Download Plant Insecticidal Proteins and their Potential for Developing

Indian Journal of Biotechnology
Vol 2, January 2003, pp. 110-120
Plant Insecticidal Proteins and their Potential for Developing
Transgenics Resistant to Insect Pests
K R Koundal* and P Rajendran
National Research Centre on Plant Biotechnology,
Indian Agricultural Research Institute, New Delhi 110012, India
Insects cause heavy damage to cultivated crop plants. Production of proteinaceous inhibitors that interfere with
the digestive biochemistry of insect pests is one of the naturally occurring defence mechanisms in plants. These proteins include lectins, arcelins and inhibitors of alpha amylases and proteases of various larvae pests. Use of plant
genes encoding effective inhibitors of major digestive enzymes, such as protease and a-amylase inhibitors of the target pest species is emerging as viable approach for the production of pest resistant transgenic crop plants. Therefore,
it is important to characterize proteins and their genes from our indigenous crops in order to strengthen and
broaden our gene bank for pest control manipulations. The availability of diverse insecticidal proteins and their
genes from different plant species will make it easier to use one or more genes in combination to develop resistant
crop plants. Eventually, insect resistant transgenic plants will certainly prove more economic than any conventional
control strategy if long-term benefit of transgenic crops especially factors such as environmental damage and human
health risks are considered.
Keywords: plant proteins/genes,
protease inhibitor, lectin, a-amylase inhibitor, transgenics, bioassays
Introduction
Pest infestation leading to massive crop damage is
the most serious limiting factor in crop productivity.
In the course of evolution, many classes of proteins
and secondary metabolic substances have been expressed in plants as effective counter measures against
herbivory and the application of these natural plant
defence traits as candidates for genetic engineering
for crop pest control is now a reality. Plant secondary
metabolites include a plethora of biochemical entities
that are synthesized through complex pathways and
therefore, the attempts to explore their biochemistry
and physiology have always remained challenging.
Broadly, they belong to totally unrelated classes of
compounds like alkaloids, f1avonoids, foliar enzymes
(polyphenol oxidases & peroxidases), foliar phenolic
acid esters (rutin & chlorogenic acid), non-protein
amino acids, peptide hormones, pyrethrins, steroids,
"Author for correspondence:
Tel: 91-011-25788783, 25711554
Fax: 91-011-25823984,25766420
E-mail: [email protected]
Abbreviations:
Bt: Bacillus thuriengiensis; a-AI: Alpha-amylase; Pi: Protease
inhibitor; CpTi: Cowpea trypsin inhibitor; Ti: Trypsin inhibitor;
Cti: Chymotrypsin inhibitor; GNA: Glanthus nivalis; cDNA:
Complementary
deoxyribonucleic
acid; CaMV: Cauli mosaic
virus; GM: Genetically modified.
and terpenoids. Yet another group, but more important from the application point of view belongs to
plant defence proteins that are more specific against
insect pests (Maqbool et at, 2001). In most plants,
they offer horizontal resistance against insect attack
(Fabrick et al, 2002). This review is exclusively focussed on the potential of these inhibitors or their encoding genes in pest control manipulations. Attempts
are made to depict the structural functional relationships of these inhibitors that direct their application to
develop transgenics resistants to various insect pests.
Plant Proteins with Insecticidal Activity
Production of proteinaceous inhibitors that interfere with the digestive biochemistry of insect pests is
one of the naturally occurring defence mechanisms in
plants. This mechanism is manifested in the form of
accumulation of one or several defence proteins such
as lectins, arcelins and inhibitors of alpha-amy lases
and protease of larval pests. As insect larvae develop,
they secrete into their guts a variety of enzymes that
break down carbohydrates and proteins present in the
ingested food. The larvae then feed on the released
components from the plant tissue rapidly causing crop
damage and yield loss. Plants respond to this invasion
by synthesizing proteins that can inhibit the action of
these enzymes. If a food source contains a supply of
digestible protein or carbohydrate, but also an inhibi-
KOUNDAL & RAJENDRAN:
PLANT INSECTICIDAL
tor that acts as a biological check, the larval enzymes
cannot digest the food and the larva will not grow and
develop through its normal cycle (Boulter, 1993; Fabrick et al, 2002).
The potential for using this natural host plant resistance in pest control across the plant genetic barriers has increased with the development of gene transfer techniques. Though exogenous natural pesticidal
agents such as Bt toxin are also effective in deterring
plant predators, in spite of their current successes,
they may create environmental safety and consumer
health debates in future in addition to a multitude of
ethical concerns (Gatehouse et al, 1998). Further,
there is growing evidence that genes derived from
plants could be excellent alternatives to the toxins
from Bacillus thuringiensis. There are now several
examples of protease inhibitors of plant origin conferring insect resistance when expressed in transgenic
plants including tobacco, rice, cotton, strawberry,
poplar and peas (Ussuf et al, 2001). Plant derived
genes target different sites in insects than the synthetic chemicals and may be deployed in combination
with the bacterial insecticidal genes for complete insect control. However, this approach can be exploited
effectively if encoding genes for proteins with appreciable inhibitory properties on the target pest's enzymes are available for genetic manipulations. Thus,
it is imperative to evaluate as many plant sources as
possible for identifying the presence of proteins with
ideal insecticidal properties (Sparvoli et 01, 2001). It
is equally important to characterize these proteins and
their encoding genes to strengthen and broaden the
resistance gene pool. Among them, alpha-amylase
inhibitors, protease inhibitors and lectins have undergone extensive investigations particularly in the last
two decades and the research on these proteins and
their encoding genes has proved to be rewarding. In
addition, some related but less studied proteins like
arcelins and vicilins have also invoked research attention in recent years.
(i) Alpha-amylase
Inhibitors
Alpha-amylase a ubiquitous enzyme, catalyses the
endolytic cleavage of 1~4 linked glucose polymers,
mainly starch to give hydrolytic products with [he aconfiguration. This digestive enzyme has been of interest to plant biologists for many years because of its
agricultural and industrial importance. In plants, aamylase inhibitors are stored in seeds and tubers towards the end of the life cycle in copious amounts.
PROTEINS FOR TRANSGENICS
RESISTANT TO PESTS
1II
They are also synthesized in plants almost at the same
time and usually in the same organs that store the
starch and amylases. Their role is mainly defence
through inhibition of a variety of alpha-amylases from
diverse sources by forming irreversible complexes
with them. Chrzaszez & Janicki (1934) were the first
to report the presence of a-Als in buckwheat. Thereafter, a-AI has been isolated from many plants,
mainly cereals and legumes. a-AI inhibitors are primarily the products of single gene, and are small, low
molecular weight (in the 14-8 kDa range) monomeric
proteins, a-Als from bean contain two or more subunits (Ho & Whitaker, 1993) and a-Als in wheat,
barley and rye are tetrameric in nature (Garcia et al,
1996; Sanchez-Monge et 01, 1996). Several a-AI
proteins that have been isolated from wheat kernels
strongly inhibit alpha-amylases from insects with null
or weak effect on mammalian amylases (Feng et 01,
1996). These properties make them attractive candidates for plant genetic manipulations for imparting
resistance to insect pests. Incorporation of increasing
amounts of a-AI into the diet of the pea weevil
iBruchus pisorum) was directly correlated with delayed larval development time (Ishimoto & Kitamura
et al; 1992). The resistance of common bean
tPhaseolus
vulgaris) seeds to bruchids like cowpea
weevil and adzuki bean weevil has largely been attributed to the presence of a-Als (Huesing et al,
1991). The level of a-Als in their seeds has been
found sufficient to inhibit the development of the larvae of those bruchid species that normally do not feed
on common bean.
