Trypsin Inhibitor Gene Research Articles

Assessment of rhizospheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibitor (CpTI) gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1:10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem.

Identification and Quantification of Three Genetically Modified Insect Resistant Cotton Lines Using Conventional and TaqMan Real-Time Polymerase Chain Reaction Methods

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

Molecular Population Genetics of Herbivore-induced Protease Inhibitor Genes in European Aspen (Populus tremula L., Salicaceae)

Plants defend themselves against the attack of natural enemies by using an array of both constitutively expressed and induced defenses. Long-lived woody perennials are overrepresented among plant species that show strong induced defense responses, whereas annual plants and crop species are underrepresented. However, most studies of plant defense genes have been performed on annual or short-lived perennial weeds or crop species. Here I use molecular population genetic methods to survey six wound-inducible protease inhibitors (PIs) in a long-lived woody, perennial plant species, the European aspen (Populus tremula), to evaluate the likelihood of either recurrent selective sweeps or balancing selection maintaining amino acid polymorphisms in these genes. The results show that none of the six PI genes have reduced diversities at synonymous sites, as would be expected in the presence of recurrent selective sweeps. However, several genes show some evidence of nonneutral evolution such as enhanced linkage disequilibrium and a large number of high-frequency-derived mutations. A group of at least four Kunitz trypsin inhibitor genes appear to have experienced elevated levels of nonsynonymous substitutions, indicating allelic turnover on an evolutionary timescale. One gene, TI1, has enhanced levels of intraspecific polymorphism at nonsynonymous sites and also has an unusual haplotype structure characterized by two divergent haplotypes occurring at roughly equal frequencies in the sample. One haplotype has very low levels of intraallelic nucleotide diversity, whereas the other haplotype has levels of diversity comparable to other genes in P. tremula. Patterns of sequence diversity at TI1 do not fit a simple model of either balancing selection or recurrent selective sweeps. This suggests that selection at TI1 is more complex, possibly involving allelic cycling.

Pancreatic Secretory Trypsin Inhibitor (SPINK1) Gene Mutations in Patients With Acute Pancreatitis

Mutations in the secretory trypsin inhibitor (SPINK1) gene have been found to be associated with hereditary and chronic pancreatitis. There are no previous reports on SPINK1 mutations in patients with acute pancreatitis (AP). The study population consists of 371 patients with AP, of which 207 patients had mild and 164 had a severe form of the disease. The etiologies of AP were identified. Four hundred fifty-nine blood donors served as controls. SPINK1 N34S and P55S mutations were detected by minisequencing and confirmed by direct sequencing. The N34S mutation was found in 29 (7.8%) of the patients and in 12 (2.6%) of the controls (P < 0.0001, Fisher exact test). There was no difference in the frequency of the P55SS mutation between the groups. A majority of the patients (n = 229; 61.7%) had alcohol-induced AP. The frequency of the N34S mutation was higher in the subgroups of severe AP (15/164; 9.1%) and alcohol-induced AP (21/229; 9.2%), but the differences were not statistically significant. No differences in age at admission and number of attacks of AP were observed between the groups. SPINK1 N34S mutation enhances the susceptibility of AP.

Reduced CFTR Function and the Pathobiology of Idiopathic Pancreatitis

Idiopathic chronic pancreatitis (ICP) is the leading cause of chronic pancreatitis in children and nonalcoholic adults. The risk of developing ICP is increased in individuals who have mutations of the cystic fibrosis gene (CFTR) and of a trypsin inhibitor gene (PSTI). In studies from the United States and France, the risk of ICP is increased about 40-fold by having two abnormal copies of the CFTR gene, about 14-fold by having the N34S PSTI mutation, and about 500-fold by having both. When ICP patients have two abnormal copies of the CFTR gene, there is also evidence of reduced residual CFTR protein function in extrapancreatic tissues based on clinical findings and nasal ion transport responses. Thus, pancreatitis risk is highest in individuals who have abnormalities in both the pancreatic ducts (CFTR) and acini (PSTI). These findings indicate that PSTI is a modifier gene for CFTR-related ICP and have implications for the diagnosis and pathogenesis of pancreatitis.

