial seed production is typically 95%
to 100% homozygous, and preferably 98% to 100% homozygous,
as measured by RFLP analysis using 50 to 200 probes well
distributed across the genome. If necessary, an RFLP-guided
process of self-pollination and selection can be used to
achieve this degree of genetic uniformity.
~lS4576`
WO94/16565 PCT~S94/00630
r
By the term "taxon" herein is meant a unit of botanical
classification of genus or lower. It thus includes genus,
species, cultivars, varieties, variants, and other minor
taxonomic groups which lack a consistent nomenclature.
It will also be appreciated by those of ordinary skill
that the plant vectors provided herein can be incorporated
into Agrobacterium tumefaciens or Agrobacterium rhizogenes,
which can then be used to transfer the vector into
susceptible plant cells, primarily from dicotyledonous
species. Thus, this invention provides a method for
imparting insect resistance in Agrobacterium-susceptible
dicotyledonous plants in which the expression cassette is
introduced into the cells by infecting the cells with an
Agrobacterium species, a plasmid of which has been modified
to include a plant expression cassette of this invention.
Finally, it has now been determined that aprotinin and
highly homologous serine proteinase inhibitors strongly
potentiate the insecticidal activity of lectins such as
wheat germ agglutinin. This effect is surprising and
unexpected, since many natural insecticides target the same
structures or molecules within the insect and as a result
many combinations of such natural insecticides are at best
additive and more often competitive, with little or no
increase in insecticidal activity, as shown in the
experimental results below. In addition to the increased
level of activity which this result provides, there are also
the practical benefits of increased technical flexibility
and feasibility, since the synergy or potentiation between
these two groups of plant-expressible materials permits the
attainment of effective insect or larval control with lower
levels of expression of each component. Since plant cell
systems are more amenable to such lower levels of
expression, this offers the biotechnological entomologist
greater flexibility in formulation and greater likelihood of
achieving complete insect suppression in host plants with
less-than-maximal levels of gene expression.
The combination of two different, synergistic
insecticidal materials in a single system to provide
WO94/16565 PCT~S94100630
~ ~S ~S~ 6 _ 12 -
effective insect control also reduces the potential for
development of resistant insects. Since such resistances
typically arise by spontaneous mutation, developing
resistance to a binary system such as aprotinin plus wheat
germ agglutinin would require twn mutations, one in each
target structure or molecule. `The probability of such a
double mutation is potentially (if there is no association
between the mutations) as low as the product of the
probabilities of the individual mutations, which would be
quite low indeed -- perhaps l.0 x l0-1, or less than fifty
potentially survivable individuals per year in the entire
United States. Also, the low or reduced selection pressure
for each individual mutation further reduces its spread
within the population.
Accordingly, this invention also provides a method for
killing European corn borer and corn rootworm, comprising
administering enterally to the larvae of those species a
larvicidal combination of (a) aprotinin, a serine proteinase
inhibitor having at least 90% homology to aprotinin by amino
acid sequence, or a combination thereof; and (b) an
insecticidal lectin.
The following description further exemplifies the
compositions of this invention and the methods of making and
using them. However, it will be understood that other
methods, known by those of ordinary skill in the art to be
equivalent, can also be employed.
In the following examples the wide variety of insects
screened has resulted in several different bioassays being
used to determine the effect of aprotinin and combinations
of aprotinin plus lectin on larval growth and survivorship.
However, all of the bioassays allow the test materials to be
enterally administered to the insect. In vitro bioassays
for the European corn borer (Ostrinia nubalis), and southern
corn rootworm (Diabrotica undecimpunctata howardii) were
done by incorporating the test protein into the artificial
diet. This is referred to herein as an "Incorporated
Bioassay". This was accomplished by making up a standard
artificial diet at 90% of the original water and adding a
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WO94/16565 PCT~S94/00630
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solution of the test protein to this mixture.
Concentrations of the protein in this diet are recorded as
mg or ~g of protein per ml of diet. Weight and mortality
are recorded after seven days. Specific assays and
variations are described in the individual examples.
Éxample l
EUROPEAN CORN BORER
Aprotinin was the most effective protease inhibitor
against European corn borer with high mortality occurring
during a replicated 7-day incorporated bioassay. The
results are shown in Table l.
Table l.5 Effect of aprotinin and other protease inhibitors (PI)
on European corn borer neonate larvae
in incorporated bioassays
Treatment Weight Reduction (fold) ~Mortality
20 Control 5.2 -- 0*
Aprotinin -- -- lO0
Soybean PI (Bowman-Birk) 4.7 _ 0
Soybean PI (Kunitz) 3.9 1.3 9
Chicken PI (Type IV) 2.9 l.8 05
*Corrected mortality
All materials were tested at 20 mg PI/ml of diet
SOU.~KN CORN ROOTWORM
Aprotinin also showed effectiveness against Southern
corn rootworm neonate larvae in incorporated bioassays, as
seen in Table 2.
W094/16565 215 457 6 PCT~S94/00630
- 14 -
..,
Table 2
Effect of aprotinin and other protease inhibitors (PI)
on Southern corn rootworm neRnate larvae
in incorporated bioa~`6says
Treatment Weight Reduction (fold) %Mortality
Control 3.3 -- o*
Aprotinin 0.4 8 60
Soybean PI (Bowman-Birk) 2.4 1.5 50
Cystatin 2.5 1.4 30
*Corrected mortality
All materials were tested at 20 mg PI/ml of diet
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Example 2
Combination of Aprotinin plus Wheat Lectin
Tests were-,performed employing Wheat Germ Agglutinin
(WGA), aprotinin, ~ap,d combinations of the two in 7-day
incorporated bioassays~., The results are shown in Table 3.