Though the cereal and leguminous a-amylase inhibitors share a similar mode of action, studies on
their structure and evolutionary relationships have
clearly shown that they belong to entirely different
classes of proteins that might have evolved independently in monocotyledon and dicotyledon in a convergent manner. The cereal trypsin/ a-AI proteins are
members of a super family of seed proteins with limited sequence homology. Homologous relationships
of the cereal o-Als were established with the 2S storage proteins from castor bean and the Kazal secretary
trypsin inhibitor from bovine pancreas (Odani et 01,
1983), thus showing that this protein super family is
distributed beyond the plant kingdom. The C_N-1._pr~-_
teins from wheat, barley and rye were eventually
found to be members of the trypsin/a-AI family
(Shewry et 01, 1984) and the suBtilisin/endogenous
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INDIAN J BlOTECHNOL,
a-Als from cereals were found to be homologous to
the soybean trypsin inhibitor (Maeda, 1986).
On the other hand, the common bean a-amylase
inhibitor belongs to a family of vacuolar glycoproteins that comprises phytohemglutinins (PHA), arcelins and a-Als (Moreno & Chrispeels, 1989; Chrispeels & Raikhel, 1991). PHA is the true lectin
whereas arcelins may retain a weak carbohydrate
binding activity and a-Als is completely devoid of
such activity.Their genes are tightly linked, behave as
a single Mendelian locus, and have likely arisen by
duplication and divergence of an ancestral gene (Nodari et al, 1993).Three dimensional models of a-AIs,
arcelins and lectins have shown that arcelins and aAIs are truncated lectins (Rouge et al, 1993). In the
course of evolution, the loss of one or two loops located in the region of monosaccharide binding site in
lectin led to the development of arcelins and a-IUs,
respectively. This might be the reason why arcelins
exhibit a weak agglutinating activity, while a-Als
cannot agglutinate erythrocytes. Further, an a-AI-like
protein, YBP 22, isolated from the tuberous roots of
the Mexican yam bean [Pachyrrhizus erosus (Linn.)
Urban] showed sequence homology with several
known protease inhibitors, and a polyclonal antibody
raised against the protein cross-reacted with soybean
trypsin inhibitor (Gomes et al, 1997). The cDNA
clones of lectin related seed proteins isolated from
Lima bean showed 93.7% nucleotide identity and encoded an arcelin-like and an a-AI-like protein respectively (Sparvoli et al, 1998).
(ii) Protease Inhibitors (PIS)
Read and Haas (1938) reported the presence particularly in legume seeds of proteases and their biochemistry as protein molecules was confirmed by Kunitz who isolated and crystallized the trypsin inhibitor
from soybean (Kunitz, 1945). Protease inhibitors is
the largest class of proteins that have undergone extensive investigations and consequently their structure, properties, function and metabolism have been
well documented. They are almost ubiquitous in
plants and those present in the Leguminosae, Graminae and Solanaceae have been studied and characterized in greater detail (Richardson, 1977; Connors et
al, 2002). Although, some of them may playa role in
endogenous protein metabolism, most of the protease
inhibitors that have been characterized from plants do
not inhibit endogenous plant proteases, but have
specificities for animal or microbial enzymes (Las-
JANUARY 2003
kowski & Kato, 1980; Laskowski et al, 1988).111 vitro
feeding trials using artificial diets containing the inhibitors have confirmed the protective role for protease inhibitors against several crop pests. The effects
of Pis on susceptible insects are generally seen as an
increase in mortality, decrease in growth rate and
prolongation of developmental period of the larvae.
These detrimental effects are accomplished by
blocking insect midgut proteinases thus impairing
protein digestion, which inhibits or at least delays (in
the case of weak inhibitors) the release of peptides
and amino acids from dietary protein. The presence of
inhibitor thus leads to the loss of nutrients particularly
sulphur containing amino acids, and thereby weak and
·stunted growth and ultimate death (Gatehouse et aI,
1992).
A systematic classification of Pis is not viable due
to their diversity in terms of source, structure, specificities and size. However, these are mainly grouped in
the four specificity groups, viz. serine, cysteine, metallo and aspartic protease inhibitors (inhibiting serine,
cysteine, metallo and asparty I proteases, respecti vely)
(Garcia-Olmedo et at, 1987). Among them, the potato
inhibitor 1 and 11 families, the Bowman-Birk inhibitor (BB!) family, and the soybean trypsin inhibitor
(Kunitz) family are very important due to their inhibitory potential on gut enzymes of major crop pests.
(iii) Potato Inhibitor 1 and 11 Families
Among the Pis, the wound-inducible inhibitors
from potato and tomato represent a unique group with
insecticidal properties due to several interesting features of these proteins and their encoding genes. They
comprise a non-homologous gene family in which
members have been identified mainly from the solanaceous plants. Among them, potato inhibitor I and II
and tomato protease inhibitor I and II have been well
characterized (Plunkett et al, 1982). The unique and
most striking feature of their encoding genes is the
presence of introns, two each in inhibitor I genes and
one in the gene encoding potato inhibitor II. In fact,
they are the only protease inhibitor genes reported so
far to contain introns. In potato alone, a mixture of ten
or more isoinhibitors of protease inhibitor I and at
least three forms of inhibitor II have been identified.
In addition, homologues of the inhibitor have been
found in some non solanaceous plants like alfalfa.
broad bean, clover, cowpea, cucumber, French bean.
grape, squash, strawberry, barley and buckwheat
(Lorene et al, 2001; Ye et al, 2001). In leaves of to-
KOUNDAL & RAJENDRAN:
PLANT INSECTICIDAL
mato and potato, they are expressed constitutively at
low levels during plant growth and development. In
response to wounding by insects or other mechanical
damage, their concentration increases dramatically
even in the unwounded leaves of the same plant, and
within a few hours of injury, their levels often exceed
10% of total soluble proteins. In potato tubers, they
accumulate throughout the course of tuber development, and represent a substantial fraction of the soluble protein. Thus, unlike other plant protease inhibitor
genes, these genes are regulated environmentally as
well as developmentally, and their expression is believed to be under a complex control involving several cis and transacting factors making them excellent
models for study of plant gene regulation (Keil et al,
1986; Kouzuma et al, 2001).