Screening of a Peanut (Arachis hypogaea L.) cDNA Library To Isolate a Bowman−Birk Trypsin Inhibitor Clone

Peanut crop losses due to insect and pest infestation cost peanut farmers nearly 20% of their annual yields. The conventional use of chemicals to combat this problem is costly and toxic to humans and livestock and leads to the development of resistance by target insects. Transgenic plants expressing a trypsin inhibitor gene in tobacco and cowpea have proven to be efficient for resistance against insects. Therefore, a transgenic peanut overexpressing a trypsin inhibitor gene could be an alternative solution to the use of toxic chemicals. Five Bowman-Birk trypsin inhibitor (BBTI) proteins were previously isolated from peanut. However, to date, neither cDNA nor genomic DNA sequences are available. The objective of this research was to screen a peanut cDNA library to isolate and sequence at least one full-length peanut BBTI cDNA clone. Two heterologous oligonucleotides were constructed on the basis of a garden pea (Pisum sativa) trypsin inhibitor nucleotide sequence and used as probes to screen a peanut lambda gt-11 cDNA library. Two positive and identical cDNA clones were isolated, subcloned into a pBluescript vector, and sequenced. Sequence analysis revealed a full-length BBTI cDNA of about 243 bp, with a start codon ATG at position +1 and a stop codon TGA at position +243. In the 3' end, two poly adenylation signals (AATAAA) were identified at positions +261 and +269. The isolated cDNA clone encodes a protein of 80 amino acid residues including a leader sequence of 11 amino acids. The deduced amino acid sequence is 100% identical to published sequences of peanut BBTI AI, AII, BI, and BIII and 81% identical to BII.

Expression of the soybean (Kunitz) trypsin inhibitor in leaves of white clover ( Trifolium repens L.)

The coding region of the Ti a allelic form of the soybean (Kunitz) trypsin inhibitor (SBTI) gene ( KTi3) has been introduced, as a transcriptional fusion with the CaMV 35S promoter, into white clover ( Trifolium repens L.). Southern analysis of DNA extracted from primary transformants confirmed that an intact copy (or copies) of the gene is integrated into the white clover genome. Gel filtration column chromatography of transgenic leaf tissue revealed a higher molecular mass (ca. 22 kDa) trypsin inhibitory fraction, and a lower molecular mass (ca. 15 kDa) trypsin inhibitory fraction. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using an anti-SBTI monoclonal antibody (MAb) determined that SBTI occurred in the higher molecular mass fraction as a protein of ca. 22 kDa. Gel filtration column chromatography revealed that the lower molecular mass trypsin inhibitory fraction also occurred in non-transgenic leaf tissue. Further purification of the SBTI inhibitory fraction using reverse-phase column chromatography revealed two major trypsin inhibitory peaks, only one of which was recognised by the SBTI MAb. N-terminal sequencing confirmed that this antibody-recognised inhibitor is the KTi3 gene product, and the protein is processed by the removal of the N-terminal leader sequence. N-terminal sequencing of the major protein in the second reverse-phase trypsin inhibitory fraction produced no matches to any inhibitors in the database. Further purification of the lower molecular mass trypsin inhibitory protein using reverse-phase chromatography from either transgenic or non-transgenic leaf tissue revealed a single trypsin inhibitory fraction (designated WCTI-1). N-terminal sequencing produced a match with non-specific lipid transfer proteins from higher plants. Kinetic analysis of WCTI-1 determined an equilibrium constant ( K I value) of 1.2 × 10 −8 M for trypsin, and showed that the inhibitor was effective against trypsin-like activity of black field cricket ( Teleogryllus commodus) and grass grub ( Costelytra zealandica), but ineffective against trypsin-like activity from light brown apple moth ( Epiphyas postvittana) and porina ( Wiseana copularis).

25 Contribution of the pancreatic secretory trypsin inhibitor gene (SPINK1) to the etiology of CFTR-related recurrent pancreatitis

25 Contribution of the pancreatic secretory trypsin inhibitor gene (SPINK1) to the etiology of CFTR-related recurrent pancreatitis

Phylogenetic analysis of Theobroma (Sterculiaceae) based on Kunitz-like trypsin inhibitor sequences