Table 3
Effect of WGA and aprotinin on European corn
borer neonate larvae in incorporated bioassays
Treatment Expected mortality from
%Mortality aprotinin addition
Control 0
1. WGA 0.10 mg/ml 10
2. WGA 0.15 mg/ml 15
3. WGA 0.20 mg/ml 20
4. WGA 0.25 mg/ml 25
5. Aprotinin 0.25 mg/ml15
6. Aprotinin 0.5 mg/ml 10
7. Aprotinin 1.0 mg/ml 25
8. Aprotinin 2.0 mg/ml 30
Combinations
4 + 8 90 55
3 + 7 80 45
2 + 7 75 40
3 + 5 70 35
3 + 6 70 30
2 + 6 55 25
When the wheat lectin was replaced with Bauhinea
purpùrea lectin, similar results were also obtained.
Replicated 7-day bioassays were also performed to measure
effects on growth. Results are shown in Table 4.
WO94/16565g5 ~ 6 16 - PCT~S94/00~0
Table 4
Effect of Aprotinin and WGA Combinations
on ECB growth
TreatmentWeight Reduction
Weight (fold)
Control l0.5 --
l. Aprotinin 0.l mg/ml 7.8 l.3
2. WGA 0.l mg/ml l0.3 --
l + 2 2.5 4.2
The weight and weight reduction is significantly
different from all other weights and reductions at p< 0.05.
The foregoing results indicate a synergy between aprotinin
and insecticidal lectins in combination, both in terms of
mortality and growth inhibition. Based on results with
other combinations of insecticidal compounds, an additive or
neutral effect would have been expected.
Industrial Applicability
I. Isolation of the protein gene and insertion into
bacteria
In order to isolate the coding sequence for the
protein, it is necessary to have nucleotide sequence data
which establishes an open reading frame (i.e., the correct
triplet code for translation which should have only one
"stop" signal at the very end of the gene.) It is also
necessary to have an indication of where to look for the
protease cleavage junction between the protein and the
replicase which precedes it in the sequence. This can be
determined from the peptide sequence of the N-terminal
portion of the protein or by comparing the protein sequence
with that of other homologous proteins. This can generally
be accomplished and the necessary information obtained
without sequencing the entire gene. Once the sequence at
both ends of the gene has been determined, the remainder of
the gene can be cloned using restriction enzymes that flank
the protein coding region or, more preferably, by cloning
the precise protein coding region by oligonucleotide-
W094/16565 _ ;7 _ PCT~S94tOO~O
directed amplification of DNA (polymerase chain reaction orPCR).
Once the gene has been isolated, it can be cloned into
a bacterial expression vector with linkers added to create
all three reading frames (using 8mer, lOmer, and 12mers each
of which contain an ATG translational start site). The
resulting vectors, containing the fragments of interest, can
be inserted into, for example, BRL's Maximum Efficiency DH5
F' IQ transformation competent E. coli cells. All three
transformations, one for each linker, are then screened via
minipreps for the presence and orientation of insert.
Appropriate clones are then chosen to test for expression of
the protein gene.
Clones containing the properly oriented inserts are
grown in culture medium conducive to the induction of the
gene (LB medium with added IPTG). The cells are lysed and
bacterial proteins are subjected to electrophoresis in SDS
polyacrylamide gels and then transferred to nitrocellulose.
The resulting protein blots are easily screened for presence
of protein using rabbit polyclonal and mouse monoclonal
anti-protein antibody.
Having determined the proper reading frame, it is then
necessary to remove the gene from the bacterial expression
vector. The linker at the start of the gene region supplies
the necessary start codon.
II. Expression of the Protein Gene in Plants
A plant expression cassette, employing the regulatory
sequences developed by Beach, et al., and containing the
protein gene, is constructed. The restriction map of the
preferred plasmid, designated pPHI414, is illustrated in
Figure 1. This plasmid contains an enhanced 35S promoter
spanning nucleotides - 421 to +2 of Cauliflower Mosaic virus
with the region from - 421 to - 90 duplicated in tandem, a
79 bp HindIII Sall fragment from pJII101 spanning the 5~
leader sequence of Tobacco Mosaic Virus, a 579 bp fragment
spanning the first intron from maize AdH1-S, and a 281 bp
W094/16565 215 4 5 7 6 PCT~S94/00~0
- 18 -
fragment spanning the polyadenylation site from the nopaline
synthase gene in pTiT37.
Another construct which can be used as an expression
cassette is the pPHI412 plasmid shown in Eigure 2. It
differs from pPHI414 in that it lacks the AdH intron
segment. However, like pPHI414, it is constructed to have
numerous restriction sites between the O' segment and the
NOS segment, which sites can be conveniently used for
splicing any desired protein structural gene into position.
This vector can be cotransformed with a similar
plasmid containing a selectable marker for antibiotic
resistance into Black Mexican Sweet corn protoplasts by
electroporation. These protoplasts can then be induced to
regenerate cell walls and develop into callus by
conventional techniques. Likewise, this callus can then be
subjected to antibiotic selection to select for transformed
colonies, and these colonies can be tested for expression of
protein with antisera for the appropriate protein using
known methods. The efficiency of protection can be measured
by infesting callus (or suspension cultures derived from
callus) with the target insect and measuring survival
percentages.
The protein gene can be introduced into embryogenic
maize callus by methods similar to those used for slack
Mexican Sweet. Embryogenic callus can be regenerated to
whole fertile plants. The insect resistance imparted by the
endogenous production of the protein is a simply inherited,
dominant trait and can, if desired, be introduced into other
plant varieties of the species by simple crossing or
backcrossing.
Using the foregoing techniques, aprotinin has been
expressed in maize suspension cells as determined by
transient assays.