All these proteins belong to the serine protease
(endopeptidases) inhibitor group and are relatively
small in size. Potato inhibitor I is a pentameric protein
of Mr 41000 and is made up of monomers of Mr 8100
with single reactive site to inhibit chymotrypsin where
as the corresponding inhibitor species in tomato,
which is also a pentamer, with subunits of Mr 7800
each. Their cysteine content is low and they either
have one disulfide bridge or none and the presence of
cysteine seems to be not essential for their activity. In
other inhibitor families, disulfide bridges stabilize the
three dimensional structure and are believed to be essential for their activity. In the inhibitor II family,
both in tomato and potato, the mature protein is a dimer of about Mr 25000 made up of two promoters of
identical size, with five disulfide bonds and two reactive sites per monomer, which inhibit chymotrypsin
and trypsin respectively. Wound induction of these
inhibitor proteins is triggered by a small (18-mer)
peptide hormone, systernin or Protease Inhibitor Inducing Factor (PUP), released into the vascular system in response to wounding which in turn is transported rapidly to other tissues of the plant where it
initiates the transcription of the PI genes. In addition,
various factors including fungal elicitors, bacterial
infection, sucrose, abscisic acid, and jasmonic acid
have direct or indirect role in the increased synthesis
of these inhibitors in potato. Nuclear runoff assays
have shown that the accumulation of inhibitors I and
II in tomato is transcriptionally regulated (Graham et
al, 1986). Once synthesized, the inhibitor I and U
proteins are sequestered into the central vacuole,
which ensures their way long half lives. Analysis of
the genomic clone encoding the potato protease in-
PROTEINS FOR TRANSGENICS
RESISTANT TO PESTS
113
hibitor II has revealed the presence of sequences
characteristic of typical plant signal peptides located
immediately downstream of the only intron present in
the gene (Keil et al, 1986). In fact, signal peptides of
molecular weight around 2000-3000 daltons have
been detected in newly translated inhibitor proteins.
Moreover, simulation of the inhibitor induction was
possible in transgenic tomatoes carrying the prosysternin gene.
(iv) Bowman-Birk
Inhibitor (BBI) Family
The trypsin subclass of serine protease inhibitors
from legume seeds exhibit insecticidal effects against
several crop pests belonging to the orders of Lepidoptera, Coleoptera and Orthoptera (Shukle & Murdock, 1983). Many of these inhibitors are products of
multigene families with varying specificities towards
different proteases (Ryan, 1990). These inhibitors are
cysteine-rich with a molecular mass of 8-20 kDa
(Chen et ai, 1997).
The Bowman-Birk inhibitor (BB!) and its related
family of isoinhibitors comprise a closely related
group of serine Pis. The protein was first identified
and isolated from soybean seeds by Bowman (1946)
and further characterized by Birk and associates (Birk
et al, 1963), hence the name Bowman-Birk inhibitor
(BBI). Later on, this class of inhibitors was isolated
from a large number of legumes and several other
plants including monocot. They contain higher
amounts of sulphur amino acids and have a molecular
mass less than 10,000 kDa. These proteins are classified as double-headed serine protease inhibitors due to
the presence of two reactive site domains within the
same polypeptide, one each for trypsin (Lys-Ser) and
chymotrypsin (Leu-Ser) molecules. These are products
of small gene analogues without introns (Hilder et al.
1987; Boulter et al, 1989). The BBIs from seeds are
double headed proteins of 8kDa, whereas the 8kDa
inhibitors from monocotyledonous seeds are single
headed. However, 16 kDa double-headed inhibitors
have also been reported to be present in them. Comparison of the primary structure of the 8 kDa the BBI
domains have revealed that a crucial disulfide which
connects the amino and carboxy termini of the active
site loop, while present in the double-headed inhibitors
from dicotyledons is lost monocotyledons leading to
the loss of a reactive site and making them singleheaded. Thus, the gene duplication event leading to a
16-kDa double-headed inhibitor in monocotyledons
might have occurred, probably after the divergence of
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INDIAN J BIOTECHNOL, JANUARY 2003
monocotyledons and dicotyledons and also after the
loss of a second reactive site in monocotyledons.
The cowpea trypsin inhibitor constitutes a somewhat larger gene family of four major isoinhibitors,
although the exact number of active genes is not
known. Three of the isoinhibitors are specific for
trypsin at each active site and fourth is a trypsin chymotrypsin bifunctional inhibitor. Feeding trials on
artificial diets have shown that the CpTI had inhibitory effects against a wide range of insects including
Lepidopteran like Heliothis spp. (tobacco budworm
and corn silkworm), Spodoptera (armyworms), Manduca sexta, Coleoptera (beetles) including Callosobrucus maculatus, Diabrotica sp. (corn rootworms),
Anthonomous grandis (cotton boll weevil) and a range
of other insects. This broad range is typical of many
plant based protection mechanism (Park et al, 2001).
The trypsin inhibitor extracted from cowpea was a
more effective anti-metabolite than those extracted
from a number of other legumes (Boulter, 1993).
Comparison of the content of anti-metabolites in
cowpea varieties has revealed that the seeds of the
wild cowpea (Vigna vexillata) contained higher
amounts of trypsin inhibitor and chymotrypsin inhibitor and less of lectins compared to the cultivated
accessions (Marconi et al, 1993). The high resistance
to bruchid and the high TI and CTI contents of
V.vexillata suggested that though there may not be
any direct correlation between bruchid resistance and
TI content, the protease inhibitors promote or at least
strongly influence the plant's defence system.
The cowpea protease inhibitor protein is comprised
of readily indentifiable core region covering the invariant cysteine residues and active serine centres that
are bound to highly variable amino and carboxy terminal regions. The specificity and binding properties
of CpTI have been documented as attributes related to
the first and second domains comprising the core region of the inhibitor protein. It is likely that isoforms
with striking variations in inhibition and binding
properties may be products of crucial mutations in
regions adjoining the binding sites in one or both the
domains (Laskowski et ai, 1988). An added advantage
from the biosafety point of view is that the cowpea
trypsin inhibitor is very sensitive to pepsin digestion,
which is unlikely with many trypsin inhibitors. Since
the ingested food is initially exposed to pepsin in the
mammalian digestive system, the CpTI may be completely digested by pepsin rendering it safe for consumption.
The PI genomic clone (pLK8) isolated recently in
our laboratory is from a genomic DNA library of
cowpea, cv 130 possessed an ORF which on translation gave a protein with 167 amino acids (Lawrence et
al, 2001). The molecular weight of this protein was
calculated to be 18.5 kDa; in which 18 amino acids,
strongly basic, 20 strongly acidic, 40 hydrophobic and
62 polar, with an isoelectric point of 6.5. The protein
had a N terminal signal sequence of 69 amino acids
which is important in its translocation as has been
reported in many proteinase inhibitors such as soybean (Hammond et al, 1984), cowpea, (Hilder et al,
1989) pea, (Domoney et al, 1995), maize (Rohrmeir
et al 1993), and alfalfa (McGurl et al, 1995). The
cowpea protease inhibitor had the signal peptide
cleavage site between 69-70 amino acid residues
which is comparable to the alfalfa trypsin inhibitor
(ATI) having a signal peptide of 44 or 55 amino acid
residues which may target the ATI to the ER and then
eventually deposit it in the vacuole of the cell
(McGurl et al, 1995).
