Cowpea Trypsin Inhibitor Research Articles

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.

Comparison of nutrient composition of parental rice and rice genetically modified with cowpea trypsin inhibitor in China

In order to compare the nutritional composition of parental rice and rice genetically modified (GM) with cowpea trypsin inhibitor (GM rice) that has been developed in China, the nutrient content (moisture, protein and amino acids, fat and carbohydrate, dietary fiber, ash, elements and vitamins) of parental and GM rice was analyzed and compared, using Chinese national standard methods or AOAC methods. The results showed that, except for small differences in fat and fatty acids, there was no significant difference between these two types of rice with respect to most of their nutrient composition. Therefore, we draw a conclusion from this preliminary comparison that the nutritional composition of this GM rice does not change with the insertion of a foreign gene. However, it should undergo further analyses to conclude whether it can meet the requirement of “substantial equivalence” in nutrient composition.

Feeding behaviour of Helicoverpa armigera larvae on insect‐resistant transgenic cotton and non‐transgenic cotton

Abstract: Feeding behaviour of Helicoverpa armigera Hübner (Lep.; Noctuidae) larvae on non‐transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.), Zhong 30, and transgenic cowpea trypsin inhibitor (CpTI)‐Bt cotton, SGK 321, and non‐transgenic cotton, Shiyuan 321, was investigated in both choice tests and no‐choice tests. The results of choice tests suggested that neonates have the ability to detect and avoid transgenic cotton. In the choice tests of neonates with both transgenic and non‐transgenic cotton leaves, a significantly greater proportion of larvae and higher consumption were observed on non‐transgenic cotton than on the transgenic Bt or CpTI‐Bt cotton. In the choice tests with leaves of two transgenic cotton lines, the proportion of neonates on leaf discs of the two lines was not significantly different, but there was significantly higher consumption on CpTI‐Bt transgenic cotton than that on Bt transgenic cotton. In addition, significantly more neonates were found away from the leaf discs, lower consumption and higher mortality were achieved in the choice test with two transgenic cotton leaves than in the choice tests containing non‐transgenic cotton leaves. Leaves and buds were examined in choice tests of fourth instars. It appeared that fourth instars were found in equal numbers on transgenic and non‐transgenic cotton, except when larvae were exposed to leaves for 3 h. However, the total consumption on transgenic cotton was lower than that of the non‐transgenic cotton, so fourth instars may still have the capacity to detect transgenic cotton and reduce feeding on it, although they showed no preference on either transgenic or non‐transgenic cotton. More larvae were found off diet in the treatments with leaves than that of buds, and the number of injured leaf discs by per fourth instar was significantly higher than that of buds in choice tests, suggesting that leaf is a less preferred organ for H. armigera larvae, elicited more larval movements. Similarly, in no‐choice tests of fifth instars, significantly fewer feeding time and more moving time occurred on leaf than that of bud, boll and petal. When cotton line was considered, compared with non‐transgenic cotton, significantly lower feeding time and higher resting time occurred on the two transgenic cottons. Overall, H. armigera larvae have the ability to detect the transgenic Bt and CpTI‐Bt cottons or the less preferred organs and selectively feed more on the non‐transgenic cotton or the preferred organs, especially the neonates, which have a high capacity for avoiding transgenic cotton.

Impact of Genetically Modified Potato Expressing Plant-Derived Insect Resistance Genes on the Predatory Bug Podisus maculiventris (Heteroptera: Pentatomidae)

The effects of the transgene proteins Galanthus nivalis agglutinin (GNA) and cowpea trypsin inhibitor (CpTI) on the predatory stinkbug Podisus maculiventris were studied under laboratory conditions. When the P. maculiventris were provided with tomato moth (L. oleracea) larvae injected with either GNA or CpTI at a dose of 10 μg day−1, growth was significantly reduced and, as a consequence, female adult weight was also significantly reduced. For GNA-fed and CpTI-fed P. maculiventris, this weight reduction was 11.3 and 16.6%, respectively. Males, however, were not significantly affected. Female bugs that had not been exposed to the transgene proteins as nymphs, however, showed no reduction in fecundity when these adults were provided with prey injected with either GNA or CpTI at this same dose. When provided with hosts that had been reared on transgenic plants expressing either GNA or CpTI, no effects on the survival of nymphs were observed and only small, largely non-significant, reductions in weights were recorded throughout preadult development. Male nymphs fed on the GNA-fed prey did, however, exhibit a significant lengthening of preadult development of 0.8 days. The subsequent adults showed significantly reduced egg production for the GNA treatment. The results indicate that P. maculiventris may suffer some indirect adverse effects from foraging for prey in crops expressing either GNA or CpTI, due to prey being of inferior quality, rather than to direct toxicity of the transgene products themselves.