Trypsin inhibitor gene sequences were used to investigate the phylogenetic relationships among Theobroma and Herrania species, considered as sister-groups, with particular interest on the monophyly and infra-generic relationships of Theobroma. The presumed amino acid sequences of 23 analyzed samples, from 11 Theobroma and three Herrania species, comprising all sections from both genera, demonstrated a high similarity with a previously characterized T. cacao Kunitz trypsin inhibitor. The trypsin inhibitor gene accumulated mutations at faster rate than prior analyzed nuclear or chloroplastic genes. None of the sequences presented introns. The phylogeny of the trypsin inhibitor sequences was congruent with the phylogenetic hypotheses of the Theobroma and Herrania species based on morphology. The monophyly of Theobroma was not strongly supported, corroborating previously described absence of obvious synapomorphies for Theobroma. The species grouped consistently according to genus and section. The monophyly of all Theobroma sections was supported, except for section Glossopetalum, which was paraphyletic to section Andropetalum. Evidences sustain that T. mammosum may be included into section Glossopetalum. The potential use of trypsin inhibitor gene sequences in phylogenetic studies of Theobroma was demonstrated.

Insect resistance to Nilaparvata lugens and Cnaphalocrocis medinalis in transgenic indica rice and the inheritance of gna+sbti transgenes

Molecular genetic analysis and insect bioassay of transgenic indica rice 'Zhuxian B' plants carrying snowdrop lectin gene (gna) and soybean trypsin inhibitor gene (sbti) were investigated in detail. PCR, 'dot' blot and PCR-Southern blot analysis showed that both transgenes had been incorporated into the rice genome and transmitted up to R3 progeny in most lines tested. Some transgenic lines exhibited Mendelian segregation, but the other showed either 1:1 (positive: negative for the transgenes) or other aberrant segregation patterns. The segregation patterns of gna gene crossed between R2 and R3 progeny. In half of transgenic R3 lines, gna and sbti transgenes co-segregated. Two independent homozygous lines expressing double transgenes were identified in R3 progeny. Southern blot analysis demonstrated that the copy numbers of integrated gna and sbti transgenes varied from one to ten in different lines. Insect bioassay data showed that most transgenic plants had better resistance to both Nilaparvata lugens (Stahl) and Cnaphalocrocis medinalis (Guenee) than wild-type plants. The insect resistance of transgenic lines increased with the increase in transgene positive ratio in most of the transgenic lines. In all, we obtained nine lines of R3 transgenic plants, including one pure line, which had better resistance to both N lugens and C medinalis than wild-type plants.

Two novel severe mutations in the pancreatic secretory trypsin inhibitor gene (SPINK1) cause familial and/or hereditary pancreatitis.

Mutations in the serine protease inhibitor Kazal type 1 gene (SPINK1) encoding pancreatic secretory trypsin inhibitor (PSTI) have recently been found to be associated with chronic pancreatitis. Nevertheless, knowledge of severe mutations is particularly scarce, both in terms of number and in the extent of clinical information. The aim of this study was to expand the known spectrum of such mutations. 46 unrelated families, each including at least two pancreatitis patients and carrying neither cationic trypsinogen (PRSS1) mutations nor the frequent SPINK1 N34S mutation, participated in this study. The four exons and their flanking sequences of the SPINK1 gene were screened by denaturing high performance liquid chromatography analysis (DHPLC); and mutations were identified by direct sequencing. A heterozygous microdeletion mutation (c.27delC), which occurs within a symmetric element, was identified in two families. In one family, c.27delC showed segregation with the disease across two generations, with a penetrance of up to 75%. But in the other family, however, the same mutation manifested as a low-penetrance susceptibility factor. In addition, a novel heterozygous splicing mutation, c.87+1G>A (G>A substitution at nucleotide +1 of intron 2) was found in one family with familial pancreatitis. Our results also helped to resolve the sharply differing views about PSTI's role in pancreatitis.

Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata.

Research into the molecular basis of plant-insect interactions is hampered by the inability to alter the expression of individual genes in plants growing under natural conditions. The ability of virus-induced gene silencing (VIGS) to silence the expression of two jasmonate-induced genes known to mediate the expression of two potent direct defences (nicotine and proteinase inhibitors) that are produced in different tissues (roots and shoots, respectively) in Nicotiana attenuata is documented here. Fragments of consensus sequences of N. attenuata's putrescine N-methyltransferase (PMT) and trypsin inhibitor (TI) genes were cloned in sense, anti-sense and inverted repeat orientations into the Tobacco Rattle Virus (TRV) to trigger post-transcriptional gene silencing by Agrobacterium-mediated inoculation in plants previously elicited with methyl jasmonate (MeJA) or left as controls. MeJA treatment elicited 2.4- and 9.8-fold increases in the concentrations of nicotine and proteinase inhibitors, respectively, and inoculation with constructs containing appropriate genes inhibited these MeJA-induced increases and halved constitutive accumulations, regardless of the orientation of the gene fragment. Root PMT transcript levels were significantly elevated in MeJA-treated plants 10 h after elicitation, but not in plants inoculated with the appropriate TRV constructs 9 d prior to MeJA treatment, demonstrating that VIGS was responsible for the inhibition of these potent direct defences. While additional research is required to minimize the effects on plant growth and the risks of using such constructs in natural settings, it is concluded that VIGS has a potential to manipulate the expression of genes important for ecological interactions.