(v) Lectins
Lectins are defined as proteins possessing at least
one non-catalytic domain that reversibly binds to specific carbohydrate(s). They are a heterogeneous group
of proteins differing from each other with respect to
their molecular structure, carbohydrate-binding specificity and biological activities. In most plant species,
they are found, at least in low amounts, in storage tissues of seeds, stems or bulbs (Esteban et al, 2002).
There are four major sub-groups of plant lectins: the
legume lectins (arachis, soybean, lentil, kidney bean,
pea, and faba bean), the monocotyledons man nose
binding lectins (onion, leek, taro & garlic), the chitin
binding lectins (barley, rice, wheat, rye, tomato &
potato) and the type 2 ribosome inactivating proteins
(castor bean & elderberry). However, heterologous
lectins outside these groups have been reported from
banana, jackfruit and pumpkin. Among them, those
from leguminous plants form a large family of homologous proteins with strong similarity at the level
of their amino acid sequences and tertiary structures,
but with highly variable carbohydrate specificities and
quaternary structures.
Over two decades ago, it was recognized that leetins possessed insecticidal properties. Lectins exert
their inhibitory effect by binding to glycoproteins embedded within the peri trophic matrix lining the insect
midgut and in turn disrupting digestive processes and
KOUNDAL & RAJENDRAN:
PLANT INSECTICIDAL
nutrient assimilation. Ingestion of lectin results in
both binding of the lectin to membrane proteins in the
insect gut epithelium, and transport to the haemolymph, and may result in systemic effects on insect
development. Wheat germ agglutinin was the most
effective among 17 plant lectins screened for their
inhibitory effect on larval development of C. maculatus (Murdock et al, 1990). While, the E-type lectins
(erythrocyte-binding type) from common bean retarded the larval growth of C. maculatus (Gatehouse
et al, 1984), the lectin from kidney bean did not thoroughly inhibit the larval growth of the pest (Ishimoto
et al, 1996) ..
The mannose-specific lectin (GNA) derived from
the snowdrop, Galanthus nivalis, has been found to be
toxic to major sap-feeding insect pests of agronomically important crops like rice (against Nilaparvata
lugens & Nephotettix virescens), and pea (against
Callosobruchus maculatusi, and has been expressed
in at least nine food crops including oil seed rape,
potato, rice, and tomato (Sauvion et al, 1996; Gatehouse et al, 1996; Zhang et al, 2000). The range of
toxicity of GNA has recently been extended to the
Lepidopteran, Lacanobia oleracea (tomato moth),
where it was observed that the mean larval biomass
was reduced by approximately 50% when reared upon
transgenic potato (Fitches et al, 1997). In transgenic
tobacco expressing the pea lectin (P-lec) gene, the leaf
damage caused by the larvae of Heliothis virescens
was greatly reduced (Boulter, 1990; Ye & Ng, 2001).
The snowdrop lectin gene has also been successfully
expressed in potato to give protection against aphids
(Hilder et al, 1995), lepidopteron pests, and in rice
against the rice brown plant hopper tNilaparvata
lugensi.
Potential of Insecticidal Proteins for Developing
Transgenics Resistant to Insect Pests
In general, transgenic plants developed using protein inhibitors of insect digestive enzymes with a view
to control crop pests are designed not to kill the insects that feed, but to retard their development. And
presumably, this is the fundamental difference between this strategy and the chemical pest control or
use of Bt toxins that are aimed at complete control
through pest mortality. Thus, perceived effects of the
inhibitors on a pest population are usually much less
dramatic than in the case with synthetic chemical
pesticides. Complete control of insects cannot be expected in any realistic trial, tending rather to increase
PROTEINS FOR TRANSGENICS
RESISTANT TO PESTS
115
mortality to a limited extent but to retard insect
growth and development significantly. However, in
an integrated pest management programme, crop
protection is accomplished through the concerted effects of several complementing control measures.
Moreover, the inhibitory effect of PIs could improve
the efficiency of defence proteins like Bt toxins or the
plants' own defence proteins by preventing their degradation by the target pest proteases (Jongsma &
Bolter, 1997). Therefore, even in situations where
transgene expression does not keep the pest population below the threshold for intervention, it should
allow a much wider window within which intervention can besuccessfully employed.
The first ever transgenic plants were produced by
Hilder et al (1987) using cowpea trypsin inhibitor
cDNA clone. The transgenic plants were resistant
against herbivorous insects such as collosobrchus
maculatus, Heliothis spodoptera and Diabrotica sp
and Tribolium sp. Johnson et al (1989) transformed
tobacco plants with gene coding tomato and potato
inhibitor proteins and the transgenic plants found resistant to M. sexta.
Sane et al (1997) amplified the cowpea genomic
DNA by polymerase chain reaction and cloned the
fragment in a plant expression vector coupled with
CaMV 35S promoter and NOS terminator and used
for tobacco transformation. The transgenic tobacco
plants were tested against Spodoptera litura and were
found resistant. Recently, a protease inhibitor gene
isolated from a native variety of cowpea in laboratory
was used to transform pigeon pea through Agrobacterium tumefaciens-mediated
genetic transformation
(Lawrence et al, 2001). The gene was driven by
CaMV 35S promoter containing kanamycin resistance
as plant selection marker. Molecular analysis of the in
vitro cultured transgenic pigeon pea plants confirmed
the integration and stable expression of the protease
inhibitor gene (Lawrence & Koundal, 2001). The first
generation of these transgenic pigeon pea plants is
being maintained in glass houses for insect feeding
experiments and preliminary insect bioassays showed
encouraging results against pod borer tHelicoverpa
armigera). Duan et al (1996) produced transgenic
plant of rice by using wound inducible expression of
potato proteinase inhibitor II. Bioassay of fifth generation transgenic rice plants showed increased resistance to major rice pest, pink stem borer. Similarly,
lectin genes have isolated and transformed into crop
plants. Transgenic plants expressing snowdrop lectin
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INDIAN J BIOTECHNOL,
(Gatehouse et al, 1997), pea lectin (Boulter et al,
1990) are found resistant to sap sucking insects. Recently, we have isolated and characterized a lectin
cDNA clone from cowpea (Datta et al, 2000). The
gene was introduced into Brassica using an expression
cassette. Comprising the CaMV S35 promoter, GUS
and NOS terminator. Other moelcular analysis of
transgenics and in vitro feeding trails for testing their
efficacy against pea aphids showed significant inhibitory effort on the growth and development (unpublished). However, efforts are being made to isolate
and characterize lectin from other legumes for identifying the novel genes that affect the insect pests but
have no effect on non-target organisms but present no
risks in human health.