Seasonal abundance of the mirids, Lygus lucorum and Adelphocoris spp . (Hemiptera: Miridae) on Bt cotton in northern China

Lygus lucorum Meyer-Dür, Adelphocoris fasciaticollis Reuter and Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) are important secondary insect pests in cotton fields in northern China. The seasonal dynamics of their mixed populations on a transgenic variety expressing the insecticidal Bt protein Cry1A, and a cotton line expressing proteins of Cry1A and CpTI (cowpea trypsin inhibitor gene) were compared to seasonal dynamics on similar but non-transgenic varieties from 1998 to 2001. No significant differences were detected between population densities of these bugs on unsprayed normal cotton and unsprayed transgenic cotton. However, mirid damage on unsprayed transgenic cotton was significantly higher due to a reduced number of insecticide sprays against Helicoverpa armigera compared with the number of sprays in the normal cotton. This suggests that the mirids have become key insect pests in transgenic cotton fields, and that their damage to cotton could increase further with the expansion of the area planted to transgenic cotton if no additional control measures are adopted.

The effect of genetic transformations for pest resistance on foliar solanidine‐based glycoalkaloids of potato (Solatium tuberosuni)

SummaryFoliage of potato cv. Desiree was harvested from glasshouse‐cultivated plants of five experimental transgenic lines expressing three different insecticidal proteins (snowdrop lectin, Galanthus nivalis agglutinin (GNA); jackbean lectin, Concanavalin A (Con A), cowpea trypsin inhibitor; (CpTi)), tissue‐cultured control plants and standard control (non‐tissue cultured) plants. The foliage was subdivided into stems, upper, middle and lower leaves and analysed separately by HPLC for the solanidine‐based glycoalkaloids a‐solanine and a‐chaconine. The results demonstrate that one or more stages in the plant transformation process (i.e. insecticidal‐ and marker‐gene insertions, gene expression and tissue culture) resulted in a lower level of leaf glycoalkaloids than that found in either the tissue‐cultured controls or standard controls, based on the selected potato lines transformed for insecticidal protein expression. However, the distribution of glycoalkaloids throughout the plant foliage was unaffected by genetic transformation and tissue culture, with the highest glycoalkaloid levels being observed in the top third of the plant. The importance of investigating unexpected effects of genetic engineering on plant secondary metabolism is discussed from an ecological viewpoint.

The effect of the Cowpea trypspin inhibitor in strawberry on damage by vine weevil under field conditions

SummaryTransgenic strawberry lines constitutively expressing the Cowpea trypsin inhibitor (CpTi) were examined under field conditions to determine if this gene offered protection against the feeding of vine weevil (Otiorhynchus sulcatus F.). Data are presented on plant performance over three seasons (years). Field results for two years confirmed previous glasshouse findings and demonstrate protection in terms of attack by vine weevil larvae on all the transgenic lines. Over three years, two ‘Melody’ lines and three ‘Symphony’ lines continued to demonstrate significantly improved plant growth compared with control lines. The genotype into which CpTi was inserted had a significant effect on performance. The CpTi has a significant effect on vine weevil in terms of a reduction in the number of pupae. The numbers of Carabid and other non-target arthropods were assessed over the duration of the trial, and were found not to be significantly affected by the CpTi transgenic lines.

Effects of two kinds of transgenic poplar on protective enzymes system in the midgut of larvae of American white moth

