|High level expression of soybean trypsin inhibitor gene in transgenic tobacco plants failed to confer resistance against damage caused by Helicoverpa armigera|
Ashis Kumar Nandi*,§, Debabrata Basu, Sampa Das* and Soumitra K Sen
IIT-BREF Biotek, Indian Institute
of Technology, Kharagpur 721 302, India
*Plant Molecular and Cellular Genetics, Bose Institute, P1/12, CIT Scheme VII-M, Calcutta 700 054, India
§Present address: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
Corresponding author (Fax, 91-3222-77980; Email, firstname.lastname@example.org).
Helicoverpa armigera is a major pest of many tropical crop plants. Soybean trypsin inhibitor (SBTI) was highly effective against the proteolytic activity of gut extract of the insect. SBTI was also inhibitory to insect growth when present in artificial diet. The gene coding for SBTI was cloned from soybean (Glycine max, CV Birsa) and transferred to tobacco plants for constitutive expression. Young larvae of H. armigera, fed on the leaves of the transgenic tobacco plants expressing high level of SBTI, however, maintained normal growth and development. The results suggest that in certain cases the trypsin inhibitor gene(s) may not be suitable candidates for developing insect resistant transgenic plants.
Helicoverpa armigera is a lepidopteran, polyphagous insect pest that feeds on important major crop plants like cotton, tomato, tobacco, chickpea, and pigeon pea (Jotwani and Butani 1984). Protease inhibitors are polypeptides present in many plants, which in addition to their physiological function, provide a natural defense against insect attack (Green and Rayan 1972; Laskowski and Sealock 1978; Malehorn et al 1994). Possibility of utilizing trypsin inhibitor gene(s) of plant origin as bio-insecticide for developing insect resistant transgenic crop plant has been a fairly accepted line of approach in crop biotechnological programme. Trypsin inhibitors present in different plants show variable levels of activity towards the proteolytic enzymes of the target insects (Broadway and Duffey 1986; Purcell et al 1992). It has been reported earlier that transgenic plants with suitable trypsin inhibitor gene can resist against insect damage (Hilder et al 1986; Johnson et al 1989; Xu et al 1996).
In the present study, trypsin inhibitors were purified from seeds of soybean (Glycine max) and cowpea (Vigna unguiculata). Soybean trypsin inhibitor (SBTI) was found to be more effective than cowpea trypsin inhibitor (CPTI) in reducing the proteolytic activity of gut extracts obtained from full-grown larvae of H. armigera. Thus SBTI was selected as the candidate gene for developing the insect resistant transgenic plant. The gene coding for the SBTI peptide was cloned. Suitable chimeric construct was developed for over expression of the SBTI gene when transferred to tobacco. Through Agrobacterium tumefaciens mediated gene transfer protocol, transgenic tobacco plants showing high expression of the transferred SBTI gene were generated. Feeding tests of young larvae of H. armigera on the leaves of the highly expressive transgenic tobacco plants did not show significant level of impairment of insect growth in comparison to untransformed plants.
2. Materials and methods
Soybean seeds (G. max, CV Birsa) were obtained from Birsa Agricultural University, Ranchi and cowpea seeds (V. unguiculata, CV Black eye) were obtained from a local nursery. H. armigera insects were collected from Plant Protection Division, Central Institute for Cotton Research, Indian Council of Agricultural Research, Nagpur.
2.2 Purification of trypsin inhibitors
The trypsin inhibitors were purified from mature seeds of soybean and cowpea following the method of Gatehouse et al (1980). About 40 g dry seeds were ground into powder and extracted in 200 ml extraction buffer (0·1 M Na-acetate, 0·3 M NaCl, 0·01 M CaCl2, pH 4·0) for 16 h at 4° C. The extracts were centrifuged at 10,000 g for 30 min. The supernatant was collected and ammonium sulphate was added to the final concentration of 30%, 60% and 90% (w/v) sequentially. Most of the trypsin inhibitor activity was retained in the 60% ammonium sulphate precipitate, which was further dissolved in 50 mM Tris-Cl (pH 8·0) and passed through trypsin ligated CNBr+-sepharose column. Bound trypsin inhibitor was eluted with 0·2 M HCl. Purified trypsin inhibitors were run in SDS-PAGE following the protocol of Shagger and Jagow (1987).
For purifying SBTI from transgenic tobacco plants,
1 g leaf tissue from different transgenic tobacco plants was extracted with
10 ml extraction buffer, and centrifuged
at 4° C for 15 min at 48,000 g. To the supernatant, 1 M Tris-Cl (pH 8·0, 1·0 ml) and trypsin ligated CNBr+-sepharose beads (0·5 ml) were added, mixed thoroughly for 34 h at 4° C. SBTI was eluted from the beads by 0·2 M HCl. Total protein in each fraction was determined by Bradford (1976) assay, and the amount of SBTI present was determined by enzyme linked immunosorbant assay (ELISA).
2.3 Extraction of H. armigera larval midgut and proteolytic activity assay of the gut extract
Midguts of 20 full grown H. armigera larvae were extracted in 1 ml buffer containing 0·1% NaCl and 50 mM Tris-HCl (pH 8·0). The extract was centrifuged at 10,000 g for 30 min at 4° C. The supernatant was diluted 50-fold with the same buffer. To 100 m l of the diluted stock, 100 m l of 5 mM CaCl2 in 50 mM Tris-HCl (pH 8·0) was added along with different concentrations of the purified inhibitors. The volume was made up to 450 m l with water and incubated at 37° C for 30 min. Subsequently, 250 m l of azocasein (2·5%) was added to each tube and incubated at 37° C for 10 min. The reaction was stopped by adding 700 m l of 10% TCA. The reaction mixtures were centrifuged, supernatants were mixed with equal volume of 0·5 N NaOH and the absorbance at 428 nm was recorded.
2.4 Cloning of SBTI gene and construction of plant expression vector
Total genomic DNA from the germinated seeds of soybean was isolated following the method of Rogers and Benedich (1988). SBTI gene was amplified by polymerase chain reaction (PCR) by using 100 ng of DNA, 2 m M of each primer, 0·2 mM of dNTPs, 1 × Taq buffer, 2 mM MgCl2 and 10 U Taq DNA polymerase in 100 m l reaction volume. Thermo-cycling was carried out at 94° C 1 min, 55° C 1 min and 72° C 1 min for 30 cycles with a pre-heating at 94° C for 4 min and extension at the end for 7 min at 72° C. The amplified product was cloned into pUC18 vector at BamHI and SmaI restriction sites and subsequently subcloned into the plant expression vector, pBI121 (Jefferson et al 1987) at BamHI and SacI restriction sites. All molecular biological methods adopted were as described in Sambrook et al (1989).
2.5 Development of transgenic tobacco plants
A. tumefaciens, strain LBA4404 was transformed with the plant expression vector by freeze-thaw method of Hofgen and Willmitzer (1988). Tobacco leaf discs were co-cultivated with overnight grown A. tumefaciens culture and transformed plants were regenerated (Horsch et al 1985) on plates containing 50 mg/l kanamycin.
2.6 Analysis of transgenic tobacco plants
Transgenic tobacco plants were analysed in search for the presence of the SBTI gene following Southern blot analysis (Southern 1975). Total genomic DNA was isolated from several independent transgenic lines and digested with restriction enzyme EcoRV, run in 0·9% agarose gel and transferred to nylon membrane. The presence of SBTI gene was monitored through the use of radiolabelled probe. Expression of SBTI gene in transgenic plants was detected by Western blot analysis (Towbin et al 1979). Total protein was extracted by trypsin extraction buffer (pH 4·0) and separated in a 15% SDS-PAGE (Laemmli 1970) and transferred to nitrocellulose membrane by electro-transfer method. SBTI band was detected through the use of anti-SBTI rabbit polyclonal antibody and HRP linked goat anti-rabbit IgG antibody.
2.7 Feeding assay of H. armigera larvae
Adults of H. armigera were collected and reared for two generations in the laboratory on artificial diet before using them for feeding assay. About 250 g of redgram flour was boiled for 15 min in a pressure cooker with 250 ml of water and homogenized. To the warm mixture, methyl-p-benzoate (1·65 g), sorbic acid (0·85 g), ascorbic acid (2·65 g), streptomycin sulphate (0·12 g) 10% formaldehyde (6·75 ml) and multivitamin complex (800 mg) were added with the addition of SBTI and/or casaminoacid, whenever needed. The mixture was finally distributed into individual vials. Thirty larvae (three days old) were fed in each experiment set. Parallel feeding assay of the larvae (three days old) was conducted on the fresh leaves of transgenic and untransformed tobacco plants. In all the cases body weight of individual larva was recorded at regular interval.