When ex-AI was expressed at high levels in transgenic peas, weevil larval development was completely
blocked and the effects of weevil damage on overall
seed yield was significantly reduced (Schroeder et al,
1995). Larval development of several species of CaZlosobruchus beetles, a pest of adzuki bean, was completely inhibited when fed transgenic pea seeds expressing the alpha-amylase inhibitor from common
bean. At the same time, Zabrotes subfasciatus, a natural bruchid pest of common bean, could freely feed on
the transgenic plant (Ishimoto et al, 1996). Recently,
an international
collaboration
between Maarten
Chrispeels, University of California and T J Higgins,
CSIRO, Australia conducted the first field trials with
transgenic pea plants carrying ce-Al genes from
Pliaseolus vulgaris. The genes encoding two ex-AI
isoforms, alpha AI-l isolated from the cultivated
common bean (P. vulgarisi and alpha AI-2 gene from
a wild accession, were introduced into pea (Pisum
sativum) cultivars by using an Agrobacterium system
(Morton et al, 2000). These transgenic plants expressed high levels of ex-AIs in their seeds. Significant
mortality of the first and second instar grubs was
found with the ex-AI-l lines resulting in less weevil
emergence than the control plants. Emergence was
delayed in the ex-AI-2 lines but further analysis
showed that the exAI-2 had no effect on overall mortality. Initial safety studies on mammals showed that
300 or 700 g of peas Kg-I of feed had no impact on
the nutritional value of transgenic peas fed to rats
(Puzstai et al, 1999) have also demonstrated that the
o-Als cause minimum mammalian toxicity in comparison with the other plant inhibitor proteins. The
toxic effects of alpha-amylase inhibitors vary with the
species of insect amylase. The variation in sensitivity
JANUARY 2003
towards the inhibitor may be attributed to either the
presence of inhibitor-insensitive amylases or to the
secretion of proteases that can degrade the inhibitor in
the larval midgut (Bown et al, 1997; Gatehouse et al,
1997). The availability of such genes from different
plant species make it easy to use one or more genes in
combination to develop transgenics for durable resistance against insect pests. The transgenic technology
may not replace the use of chemical pesticides in future but effectively complement it.
Constraints of Transgenic Crops
There are several constrains and apprehensions regarding genetically modified GM food crops. These
include: toxicity, allergenicity, carcinogenicity, use of
antibiotic resistant genes and nutritional value. Decision regarding safety should be based on the nature of
the product rather than other method by which it has
modified. It is more important to bear in mind that
many of the crop plants used today, contain natural
toxins and allergens health hazards from food and
how to reduce them is an issue. Every effort should be
made to avoid the introduction of known allergens
into food crops and foods have to be clearly tabelled
worldover. The concerns have been raised that the
widespread use of antibiotics resistance and genes as
selective markers in plant could increase the antibiotic
resistance in human pathogens. Plant breeders should
continue to remove all such markers from GM plants
and utilize alternative markers for the selection of
new varieties.
Most of the environmental concerns about GM
technology in plants have derived from the possibilities of gene flow to close relative!' of transgenic plants
creating super weeds or causing gene pollution among
other crops. There is a need for thorough risk assessment of likely causes of concern, at an early stage in
the development of transgenic plants varieties and
evaluate these risks in subsequent field tests and releases. Internal agricultural community of every nation should support serious efforts to establish a system to adequately assess and also avoid human health
and environment risks. India is among a few countries
where regulation and guidelines on research and
monitoring of transgenic plants and their food safety
assessment have been are developed by the Department of Biotechnology, Govternment of India. However, certain amount of risk is inherent in every new
technology and a careful risk benefit analysis is necessary for making balance decision.
KOUNDAL & RAJENDRAN:
PLANT INSECTICIDAL
In spite of all the ideological dispute and ensuing
democratic disagreements on the acceptability of genetically modified plants, genetic engineering for pest
control has now been realized as an environmentally
benign, flexible technology that can be safely fitted in
any integrated pest management protocol. Furthermore, it allows the possibility of developing entirely
new biological insecticides that retain the advantages
of classical biological control agents, but have fewer
of their drawbacks. In reality, insect resistant transgenic plants will certainly prove to be more economic
than any conventional control strategy if the longterm benefits of transgenic crops especially factors
such as environmental damage and health risks are
considered.
Future Strategies
The use of insect resistant transgenic plants is a viable means of producing crops with significantly enhanced level of resistance. Several transgenic plants
expressing plant-borne inhibitor proteins have been
developed in the last decade. Various approaches that
are being proposed and tried by different research
groups include:
(i) Gene Combinations/Packaging/Pyramiding
The protective efficacy, spectrum of activity and
the durability of resistance offered by the introduced
genes can be greatly enhanced through careful design
of packages of different genes that contain components which would act on quite different target insects. Protease inhibitors may have a major role in
such gene pyramiding approaches. Apart from their
inherent insecticidal property, they would protect
other introduced gene products from premature digestion in the insect gut and improve the overall performance through their mutually complementing or
synergistic effects. The first demonstration of this approach has been the introduction of both cowpea trypsin inhibitor and pea lectin in transgenic tobacco
plants where the two gene products had an additive
effect on tobacco bud worm caterpillars (Boulter et al,
1990). It may be a useful approach to combine genes
that encode proteinase inhibitors among themselves or
along with suitable lectin, a-AI and/or Bt genes so
that multiple pest resistance may be achieved in a single event in agronomically important crop plants.
Cross breeding of primary transformants carrying the
desirable gene combinations would also prove useful
in terms of enhanced insect resistance (Schuler et al,
1998).
PROTEINS FOR TRANSGENICS
RESISTANT TO PESTS
117
(ii) Protein Engineering
In-depth exploration of protein structure and function may allow researchers to use protein engineering
as a strong tool for designing novel chimeric proteins
for insect control. These chimeras are constructed by
tailoring together the sequences that encode discrete
domains of the protein intended to act on defined targets. In vitro mutagenesis can be exploited for creating very effective chimeric genes carrying desirable
domains with defined activity spectrum. The longterm goal of protein engineering would be the construction of modular protein that will target specific
pests without any harmful effects on the beneficial
organisms. In principle, any domain from any protein
can be used in this modular system to construct proteins with a given set of attributes. Although still in its
infancy, protein engineering will allow us to design
proteins for use against the most insect pests.
(iii) Single-chain Antibodies
This approach makes use of engineering antibodies
or antibody fragments specific to the target pest's essential protein and expressing it in the crop plant so
that both specificity and efficacy of action can be incorporated in a single event (Hilder & Boulter, 1999).
Besides, it has an additional advantage of avoiding
action on the non-target organisms, particularly
predators.