The leaves of Bt (Bacillus thuringiensis) transgenic poplar (Populus nigra L.) and CpTI (Cowpea trypsin inhibitor) transgenic poplar ((P. tomentosa×P. bolleana)×P. Tomentosa) were taken to feed the 4th-5th-instar larvae of American white moth (Hyphantria cunea (Drury)) for determination of the activities of the protective enzyme system inside larvae's body. The physiological and biochemical effects of the transgenic poplars on the larvae were studied. The results showed that the two kinds of transgenic poplars had similar effects on the protective enzyme system in the midgut of larvae. The activities of superoxide dismutase, catalase, and peroxidase in midgut of the larvae increased gradually, reached the highest value at a certain time, and then decreased suddenly. For the larvae that were fed with the leaves of Bt transgenic poplar, the peak value of superoxide dismutase and catalase presented at the time of 24-h feeding, while the peak of peroxidase took place at the time of 12-h feeding. The activities of these protective enzymes for the larvae that were fed with leaves of CpTI transgenic poplar peaked 12 h later than that of those fed with leaves of Bt transgenic poplar. The comparison of activities of the protective enzymes was also carried out between the larvae with different levels of intoxication. It was found that the activities of protective enzyme of the seriously intoxicant larvae were higher than that of the lightly intoxicant larvae. This difference was more obvious in the group treated with CpTI transgenic poplar.

Nucleotide sequence of a genomic clone encoding a cowpea (Vigna unguiculata L.) trypsin inhibitor

A protease inhibitor gene has been isolated from native cowpea ( Vigna unguiculata L.) cv. 130 from l-ZAP II genomic library. The nucleotide sequence of this genomic clone shared 86% homology with a cowpea trypsin inhibitor f IV mRNA, and 81% with Bowman-Birk protease inhibitor genes of soybean. The isolated gene had TATA and CAT signals in the 5' upstream region. The longest open reading frame had 504 bases, encoding 167 amino acids from the predicted coding region. The active serine centers of this protein was found between 105-158 amino acid residues, with a 69 amino acids long signal peptide in its N terminal.

Effect of dietary cowpea trypsin inhibitor (CpTI) on the growth and development of the tomato mothLacanobia oleracea (Lepidoptera: Noctuidae) and on the success of the gregarious ectoparasitoidEulophus pennicornis (Hymenoptera: Eulophidae)

Cowpea trypsin inhibitor (CpTI) was shown to have a deleterious effect on the growth and development of larvae of the tomato moth, Lacanobia oleracea, when incorporated in artificial diet (2.0% of soluble protein) and expressed in transgenic potato leaf (up to 1.0% of soluble protein). The effect of CpTI on parasitism of L oleracea by the ectoparasitoid Eulophus pennicornis was investigated. The parasitic success of the wasp was reduced by the presence of CpTI in the diet of the host and, in the case of transgenic potato leaves expressing the transgene protein, was collated with the length of time the host fed on the diet prior to parasitism. In all cases the proportion of hosts parasitised when fed CpTI-containing diets was reduced when compared with controls, although these differences were only significant when hosts were fed from the third instar on the transgenic potato leaves. Parasitoid progeny that developed on L oleracea reared on CpTI-containing diets, however, were not adversely affected. These results show that, whilst expression of CpTI in transgenic potato plants confers resistance to the lepidopterous pest L oleracea, adverse effects on the ability of the ectoparasitoid E pennicornis to parasitise this moth species successfully may also occur. These results are discussed in relation to the potential impact of transgenic crops on beneficial biological control agents.

Transgenic resistance to the nematode Rotylenchulus reniformis conferred by Arabidopsis thaliana plants expressing proteinase inhibitors.

The level of resistance to the reniform nematode Rotylenchulus reniformis conferred on Arabidopsis thaliana by transgenic expression of proteinase inhibitors (PI) was studied over 40 days of infection. A cysteine PI differing from Oc-I expressed in rice seed by one amino acid (Oc-IΔD86) had a considerable effect. It reduced parasite density and fecundity to 65±4% and 31±8% respectively, relative to values for untransformed plants when expressed at ca. 0.4% total soluble protein. The overall reproductive success was only 19±5% relative to the controls. Similar expression levels of cowpea trypsin inhibitor, a serine PI, reduced the density of the females on the plants to 77±3% of control values but did not reduce their fecundity. There was no evidence for additive effects when both PIs were expressed as a fusion protein using a peptide linker. Both PIs in the fusion PI protein are known to be functional from our previous work. When developing on A. thaliana plants expressing the transgenic proteins the intact PI fusion protein of 22 kDa was detected in nematodes by western blot analysis. Similarly, R. reniformis females were observed to have ingested Green Fluorescent Protein (28 kDa). Demonstrating the ingestion of the fusion protein, which has been associated with additive effects of the two PIs against other nematodes, supports the observation that serine PIs are of less value than cysteine PIs for R. reniformis control. The effect of Oc-IΔD86 on R. reniformis was dependent on the level of expression of different A. thaliana lines in the range of 0.1–0.4% total soluble protein indicating that the upper level of expression is required for an effective defence. Virtual northern analysis with cysteine and serine proteinase gene probes from Heterodera glycines showed high transcript abundance of a serine proteinase in both pre-parasitic and parasitic stages whereas expression of the cysteine proteinase was more abundant in the feeding adult female. This study provides further evidence that cystatins represent a valuable source of control against a range of economic nematodes including Globodera, Heterodera and Meloidogyne and now including R. reniformis. Preliminary screening in A. thaliana provides a convenient guide for selection of transgenes against nematode pathogens of crops such as pineapple that have a prolonged regeneration period.

Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs.

Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-I delta D86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.

Impact of Proteins Used in Plant Genetic Engineering: Toxicity and Behavioral Study in the Honeybee

Genetically modified oilseed rape, Brassica napus L., expressing pest and fungi resistance has been assessed for its impact on the environment before field testing and placing on the market. The evaluation of rape lines for their inocuousness to beneficial insects such as the honeybee, Apis mellifera L., were investigated because changes in the composition of nectar and pollen may occur that affect plant-honeybee interactions. The effects of these plants on bees were evaluated by acute toxicity testing of the gene products and behavioral studies of bees. Acute toxicity was determined by evaluating the mortality over 24- and 48-h periods after ingestion or injection of the gene products. For the bee behavioral study, methods used to study learning processes and foraging behavior were adapted to test the effect of the gene products at both individual and colony levels under confined conditions. The study focused on the effects of proteins inducing resistance to pest insects, cowpea trypsin inhibitor (CpTI), and resistance to fungi, chitinase, and β-1,3 glucanase. Under our experimental conditions, the 3 proteins were shown to be nontoxic at the doses tested. At the individual level, behavior experiments, based on a conditioned proboscis extension response, had the following 3 effects depending on the protein tested; (1) chitinase did not affect learning performance; (2) β-1,3 glucanase affected the level of conditioned responses, with the extinction process occurring more rapidly as the concentration increased; and (3) CpTI induced marked effects in both conditioning and testing phases, especially in high concentrations. The decrease in learning performance induced by CpTI observed at the individual level was confirmed at the colony level. Given their relative simplicity, it is suggested that such bioassays could be used for screening in the development of genetically modified plants.

Characterization of cDNAs encoding serine proteinases from the soybean cyst nematode Heterodera glycines

Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226–296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.

The effect of the CpTi gene in strawberry against attack by vine weevil (Otiorhynchus sulcatus F. Coleoptera: Curculionidae)

SummaryThe degree of protection against insect feeding conferred upon transgenic strawberry lines expressing the Cowpea trypsin inhibitor was evaluated under glasshouse conditions. Insect bioassays were carried out using vine weevil (Otiorhynchus sulcatus) in two experiments and in both experiments there was a highly significant reduction in damage by weevil larvae on the transgenic lines.

Enhanced Resistance to West Indian Sweetpotato Weevil (Euscepes Postfasciatus) in Transgenic `Jewel' Sweetpotato with Cowpea Trypsin Inhibitor and Snowdrop Lectin

Euscepes postfasciatus is one of the most important sweetpotato pests in the South Pacific, Caribbean basin, and some countries of Central and South America. Development of host resistance will greatly improve the effects of integrated pest management (IPM) for this pest. Ten transgenic clones of `Jewel' sweetpotato with cowpea trypsin inhibitors and snowdrop lectin, developed by Axis Agri. Genetics, Ltd., were assayed for weevil resistance using a no-choice bioassay. A replicated experiment was conducted in the screenhouse. Five storage roots from each clone were infested with five pairs of adults. Non-transformed `Jewel' was used as a check. Resistance was assessed 60 days after infestation by estimating the percentage of internal damage and the weevil population in the storage roots. A five-grade damage index was recorded. The experiment was repeated twice. Significant enhancement of resistance was found in the transgenic clones. Clone CTI-13 with cowpea trypsin inhibitor and clone PCG-7 with both cowpea trypsin inhibitor and snowdrop lectin demonstrated moderate resistance to E. postfasciatus, whereas the non-transformed `Jewel' was susceptible. This result shows that resistance to Euscepes postfasciatus can be achieved through genetic transformation.

Testing transgenes for insect resistance using Arabidopsis

One possible strategy to delay the selection of resistant insect populations is the pyramiding of multiple resistance genes into a single cultivar. However, the transformation of most major crops remains prohibitively expensive if a large number of transgene combinations are to be evaluated. Arabidopsis thaliana is a potentially good plant for such preliminary evaluations. We determined that four major agricultural pests, Spodoptera exigua, Helicoverpa zea, Pseudoplusia includens, and Heliothis virescens grew as well when feeding on ‘Landsberg Erecta’ Arabidopsis as they did on plants of ‘Cobb’ soybean. Landsberg Erecta was then transformed with either a synthetic Bacillus thuringiensis cryIA(c) gene, or the cowpea trypsin inhibitor gene. Transformed plants were crossed to produce plants transgenic for both genes. Following quantification of transgene expression, the four caterpillar species were allowed to feed on wild-type plants, plants expressing either cryIA(c) or the cowpea trypsin inhibitor gene, or plants expressing both. Both genes reduced growth of the species tested, but cryIA(c) was more effective in controlling caterpillar growth than the cowpea trypsin inhibitor gene. The resistance of plants with both transgenes was lower than that of plants expressing the cryIA(c) gene alone, but higher than that of plants expressing the only the CpTI gene. This could be due to a lower concentration of Cry protein in the hemizygous F1 plants. Thus, if the cowpea trypsin inhibitor had any potentiation effect on cryIA(c), this effect was less than the cryIA(c) copy number effect. Alternatively, expression of the trypsin inhibitor gene could be antagonistic to the function of the cryIA(c) gene. Either way, these results suggest that the combined use of these two genes may not be effective.

Transgenic potato plants with enhanced resistance to the tomato moth, Lacanobia oleracea: growth room trials

Insecticidal effects of three plant-derived genes, those encoding snowdrop lectin (GNA), bean (Phaseolus vulgaris) chitinase (BCH) and wheat α-amylase (WAI), were investigated and compared with effects of the cowpea trypsin inhibitor gene (CpTI). Transgenic potato plants containing each of the three genes singly, and in pairwise combinations were produced. All the introduced genes were driven by the CaMV 35S promoter; expression was readily detectable at the RNA level in transformants, but not detectable accumulation of WAI could be detected in transgenic potatoes containing its encoding gene. GNA and BCH were accumulated at levels up to 2.0% of total soluble protein; both proteins were expressed in a functional form, and GNA was shown to undergo 'correct' N-terminal processing. Accumulation levels of individual proteins were higher in plants containing a single foreign gene than in plants containing two foreign genes.

Characterization of intestinally active proteinases of cyst-nematodes.

Cryostat sections of juvenile and adult female stages of the soybean cyst-nematode, Heterodera glycines, were incubated with 4 different naphthylamide-linked peptide substrates to localize and characterize proteinase activity within the animal. Detected activity was restricted to the intestine and 2 distinct classes of proteinase were identified on the basis of substrate specificity and sensitivity to plant proteinase inhibitors. A cathepsin L-like cysteine proteinase activity capable of hydrolysing the synthetic substrates Z-Ala-Arg-Arg-MNA and Z-Phe-Arg-MNA but not Z-Arg-Arg-MNA or L-Arg-NA was inhibited by an engineered variant of a cysteine proteinase inhibitor from rice (Oc-I delta D86). The cleavage of Z-Phe-Arg-MNA was sensitive to inhibition by a combination of Oc-I delta D86 and cowpea trypsin inhibitor (CpTI). Degenerate oligonucleotide primers were used to amplify fragments of cysteine proteinase genes from 2 cyst-nematodes, H. glycines and Globodera pallida. Comparison of the H. glycines fragment with known genes established highest homology to cathepsin L-like genes. In contrast, the amplified G. pallida fragment displayed greatest homology to cathepsin B-like genes from Caenorhabditis elegans.

Cowpea Flour Vitamins and Trypsin Inhibitor Affected by Treatment and Fermentation with Rhizopus microsporus

ABSTRACTSoaking cowpeas (25°C, 24 hr) decreased folacin content but did not affect thiamin, niacin, and riboflavin. Boiling soaked seeds for 45 min sharply decreased thiamin, folacin, niacin, and riboflavin. Losses of folacin, niacin, and riboflavin were recovered during fermentation. Soaking reduced trypsin inhibitor activity (TIA) by ∼20%, whereas boiling of soaked seeds decreased TIA by ∼85%. A slight increase in TIA occurred in soaked, cooked cowpeas after 18 hr fermentation.