2.8 Determination of free amino acids and amide contents
Fresh leaf tissues of tobacco (200 mg) suspended in 1 ml 0·1 M Tris-Cl (pH 8·0) were homogenized in liquid nitrogen. The extracts were then centrifuged at 10,000 g for 10 min at 4° C. The supernatant was mixed with equal volume of 10% TCA, incubated on ice for 10 min and centrifuged. Each aliquot (0·1 ml) was mixed with 1·0 ml of ninhydrin reagent (Spies 1957) and boiled in water bath for 20 min. Absorption values of the developed purple colour were recorded at 570 nm. Casein acid hydrolysate (casaminoacid) dissolved in same buffer and treated in the similar manner, represented for the standard line of the experiment.
3.1 Purification of trypsin inhibitors
Trypsin inhibitors were purified from the extracts of mature seeds of soybean and cowpea by ammonium sulphate precipitation followed by affinity column chromatography and analysed by SDS-PAGE (figure 1). Very often, inhibitors like CPTI, belonging to BowmanBirk family (Birk 1961) are present in multimeric forms due to the presence of multiple SS linkages (Gennis and Cantor 1976). Boiling with 2% SDS and 2% b-mercaptoethanol was not sufficient to convert them fully to monomeric forms and a faint band of dimeric CPTI (>> mark in lane 1 figure 1) could also be found along with a major 8 kDa band (arrow). Soybean seeds contain both Kunitz type (KTi, Kunitz 1947) and BowmanBirk type (SBBI, Kay 1976; Wei 1983) trypsin inhibitors. Affinity purified SBTI (20 kDa, Kunitz type) was obtained with a small amount of co-purified proteins (<< mark in lane 3 and 4, figure 1). The molecular weights of these co-purified proteins and their ability to bind trypsin ligated sepharose column indicate that they represent BowmanBirk type inhibitor. The Kunitz type SBTI corresponds to about 99% of the purified protein.
3.2 Functional activity assay of purified trypsin inhibitors
The efficacy of CPTI and SBTI in reducing the proteolytic activity of gut extracts of H. armigera was determined. To the larval gut extract, aliquots of purified inhibitors were added (1 to 6 m g) and incubated at 37° C for 30 min. This allowed the trypsin to bind with the inhibitor. The activity of trypsin inhibitors was determined in terms of inhibition of proteolytic activity, as revealed through reduction of absorbance values of the TCA extract of the reaction mixture at 428 nm (figure 2). Similar treatments were carried out on bovine trypsin, as standard. Results indicated (figure 2) that both the purified inhibitors were effective against the proteolytic activity of bovine trypsin and H. armigera gut extract. However, SBTI was selectively more effective than CPTI against the gut extract. Hence, SBTI was selected as the target trypsin inhibitor for the present study.
3.3 Cloning of SBTI gene
Functional SBTI is coded by KTi3 gene in soybean (Jofuku et al 1989). Two primers were designed from the available information on the nucleotide sequences of KTi3 gene (Jofuku and Goldberg 1989); primer-1 (5¢ atcccgggatccATGAAGAGCACCATCTTCTT3¢ ) and primer-2 (5¢ tagggatcccgggtcACTCCATGCGAGAAAGG-CCATATTTTCT3¢ ) for amplification of the complete coding region of the gene with additional flanking sequences (lower case letters) for SmaI and BamHI restriction enzyme sites. With the help of these primers, PCR amplification was carried out by using total soybean genome, as template (figure 3A).
The amplified product was cloned into a pUC18 vector and transformed into E. coli, DH5a . It is known that soybean genome contains other non-functional members of KTi gene family (Jofuku et al 1989). By comparing the available KTi gene family nucleotide sequences, a third primer, primer-3 (5¢ AGGTCCTTCTGGTAGATC3¢) was designed from the KTi3 specific region. Plasmid DNA isolated from the independent colonies were further subjected to PCR based screening with primer-1 and primer-3 to exclude the possibility of inclusion of any such non-functional KTi3 homologue. The clones that showed amplification of 380 bp in second PCR (figure 3B), were finally selected. The cloned SBTI gene was sequenced with the help of gene specific primers by chain termination method of Sanger (1977) using [a 32P]dATP. Possibility of generating any error from mis-incorporation by Taq DNA polymerase in PCR step was eliminated by repeating the experiment several times. Nucleotide sequences were found to be identical to that of the known KTi3 gene except for three nucleotides. These three nucleotide mismatches resulted in the alteration of two amino acids, glu12 and asn13 to asp12 and ser13, respectively. Three different types (Tia, Tib and Tic) of Kunitz trypsin inhibitors with altered amino acid sequence in soybean have been reported earlier (Kim et al 1985). The functional KTi3 gene encodes for the polypeptide sequence of Tia type (Jofuku et al 1989). Tia and Tic are similar except that gly55 of Tia is replaced by glu55 in Tic. However, Tib is quite different from others with as many as 8 different amino acids, which includes conservative as well as non-conservative replacements. In the present case, glu12 of Tia is replaced by asp12 that is common in the homologous protein series but asn13 to ser13 is a rare substitution. Thus, the cloned gene may represent for a mutant KTi3 gene. It may also be likely that it may represent for a new member gene of KTi family. However, the functional properties of the cloned SBTI remained unaltered as revealed through trypsin inhibition activity of SBTI purified from the transgenic tobacco plants.
3.4 Construction of expression vectors and transformation of tobacco plant
The SBTI gene was cloned into a plant expression vector, pBI121 (Jefferson et al 1987) at BamHI and SacI restriction sites to generate pSBIN. The chimeric gene construct was composed of the SBTI gene fused with CaMV 35S promoter and the nos polyadenylation terminator sequences. The recombinant vector, pSBIN was then transformed into A. tumefaciens (strain LBA4404) and used for plant transformation. Tobacco leaf discs were transfected with A. tumefaciens strain containing the pSBIN. Plantlets were regenerated on MS medium (Murashige and Skoog 1962) containing 2 mg/l IAA, 8 mg/l kinetin and 50 mg/l kanamycin. Regenerated plantlets were transferred to 1/2 MS for rooting and finally transferred to soil.
3.5 Analysis of transgenic plants for the presence and expression of SBTI gene
The presence of the transferred SBTI gene in the tobacco genome was detected by Southern blot analysis. Genomic DNA (5 m g) from different transgenic plants were digested by restriction enzyme EcoRV and probed with [a 32P]dATP labelled SBTI DNA. There exists only one restriction site for EcoRV in the pSBIN vector construct outside the coding sequences of SBTI. Thus the number of bands in the autoradiogram (figure 4) indicated the number of independent insertion sites of the transferred SBTI gene within the tobacco genome, which varied from one to five.
Expression of the transferred SBTI gene was determined by Western blot analysis. Fresh leaves of transgenic tobacco plants were extracted with trypsin inhibitor extraction buffer. The extracted leaf protein (5·0 m g) was run on a 15% SDS-PAGE. The proteins separated in the gel were transferred to nitrocellulose membrane through electro-transfer. Detection of SBTI specific band was carried out with the help of polyclonal anti-SBTI rabbit antibody and anti-rabbit IgG goat antibody linked with horse radish peroxidase, used as primary and secondary antibodies, respectively. Western blots of five individual transgenic plants showed (figure 5) variations in expression level of the transgene.
3.6 Purification, quantitation and functional activity assay of SBTI from transgenic plants
Level of expression of SBTI in the transgenic tobacco plants was estimated through indirect ELISA, after purifying through trypsin ligated CNBr+ sepharose beads. The amount of SBTI recovered from different transgenic plants varied from undetectable to 1% (w/w) of the total protein (table 1). However, tests carried out with known amount of pure SBTI when mixed with untransformed tobacco plant extracts, revealed that 3045% of the inhibitor could only be recovered following the above mentioned protocol. This reflected that the level of SBTI expression in the transgenic plants might be in certain cases as high as 3 to 5% of the total soluble protein.
Functional activity assay of the trypsin inhibitor recovered from transgenic plants was estimated on bovine trypsin as well as on H. armigera gut extract. Results (data not shown) indicated that SBTI recovered from soybean seeds and from leaves of transgenic plants were equally effective in reducing the proteolytic activity of bovine trypsin and the insect gut extracts.
3.7 Determination of free amino acids and amides content of tobacco leaves
The feeding assay experiment (described in the next section) with the larvae of H. armigera on the leaves of transgenic tobacco plants indicated that the insect growth is favoured by a certain component of the growing plants. To determine if the free amino acids and amides of the growing plants play any role in combating antimetabolite effect of trypsin inhibitor, the level of these compounds present in the tobacco leaves was estimated. Leaves were collected from top, middle and lower parts of young tobacco plants. Amount of total ninhydrin positive compound (NPC) present in the leaves was estimated by ninhydrin reaction method (Spies 1957). The level of NPC present was estimated to be 0·8 ± 0·04% (w/w) in the top leaves. The middle and lower leaves showed 0·4 ± 0·03% and 0·3 ± 0·03% (w/w) NPC contents, respectively. NPC present in the artificial diet of H. armigera was found to be 0·15%.
3.8 Insect feeding assay
Purified SBTI was added at concentrations of 0·05% and 0·1% (w/w) to the artificial diet of H. armigera. In each set of diet, three days old 30 larvae were reared. Similarly, three days old 30 larvae were fed on leaves of transgenic plants. Changes in body-weight of the larvae were monitored at three, five and seven days of incubation. Feeding assay on the leaves of highly expressive SBTI transgenic lines showed that there was hardly any difference in the body weight of the larvae from the ones grown on leaves of untransformed plants (figure 6). Analysis of variance showed (calculated F = 0·004 against the tabulated F(0·05, 2,12) = 3·38) that the observed differences in body weights were not significant. Development and fecundity of these insects remained unaffected. On the other hand, SBTI recovered from the transgenic plants when mixed at 0·05% (w/w) with artificial diet, caused (figure 6) significant reduction in body-weight (t = 12·75, against t(0·05, 58) = 2·0).
Insect feeding assay was also carried out with the addition of SBTI and casaminoacids. Purified SBTI from transgenic tobacco plants was added to the artificial diet at 0·05% and 0·1% (w/w), along with 0·4%, 0·8% and 1·6% (w/w) of casaminoacids. The results of the insect feeding assay have been summarized in figure 6 by showing the effect of SBTI at 0·05% with or without 0·4% and 0·8% of casaminoacids, as any further higher dosages of SBTI (0·1%) or casaminoacids (1·6%) were similar to that observed with lower dosages. The results demonstrated that toxicity of SBTI to insects could be reversed by supplementing casaminoacids in the artificial diet at an equivalent level (0·8%) that is present in the young tobacco leaves.
Insecticidal properties of protease inhibitors are
well established. Transgenic plants with constitutive expression of protease inhibitors
have shown in the past to be resistant to insect pests (Hilder et al 1986;
Johnson et al 1989, 1991; Duan et al 1996; Irie et al
1996). On the contrary, our findings indicated that the growth and development of H.
armigera larvae were unaffected when fed on the leaves of transgenic tobacco plants
expressing very high levels of SBTI. Purified SBTI extracted
from the transgenic plants when mixed with artificial diet caused significant reduction in growth of H. armi-gera larvae. The SBTI produced in transgenic plants
was functionally active, as evident from its effective-ness in reducing the proteolytic activity of insect gut extract.
The mode of action of the protease inhibitor as anti-metabolite is not fully understood. Direct inhibition of digestive enzymes is not considered to be the main effect. It has been conjectured (Pusztai et al 1992) that a more important factor might be depletion of essential amino acids caused due to the presence of the inhibitors. However, parallel suggestions like insects grown on transgenic plants can produce inhibitor resistant proteolytic enzymes in the body to overcome the effect of the anti-metabolite has also been put forward (Jongsma et al 1995). Transgenic tobacco plants with giant taro proteinase inhibitor (GTPI) gene failed to cause mortality to feeding larvae of H. armigera and this resistance was associated with the production of increased level of chymotrypsin, elastage and GPTI insensitive protease (Wu et al 1997) by the insects. In the context of the present study, this however, cannot explain the differential behaviour of the insects when fed on artificial diet and on transgenic plant leaves. It is observed that insect growth is somehow favoured when feeding on growing plant parts. Free amino acids and amides present in the growing plants perhaps help the insects to overcome the stress imposed by the presence of the anti-metabolite. Our results suggest that the high levels of free amino acids and amides present in the young tobacco leaves may be sufficient to sustain growth and development of the insects, even in the presence of SBTI. The artificial diet contains much less amount of free amino acids and amides that are not sufficient for the sustenance of the insects. Insects, belonging to the order hemiptera, are known to nourish on free amino acids and amides of plants (Sogowa 1970).