(iv) Phage Display
The technique combines in vitro ruutagemsis, rapidity of molecular cloning, specificity of proteinprotein interactions, and precision of molecular
screening techniques. After isolation and cloning of
an ideal inhibitor gene, a large collection of its variants are prepared in the form of a library by altering
its sequence at every possible positions in the regions
critical for its action. In fact, the gene can be modified
for all the coding frames with every codon for each of
the 20 possible amino acids so that the resultant
changes in specificity, binding and other attributes in
each of the modified product (protein) can be examined. The cloned genes are then expressed on the surface of phage particles and the displayed proteins are
screened for the variant inhibitor protein, which exhibits the maximum affinity (binding) for the target
protease enzyme. Thus, such technique envisages the
screening of millions of cloned proteins with the desired one being physically separated from others
based upon its affinity to the target larval enzyme.
118
INDIAN
J BIOTECHNOL,
(v) Directed Protein Evolution
This technique, a variant to phage display may turn
to be very useful in screening of large population of
inhibitor variants for their specificity with greater accuracy. Here, protein and peptide libraries are used to
select interactive proteins (ligands) without prior information concerning their sequence or structure. It
can be used to construct a phage display library of
mutant proteins or peptides as fusion proteins on the
surface of the phage. The library can then be used for
screening any enzyme for its binding specificity so
that the inhibitor with maximum affinity towards the
enzyme can be easily located. The possible outcome
of the aforesaid developments would be the availability of the technology to overtake the insect's molecular potential for resistance development through more
programmed, eco-friendly and efficient plant genetic
engineering strategies.
Conclusion
The continuous use of pesticides for crop protection
had resulted in damaging impact on biological ecosystems, bio-magnification effects through pesticide residue deposition in food and feed pollution. The use of
target specific compounds with low persistence and the
exploitation of intrinsic plant resistance mechanisms
are suggested as safer alternative strategies for effective
insect pests management. Thus, insect resistant GM
plants will curtail the use of those hazardous pesticides
by engineering genes that encode natural biodegradable
proteins with no harmful effect to animals and human
beings. It is important to remember that biotechnology
tools complement and extend the traditional methods
used to enhance agricultural productivity and to develop new production systems. The progress of agriculture in developing, non-industrialized countries
where the economic activity still provides 60-80% employment and 50% of national income will be possible.
The application of plant biotechnology will be no
doubt, more meaningful in the poor and nonindustralized parts of the world where socio-economic
development will be possible only through sensible
adoption of advanced agricultural practices that help
for enhanced food, feed' and fibre production and an
overall improvement in living standard. Despite, the
opposition from the anti-GM movements all over the
world, undoubtedly, will find its more and immediate
application in insect pest control of major crops. The
availability of diverse insecticidal genes from different
plant species makes it a possibility to use one or more
genes in combination whose products are targetted at
JANUARY
2003
different biochemical and physiological processes. The
transgenic crops developed for insect resistance should
be compatible with other components of integrated pest
management programmes for pest resistance to be durable and effective. It is envisaged that it will have a
major impact on agricultural systems both in the developed and developing countries in the near future.
References
Birk Y, Gertler A & Khalef S, 1963. A pure trypsin
soya beans. Biochem J, 87, 281-284.
inhibitor
from
Boulter D, Edwards G A, Gatehouse
A M R, Gatehouse
J A &
Hilde Y A, 1990. Additive protective effects of incorporating
two different higher plant derived insect resistance
genes in
transgenic tobacco plants. Crop Prot, 9, 351-354.
Boulter D, Gatehouse
A M R & Hilder Y, 1989. Use of cowpea
trypsin inhibitor (CpTi) to protect plants against insect predation. Biotechnol Adv, 7, 489-497.
Boulter D, 1993. Insect
netically engineered
Bowman
D E, 1946.
pest control by copying nature using
crops. Biochemistry, 34, 1453-1466.
Differentiation
of soybean
antitrypsin
gefac-
tors. Proc Soc Exp Bioi Med, 63, 547-550.
Bown
D P, Wilkinson H S & Gatehouse
J A, 1997. Differentially
regulated
inhibitor-sensitive
and insensitive
protease
genes
from the phytophagous
insect pest, Helicoverpa annigera.
are members of complex multi gene families. Insect Biochem
Mol Bioi, 27, 625-638.
Chen
P, Chow S & Chen L, 1997. Nucleotide
Sequence
of a
cDNA (Accession
No. U76004)
Encoding
Rice BowmanBirk Proteinase
Inhibitor (PGR97-015).
Plal/t Physiol, 113.
663-668.
Chrispeels
M J & Raikhel
N Y, 1991.
their role in plant defence.
Lectins,
lectin
genes
and
Plant Cel!, 3, 1-19.
Chrzaszcz
T & Janicki J, 1934. The inactivation
of animal amylase by plant paralysers and the presence of inactivating
substances in solutions of animal amylase. Biochem J. 28. 196304.
Connors B J, Laun N P, Maynard
C A & Rowell W A, 2002.
Molecular
characterization
of gene encoding
a cystatin expressed in the Stems of American
Chestnut
(Castanea dentata). Planta, 215, 510-514.
Covene-kubis
I, Kowalska
J, Pochson B, ZuzlO A & Wilusz T.
2001. Isolation and amino acid sequence of a serine proteinase inhibitor
from common
flax (Linum usitatissimumi
seeds. Chembiochem, 2, 45-51.
R & Koundal K R, 2000. Construction
of cowpea
(Vigna unguiculata L) cDNA library and characterization
of
an isolated lectin gene. J Plant Biochem Biotechnol. 9. 67-
Datta S, Kansal
71.
Domoney C, Welham T, Sidebottom
C & Firmin J L. 1995. Multiple isoforms of Pisurn trypsin inhibitors result from modification of two primary gene products. FEBS Lett, 360, 15-20.
Duan R F, Xue Q, Abo-El Saad, M XuD & Wu R, 1996. Transgenic rice plants harboring and introduced
potato proteinase
inhibitor II are insect resistant. Nature Biotechnol. 14. 494498.
Esteban R, Dipico S, Munoz F J, Rorno S & Cubrador
seedling specific vegetative
lectin gene related
E. 2002. A
to develop-
ment in Cicer arietinum. Physiol Plant, 114. 619-626.
KOUNDAL & RAJENDRAN:
PLANT INSECTICIDAL
Fabrick J, Behnke C, Czapla T, Beta K, Rao A G, Kramer K J &
Reeck G R, 2002. Effects of a potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of
the southern corn root worm, Diabrotica undecimpunctata
howardi (coleaptera: chrysome lidate). Insect Biochem Mol
Bioi, 32, 405-415.
Feng G H, Richardson M, Chen M S, Kramer K J, Morgan T D &
Reeck G R, 1996. Alpha-amylase inhibitors from wheat:
Amino acid sequences and patterns of inhibition of insect
and human alpha-amylases. Insect Biochem Mol Bioi, 26,
419-426.
Fitches E, Gatehouse A M R & Gatehouse J A, 1997. Effects of
snowdrop lectin (GNA) delivered via artificial diet and
transgenic plants on the development of tomato moth (Lacanobia oleracea) larvae in laboratory and glasshouse trials.
J Insect Physiol, 43, 727-739.
Garcia-Casado G, Sanchez-Monge R, Puente, X S & Salcedo G,
1996. Divergence in properties of two closely related alphaamylase inhibitors of barley. Physiol Plant, 98,523-528.
Garcia-Olmedo F, Salcedo G, Sanchez-Monge R, Gomez L, Royo
J & Carbonero P, 1987. Plant proteinaceous inhibitors of
proteinases and a-amylases. Oxford surveys Plant Mol &
Cell Bioi, 4,275-334.
Gatehouse A M R & Gatehouse J A, 1998. Identifying proteins
with insecticidal activity: Use of encoding genes to produce
insect resistant transgenic crops. Pestic Sci, 52,165-175.
Gatehouse A M R, Boulter D & Hilder V A, 1992. Potential of
plant-derived genes in the genetic manipulation of crops for
insect resistance. in plant genetic manipulation for crop protection edited by A M R Gatehouse, V A Hilder and D
Boulter. CAB International, Wallingford, England, Pp. 155181.
Gatehouse A M R, Dewey F M, Dove J, Fenton K A & Pusztai A,
1984. Effect of seed lectin from Phaseolus vulgaris on the
development of larvae of Callosobruchus maculates mechanism of toxicity. J Sci Food Agric, 35, 373-380.
Gatehouse A M R, Down R E, Powell K S, Sauvion N, Rahbe Y,
Newell C A, Merryweather A, Hamilton W D 0 & Gatehouse J A, 1996. Transgenic potato plants with enhanced resistance to the peach-potato aphid Myzus persicae. Entomol
Exp App, 79,295-307.
Gatehouse A M R, Gatehouse J A & Boulter D, 1980. Isolation
and characterization of trypsin inhibitor from cowpea (Vigna
unguiculatas. Phytochemistry, 9,751-756.
Gatehouse L N, Shannon A L, Burgess E P J & Christeller J T,
1997. Characterization of major midgut proteinase cDNAs
from Helicoverpa armigera larvae and expression in response to four proteinase inhibitors in the diet. Insect Biochem Mol Bioi, 27, 929-944.
Gomes A V, Charren G S & Barnes J A, 1997. Major proteins of
yam bean tubers. Phytochemistry, 46, 185-193.
Graham J S, Hall G, Pearce G & Ryan C A, 1986. Regulation of
synthesis of proteinase inhibitors 1 and 11 mRNAs in leaves
of wounded tomato plants. Planta, 169, 399-405.
Hammond R W, Foard D E & Larkins B A, 1984. Molecular
cloning and analysis of a gene coding for the Bowrnan-Birk
protease inhibitor in soybean. J Bioi Chem, 259, 9883-9890.
Hilder V A & Boulter D, 1999. Genetic engineering of crop plants
for insect resistance -A critical review. Crop Prot, 18, 177191.
PROTEINS FOR TRANSGENIC~
RESISTANT TO PESTS
119
Hilder V A, Gatehouse A M R, Sheerman S E, Barker R F &
Boulter D, 1987. A novel mechanism of insect resistance engineered into tobacco. Nature (Lond), 330, 160-163.
Hilder V A, Powell K S, Gatehouse A M R, Gatehouse J A, Gatehouse L N, Shi Y, Hamilton W D 0, Merryweather A, Newell C A, Timans J C, Peumans W J, Van damme E & Boulter
D, 1995. Expression of snowdrop lectin in transgenic tobacco
plants results in added protection against aphids. Transgenic
Res, 4, 18-25.
Hilder V A, Barker R F, Samour R A, Gatehose A M R, Gatehouse J A & Boulter D, 1989. Protein and cDNA sequences
of Bowman-Birk protease inhibitors from the cowpea (Vigno
unguiculata Walp). Plant Mol Bioi, 13,701-710.
Ho M F & Whitaker J R, 1993. Subunit structures and essential
amino acid residues of white kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors. J Food Biochem, 17, 35-52.
Huesing J E, Shade R E, Chrispeels M J & Murdock L L, 1991.
Alpha-amylase inhibitor, not phytohemagglutinin,
explains
resistance of common bean seeds to cowpea weevil. Pia III
Physiol, 96, 993-996.
Ishimoto M & Kitamura K, 1992. Tolerance to the seed alphaamylase inhibitor by the two insect pests of the common
bean, Zabrotes subfasciatus and Acanthoscelides oHtectus
(Coleoptera: Bruchidae). Appl Ent Zool, 27, 243-251.
Ishimoto M, Sato T, Chrispeels M J & Kitamura K, 1996. Bruchid
resistance of transgenic azuki bean expressing seed alphaamylase inhibitor of common bean. Entomol Exp Appl. 79.
309-315.
Johnson R, Narvaez J, An G & Ryan C A, 1989. Expression of
proteinase inhibitors I and II in transgenic plants: Effects on
natural defence against Manduca sexta larvae. Proc Natl
Acad Sci USA, 86, 9871-9875.
Jongsma M A & Bolter C, 1997. The adaptation of insects to plant
protease inhibitors. J Insect Physiol, 43, 885-895.
Keil M, Sanchez-Serrano J, Schell J & Willmitzer L, 1986. Primary structure of a proteinase inhibitor II gene from potato
(Solanum tuberosum). Nucleic Acids Res, 14,5641-5650.
Kouzuma Y,Tsukigate K, Inanaqa H, Doi-Kawanok, Yamasaki N,
Kimura M, 2001. Molecular cloning and functional expression of cDNA encoding the cysteine protease inhibitor from
sunflower seeds. Biosci Biotechnol Biochem, 65, 969-972.
Kunitz M, 1945. Crystallization of a trypsin inhibitor from soybean. Science, 101, 668-669.
Laskowski M & Kato I, 1980. Protein inhibitors of proteinases.
Ann Rev Biochem, 49, 593-626.
Laskowski M, Kato I, Kohr W J, Park S J, Tashino M & Whatley
H S, 1988. Positive Darwinian selection in evolution of protein inhibitors of serine proteinases, Cold Spring Harbor
Symp Quant Bioi, 52, 545-553.
Lawrence P K & Koundal K R, 2001. Agrobacterium tumefaciens-mediated transformation of pigeon pea [Cajanus cajan (L.) Millsp.] and molecular analysis of regenerated
plants. Curr sa. 80, 1428-1432.
Lawrence P K, Nirrnala J & Koundal K R, 2001. Nucleotide sequence of a genomic clone encoding a cowpea (Vigna unguiculata L.) trypsin inhibitor. Electronic J Biotechnol, 4.
1-5.
.
Maeda K, 1986. The complete amino acid sequence of the endogenous alpha-amylase inhibitor in wheat. Biochim Biophys Acta, 871, 250-256.
120
INDIAN J BIOTECHNOL,
Marconi, E, Nig N G & Carnovale E, 1993. Protease inhibitors
and lectins in cowpea. Food Chem, 47, 37-40.
McGurl B, Mukherjee S, Kahn M & Ryan C A, 1995. Characterization of two proteinase inhibitor (AT!) cDNAs from alfalfa
leaves (Medicago sativa var. Vernema): The expression of
ATI genes in response to wounding and soil microorganisms.
Plant Mol Bioi, 27, 995-1001.
Moreno J & Chrispeels M J, 1989. A lectin gene encodes the alpha-amylase inhibitor of the common bean. Proc Natl Acad
Sci USA, 86, 7885-7889.
Morton R L, Schroeder H E, Bateman K S, Chrispeels M J, Armstrong E & Higgins T J, 2000. Bean alpha-amylase inhibitor
1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions. Proc Natl Acad Sci USA, 97, 3820-3835.
Maqbool S B, Sheikh Riazuddin, Vguyen Thi Loc, Gatehouse A
M R, Gatehouse J A & Christou P, 2001. Expression of multiple insecticidal genes confers broad resistance against a
range of different rice pests. Mol Breed, 7, 85-93.
Murdock L L, Hensing J E,Nielsen S S, Pratt R C & Shede R E ,
1990. Biological effects on plant lactins on the cowpea weevil, Phytochemistry, 29, 66-89.
Nodari R 0, Tsai S M, Gilbertson R L & Gepts P, 1993. Towards
an integrated linkage map of common bean. 2. Development
of an RFLP-based linkage map. Theor Appl Genet, 85, 513520.
Odani S, Koide T & Ono T, 1983. A possible evolutionaryrelationship between plant trypsin inhibitor, alpha-amylase inhibitor, and mammalian pancreatic secretary trypsin inhibitor
(Kazal). J Biochem, 93,1701-1704.
Plunkett G, Senear, D F, Zuroske G & Ryan C A, 1982. Proteinase inhibitor I and II from leaves of wounded tomato plants:
purification and properties. Arch Biochem Biophys, 213,463472.
Pusztai A, Bardocz G G, Alonso R, Chrispeels M J, Schroeder H
E, Tabe L M & Higgins T J, 1999. Expression of the insecticidal bean alpha-amylase inhibitor transgene has minimal
detrimental effect on the nutritional value of peas fed to rats
at 30% of the diet. J Nutr, 129, 1597-1603.
Park S H, Park J & Smith R H, 2001. Herbicide and insect resistant elite transgenic rice. J Plant Physiol, 158, 1221-1226.
Read J W & Haas L W, 1938. Studies on the baking quality of
flour as affected by certain enzyme actions. V. Further studies concerning potassium bromate and enzyme activity. Cereal Chem, 45, 59-68.
Richardson M J, 1977. The proteinase inhibitors of plants and
microorganisms. Phytochemistry, 16, 159-169.
Rohrmeier T & Lehle L, 1993. WIPI, a wound-inducible gene
from maize with homology to Bowman-Birk proteinase inhibitors. Plant Mol Bioi, 22, 783-792.
Rouge P, Barre A, Causse H, Chatelain C & Porthe G, 1993. Arcelin and alpha-amylase inhibitor from the seeds of common
bean (Phaseolus vulgaris L.) are truncated lectins. Biochem
Syst Ecol, 21, 695-703.
Ryan C A 1990. Proteinase inhibitors in plants: Genes for improving defences against insects and pathogens. Ann Rev
Phytopathol, 28, 425-449.
JANUARY 2003
Sanchez-Monge R, Garcia-Casado G, Barber D & Salcedo G.
1996. Interaction of allergens from house-dust mite and from
cereal flours: Dermatophagoides
pteronyssinus
alphaamylase (Der p 4) and wheat and rye alpha-amylase inhibitors. Allergy, 51, 176-180.
Sane V A, Nath P, Aminudden & Sane P V, 1997. Development
of insect resistant transgenic plants using plant genes: Expression of cowpea trypsin inhibitor in transgenic tobacco
plants. Curr Sci, 72, 741-747.
Sauvion N, Rahbe Y, Peumans W J, van Damme
house J A & Gatehouse A M R, 1996. Effects
other mannose binding lectins on development
of the peach-potato aphid Myzus persicae.
Appl, 79, 285-293.
E J M, Gateof GNA and
and fecundity
Entotnol Exp
Schroeder H E, Gollasch S, Moore A, Tabe L M, Craig S, Hardie
D C, Chrispeels M J, Spencer D & Higgins T J V, 1995.
Bean alpha-amylase inhibitor confers resistance to the pea
weevil (Bruchuspisorum) in transgenic peas tPisum sativum
L.). Plant Physiol, 107,1233-1239.
Schuler T H, Poppy G M, Kerry B R & Denholm r, 1998. Insect
resistant transgenic plants. Trend Biotech, 16, 168-175.
Shewry P R, Lafiandra D, Salcedo G, Aragoncillo A, GarciaOlmedo F, Lew E J L, Dietler M D & Kasarda D D, 1984. Nterminal amino acid sequences of chloroform/methanol soluble proteins and albumins from endosperm of wheat, barley
and related species. FEBS Leu, 175, 359-363.
Shukle R H & Murdock L L, 1983. Lipoxygenease, trypsin inhibitor, and lectin from soybeans: effect on larval growth of
Manduca sexta (Lepidoptera: Sphingidae). Environ Entoinol.
12, 787-791.
Sparvoli F, Gallo A, Marinelli D, Santucci A & Bollini R, 1998.
Novel lectin-related proteins are major components in Lima
bean (Phaseolus lunatus L.) seeds. Biochim Biophys Acta.
1382,311-323.
Sparvoli F, Lanave C, Santicci A, Bo!lini R & Lioc L, 2001. Lectin and lectin related proteins in Liana bean Phaseolus hawtus). Plant Mol Bioi, 45, 587-597.
Ussuf K K, Laxmi N R & Mitra R, 2001. Proteinase inhibitors:
Plant derived genes of insecticidal proteins for developing
insect resistant transgenic plants. Curr Sci, 86, 847-53.
Xu D, Xue Q, McElroy D, Mawal Y, Hilder V A & Wu R, 1996.
Constitutive expression of a cowpea trypsin inhibitor gene,
Cp'Ti, in transgenic rice plants confers resistance to two major rice pests. Mol Breed, 2, 167-173.
Ye X & Ng T B, 2001. Isolation of lectin and albumin from Pisun
sativum var. macrocarpon ser. lnt J Biochem Cell Bioi. 33,
95-102.
Ye X Y, Ng T B & Rao P F, 2001. A Bowman-birk type trypsin
chymotrypsin inhibitor from broad beans. Biochem Biophvs.
Res Commun, 289, 91-96.
Zhang W, Peumans W J, Barre A, Astoul C H, Rovira P, Rouge P.
Proost P, Traffabach P, Jalali A A & Van Darnme E J, 2000.
Isolation and characterization of Jacalin related mannose
binding lectin from salt stress rice Oryza sativa plants.
Planta, 210, 970-978.