Significance of trypsin inhibitor gene mutation in the predisposition to pancreatitis

Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed a hypothesis: if the function of PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis, and performed mutational analysis of PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were suggested to be associated with the predisposition to pancreatitis. N34S mutation was co-segregated with two intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the function of recombinant N34S protein, we could not demonstrate the loss of function of this protein. Intronic mutations rather than N34S itself (IVS1-37T>C+N34S+IVS3-69insTTTT complex) may be associated with the decreased function of PSTI. Alternatively, increased digestion of N34S in vivo may be applicable. As for R67C, the conformational alteration of the protein by forming intramolecular or intermolecular disulfide bonds with 67Cys was strongly suggested. These results, along with the brand new findings in PSTI knockout mice, suggest that genetic mutation of PSTI is one of the important mechanisms for predisposition to pancreatitis by lowering the trypsin inhibitory function.

Generation of pancreatic secretory trypsin inhibitor gene knockout mice

Pancreatic secretory trypsin inhibitor (PSTI) is synthesized in the acinar cells of the pancreas and acts as a potent protease inhibitor that inhibits intrapancreatic activation of trypsin to prevent the occurrence of pancreatitis. Recently, we and other groups reported that some patients with chronic pancreatitis had a point mutation in the PSTI gene. Genetic mutations in the PSTI gene seem to promote a predisposition to pancreatitis, possibly by lowering the threshold of pancreatitis caused by other factors. In order to prove this hypothesis, we generated PSTI knockout mice using cre/mutant lox system. Heterozygous offsprings of chimeras appeared entirely normal.

Mutational analysis of the pancreatic secretory trypsin inhibitor gene in familial and juvenile pancreatitis in Japan.

Mutations in the pancreatic secretory trypsin inhibitor ( PSTI) gene have been reported in patients suffering from chronic pancreatitis. The aim of the present study was to investigate whether similar gene mutations are present in familial and juvenile pancreatitis patients in Japan. All four exons of the PSTI gene and their flanking intronic sequences were amplified by polymerase chain reaction and sequenced for 37 familial pancreatitis patients (24 families) and 15 juvenile pancreatitis patients, distributed throughout Japan. Three types of exonic mutation in the PSTI gene were observed. The N34S mutation was found in six familial pancreatitis patients (three families) and in one juvenile pancreatitis patient, and the R67C mutation was found in one familial pancreatitis patient and one juvenile pancreatitis patient. We also found a 272C>T mutation in the 3' untranslated region of exon 4 in one familial pancreatitis patient and four juvenile pancreatitis patients. It should be noted that the N34S mutation was cosegregated with two intronic mutations, specifically, IVS1-37T>C and IVS3-69insTTTT. The same set of N34S mutations (N34S + IVS1-37T>C + IVS3-69insTTTT) that exists in other countries was found in the PSTI gene in Japanese familial and juvenile pancreatitis patients. Another unique mutation (R67C) was also observed in two patients; 272C>T was suggested to be a normal polymorphism.

Mutations in the pancreatic secretory trypsin inhibitor (SPINKI1/PSTI) gene in Japanese patients with pancreatitis

Mutations in the pancreatic secretory trypsin inhibitor (SPINKI1/PSTI) gene in Japanese patients with pancreatitis

Cationic trypsinogen and pancreatic secretory trypsin inhibitor gene mutations in neonatal hypertrypsinaemia

Neonatal hypertrypsinaemia with normal sweat chloride detected during CF screening may be related to trypsin activation. We have looked for mutations of the cationic trypsinogen (PRSS1) and pancreatic secretory trypsin inhibitor (PSTI) genes in 50 hypertrypsinaemic neonates with known CFTR genotypes and negative sweat test. No mutations were found in either gene. Two silent polymorphisms were detected in the PRSS1 gene. A polymorphism in the promoter region and an intronic polymorphism of the PSTI gene were found. No difference was observed in the frequency of PRSS1 or PSTI polymorphisms in neonates carrying or not carrying CF mutations. These results do not provide an indication for an increased frequency of mutations in the PRSS1 and PSTI genes in this group of neonates with transient hypertrypsinaemia.