There is no report on the effectivity of SBTI towards the insect H. armigera. However, transgenic tobacco plants expressing CPTI have been shown to be resistant against this insect (Hilder et al 1986). The SBTI gene has been reported to be inhibitory to the brown plant hopper in transgenic rice plants (Lee et al 1999). In contrast to our findings, a recent report (Schuler et al 1998) indicates that the serine-proteinase inhibitor KTi3 from soybean resulted up to 100% mortality of first instar cotton leafworms (Spodoptera littoralis) when expressed in transgenic tobacco. However, the fact remains that no crop expressing proteinase inhibitor transgene(s) has yet been commercialized. Thus, the present findings and the earlier report (Jongsma et al 1995) points to the possibility that the trypsin inhi-bitors may not be suitable insecticidal agents for a transgenic approach for crop protection against insect damage.
Financial assistance in the form of a fellowship by the Council of Scientific and Industrial Research, New Delhi to AKN is gratefully acknowledged.
Birk Y 1961 Purification and some properties of a highly active inhibitor of trypsin and a-chymotrypsin from soybean; Biochem. Biophys. Acta 54 378380
Bradford M M 1976 A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of dye binding; Anal. Biochem. 72 248254
Broadway R M and Duffey S 1986 Plant protease inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Helicoverpa zea and Spodoptera exiqua; J. Insect Physiol. 32 827833
Duan X, Li X, Xue Q, Abo-el-Saad M, Xu D and Wu R 1996 Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant; Nature Biotechnol. 14 494498
Gatehouse A M R, Gatehouse J A and Boulter D 1980 Isolation and characterization of trypsin inhibitor from cowpea (Vigna unguiculata); Phytochemistry 19 751756
Gennis L S and Cantor C R 1976 Double headed protease inhibitors from black eyed peas III: subunit interaction of the native and half site chemically modified proteins; J. Biol. Chem. 251 747753
Green T and Ryan C A 1972 Wound induced proteinase inhibitor in plant leaves: a possible defense mechanism against insects; Science 175 776777
Hilder V A, Gatehouse A M R, Sheerman S E, Barker R F and Boulter D 1986 A novel mechanism of insect resistance engineered into tobacco; Nature (London) 330 160163
Hofgen R and Willmitzer L 1988 Storage of competent cell for Agrobacterium transformation; Nucleic Acids Res. 16 9877
Horsch R B, Fry J, Hoffman N L, Wallroth M, Echholtz D, Rogers S G and Fraley R T 1985 A simple and general method for transferring genes into plants; Science 227 12291231
Irie K, Hosoyama H, Takeuchi T, Iwabuchi K, Watanabe H, Abe M, Abe K and Arai S 1996 Transgenic rice established to express corn cystatin exhibits strong inhibitory against insect gut protease; Plant Mol. Biol. 30 149157
Jefferson R A, Kavanagh T A and Bevan M W 1987 Gus fusion: b -glucuronidase as a sensitive and versatile gene fusion marker in higher plants; EMBO J. 6 39013907
Jofuku K D and Goldberg R B 1989 Kunitz trypsin inhibitor gene are differentially expressed during the soybean life cycle and in transformed tobacco plants; Plant Cell 1 10791093
Jofuku K D, Schipper R D and Goldberg R B 1989 A frameshift mutation prevents Kunitz trypsin inhibitor mRNA accumulation in soybean embryos; Plant Cell 1 427435
Johnson R, Narvaez J, An G and Ryan C 1989 Expression of proteinase inhibitors I and II in transgenic tobacco plant: effects on natural defense against Manduca sexta larvae; Proc. Natl. Acad. Sci. USA 86 98719875
Johnston K A, Gatehouse J A and Anstee J H 1991 In vitro and in vivo studies of the effects of plant proteinase inhibitors on Helicoverpa armigera larvae; J. Exp. Bot. 42 238
Jongsma M A, Bakker P L, Peters J, Bosch D and Stiekema W 1995 Adaptation of Spodoptera exiqua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition; Proc. Natl. Acad. Sci. USA 92 80418045
Jotwani M G and Butani D K 1984 Insect pest of crops; in Handbook of agriculture (eds) P L Jaiswal and A M Wadhwani (New Delhi: ICAR) pp 417550
Kay E 1976 Origin of circular dichroism bands in BowmanBirk soybean trypsin inhibitor; J. Biol. Chem. 251 34113416
Kim S H, Hara S, Hase S, Ikenaka T, Toda H, Kitamura K and Kaizuma N 1985 Comparative study on amino acid sequences of Kunitz type soybean trypsin inhibitors, Tia, Tib and Tic; J. Biochem. 98 435448
Kunitz M 1947 Isolation of a crystalline protein compound of trypsin and soybean trypsin inhibitor; J. Gen. Physiol. 30 311320
Laemmli U K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4; Nature (London) 227 680685
Laskowski M and Sealock R W 1978 The Enzymes vol 3 (New York: Academic Press)
Lee S L, Lee S-Ho, Koo J C, Chun H J, Lim C O, Mun H J and Song Y H 1999 Soybean kunitz trypsin inhibitor confers resistance to the brown plant hopper in transgenic rice; Mol. Breeding 5 19
Malehorn D E, Borgmeyer J R, Smith C E and Shah D M 1994 Characterization and expression of an anti fungal zeamatin like protein from Zea mays; Plant Physiol. 106 1471
Murashige T and Skoog F 1962 A revised medium for rapid growth and bio-assays with tobacco tissue cultures; Physiol. Plant 15 473497
Purcell J P, Greenplate J T and Sammons R D 1992 Examination of midgut luminal proteinase activities in six economically important insects; Insect Biochem. Mol. Biol. 22 4147
Pusztai A, Grant G, Brown D J, Stewart J C, Bardocz S, Ewen
S W, Gatehouse A M and Hilder V 1992 Nutritional evaluation of the trypsin (EC 188.8.131.52) inhibitor from cowpea (Vigna unguiculata Walp.); Br. J. Nutr. 68 783791
Rogers S O and Benedich A J 1988 Extraction of DNA from plant tissues; in Plant molecular biology (eds) S B Gelvin,
R A Schilperoort and D P S Verma (Netherlands: Kluwer Academic Publishers) pp A6:110
Sambrook J, Fritsch E F and Maniatis T 1989 Molecular cloning; A laboratory manual 2nd edition (New York: Cold Spring Harbor Laboratory)
Sanger F, Nicklen S and Coulson A R 1977 DNA sequencing with chain terminator inhibitors; Proc. Natl. Acad. Sci. USA 74 54635466
Schuler T H, Poppy G M, Kerry B R and Denholm I 1998 Insect resistant transgenic plants; Trend. Biotech. 16 168175
Shagger H and Jagow G V 1987 Tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis for the separation of proteins in range from 1 to 100 kDa; Anal. Biochem. 166 368379
Spies R J 1957 Colorimetric procedures for aminoacids; Methods Enzymol. 3 467477
Sogawa K 1970 Studies on feeding habits of the brown plant hopper I. Effects of nitrogen deficiency of host plant insect feeding; Jpn. J. Appl. Entomol. Zool. 14 101106
Southern E M 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis; J. Mol. Biol. 96 503
Towbin H, Staehelin T and Goedon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; Proc. Natl. Acad. Sci. USA 76 43504354
Wei C H 1983 Crystalization of two cubic forms of soybean trypsin inhibitor E-1, a member of BowmanBirk family; J. Biol. Chem. 258 93579359
Wu Y, Llewellyn D, Mathews A and Dennis E S 1997 Adaptation of Helicoverpa armigera to a proteinase inhibitor expressed in transgenic tobacco; Mol. Breeding 3 371380
Xu D, Xue Q, McElroy D, Mawal Y, Hilder V A and Wu R 1996 Constitutive expression of cowpea trypsin inhibitor gene CPTI in transgenic rice plants confer resistance to two major rice insect pests; Mol. Breeding 2 167173
MS received 15 October 1998; accepted 16 August 1999
Corresponding editor: Rakesh Tuli
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