Motion-Genetic Testing is Useful in the Diagnosis of Nonhereditary Pancreatic Conditions: Arguments Against the Motion

Mutations of two genes, the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the pancreatic secretory trypsin inhibitor gene (PSTI), are associated with an increase in the risk of idiopathic chronic pancreatitis. Persons who have mutations of both CFTR alleles (one severely and one mildly affected) are especially susceptible to this disease. Because these compound heterozygotes have sufficient residual CFTR function, they do not develop cystic fibrosis lung disease. One PSTI mutation, N34S, independently increases the risk of pancreatitis. Thus, the risk of pancreatitis is greatest among individuals who are CFTR compound heterozygotes and who also have the PSTI mutation. Nonetheless, most people with CFTR and PSTI mutations do not develop pancreatitis. This fact indicates that environmental influences and gene-gene interactions also affect pancreatitis risk. Although CFTR and PSTI genetic testing can identify persons at an increased risk of pancreatitis, there are several reasons why the routine screening of individuals with nonhereditary pancreatitis is not recommended at this time: most disease-associated mutations are not detected by readily available techniques, genetic counselling guidelines do not exist, most patients with mutations do not develop pancreatitis and the results of testing do not affect the clinical management of pancreatitis.

Functional analysis of recombinant pancreatic secretory trypsin inhibitor protein with amino-acid substitution.

We hypothesized that mutation of the pancreatic secretory trypsin inhibitor ( PSTI) gene may promote a predisposition to pancreatitis, possibly by reducing the inhibition of trypsin activity. Based on this hypothesis, we performed a biochemical analysis of recombinant PSTI protein. The trypsin inhibitory activity of the recombinant protein was analyzed. The activity of PSTI protein with a point mutation of the most common type: (34)Asn (AAT)-to-Ser (AGT)(101A>G N34S: N34S) in exon 3, was compared with that of the wild type. The function of N34S PSTI remained unchanged under both the usual alkaline and acidic conditions compared with the wild-type PSTI. The calcium concentration did not affect the activity of recombinant PSTI. The trypsin susceptibility of the N34S protein was not increased either. Mechanisms other than the conformational change of PSTI associated with amino-acid substitution, such as abnormal splicing, may underlie the predisposition to pancreatitis in patients with the N34S mutation.

Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor: CHARACTERIZATION, POST-TRANSLATIONAL PROCESSING STUDIES, AND TRANSGENIC EXPRESSION TO DEVELOP INSECT-RESISTANT PLANTS

A number of trypsin inhibitor (TI) genes have been used to generate insect-resistant plants. Here we report a novel trypsin inhibitor from Indian mustard Brassica juncea (BjTI) that is unique in being the precursor of a 2S seed storage protein. The inhibitory activity is lost upon processing. The predicted amino acid sequence of the precursor based on the B. juncea 2S albumin (Bj2S) gene cloned and sequenced in this laboratory (Bj2Sc; GenBank(TM) accession number ) showed a soybean-TI active site-like motif GPFRI at the expected processing site. The BjTI was found to be a thermostable Kunitz type TI that inhibits trypsin at a molar ratio of 1:1. The 20-kDa BjTI was purified from midmature seeds and found to be processed in vitro to 9- and 4-kDa subunits upon incubation with seed extract. The Bj2Sc sequence was expressed in Escherichia coli pET systems as the inhibitor precursor. The radiolabeled gene product was expressed in vitro in a coupled transcription-translation system and showed the expected processing into subunits. Two in vitro expressed pre-2S proteins, mutated at Gly and Asp residues, were processed normally to mature subunits, showing thereby no absolute requirement of Gly and Asp residues for processing. Finally, the 2S gene was introduced into tobacco and tomato plants. Third generation transgenics expressing BjTI at 0.28-0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm, Spodoptera litura. This novel TI can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption.