Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd

Inga laurina is a tree that belongs to the Mimosoideae sub-family of the Leguminosae. A protein inhibitor of trypsin (ILTI) was isolated from its seeds by ammonium sulphate precipitation, ion-exchange chromatography and rechromatography on an HiTrap Q ion-exchange column. By SDS-PAGE, ILTI yielded a single band with a Mr of 20 kDa with or without reduction. ILTI was found to be a single polypeptide chain containing 180 amino acids, the sequence of which was clearly homologous to the Kunitz family of serine protease plant protein inhibitors, and it also showed significant similarity to the seed storage proteins, sporamin and miraculin. However, ILTI displayed major differences to most other Kunitz inhibitors in that it contained only one disulfide bridge, and did not have two polypeptide chains as for the majority of other Kunitz inhibitors purified from Mimosoideae species. ILTI inhibited bovine trypsin with an equilibrium dissociation constant (K(i)) of 6 x 10(-9)M, but did not inhibit chymotrypsin, papain and alpha-amylase. Its amino acid sequence contained a Lys residue at the putative reactive site (position 64). ILTI was stable over a wide range of temperature and pH and in the presence of DTT.     

Adaptation of Spodoptera exigua (Lepidoptera: Noctuidae) to barley trypsin inhibitor BTI-CMe expressed in transgenic tobacco

Nicotiana tabacum plants were transformed with the cDNA of barley trypsin inhibitor BTI-CMe under the control of the 35S CaMV promoter. Although the transgene was expressed and the protein was active in the homozygous lines selected, growth of Spodoptera exigua (Lepidoptera: Noctuidae) larvae reared on transgenic plants was not affected. The protease activity in larval midgut extracts after 2 days feeding on transformed tobacco leaves from the highest expressing plant showed a reduction of 25% in the trypsin-like activity compared to that from insects fed on non-transformed controls. The susceptibility of digestive serine-proteases to inhibition by BTI-CMe was confirmed by activity staining gels. This decrease was compensated with a significant induction of leucine aminopeptidase-like and carboxipeptidase A-like activities, whilechymotrypsin-, elastase-, and carboxipeptidase B-like proteases were not affected.     

Inga vera trypsin inhibitor interferes in the proteolytic activity and nutritional physiology of Ephestia kuehniella larvae

Plant peptidase inhibitors provide plants with a defense strategy to inhibit insect digestive enzymes and have been studied as an alternative strategy for pest control as they interfere in normal insect physiology. We evaluated the effects of ingestion of the trypsin inhibitor from Inga vera Willd. (Fabaceae) seeds on the nutritional and digestive physiology of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) larvae. Inga vera trypsin inhibitor (IVTI) reduced the efficiency of the conversion of ingested and digested food in these larvae and increased the metabolic cost, causing an anti‐nutritional effect. In both short‐ and long‐term bioassays, the ingestion of IVTI inactivated most of the insect's trypsin activity, but increased chymotrypsin activity as a compensatory response by the insect; however, protein digestion continued to be partially blocked. Consequently, chymotrypsin‐like enzymes, which were over‐produced in the gut, were excreted more into the frass of IVTI‐fed larvae. As such, the resistance of IVTI to hydrolysis by insect midgut proteases resulted in detrimental effects to larvae. These data provide support for the use of IVTI as a biotechnological tool for pest control.     

Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura

A sweet potato (Ipomoea batatas cv. Tainong 57) trypsin inhibitor gene was introduced into tobacco plants (Nicotiana tabaccum cv. W38) by Agrobacterium tumefaciens– mediated transformation. From 30 independent transformants, three lines with high level of expression were further analyzed. The trypsin inhibitor gene, under control of the 35S CaMV promoter, led to the production of the trypsin inhibitor proteins up to 0.2% of the total protein. In insecticidal bioassays of transgenic tobacco plants, larval, growth of Spodoptera litura (F.), the tobacco cutworm, was severely retarded as compared to their growth on control plants. This observation implied that expression of sweet potato trypsin inhibitor can provide an efficient method for crop protection. Received: 29 July 1996 / Revision received: 15 November 1996 / Accepted: 8 December 1996     

A Kunitz trypsin inhibitor gene family from trembling aspen (Populus tremuloides Michx.): cloning,functional expression,and induction by wounding and herbivory

Three Kunitz trypsin inhibitor genes were isolated from trembling aspen (Populus tremuloides) by PCR and cDNA screening. Based on sequence similarity, they were grouped into two classes. Southern blots showed complex banding patterns and a high level of restriction fragment polymorphism between different aspen genotypes, suggesting that these trypsin inhibitors are members of a large, rapidly evolving gene family. One of the trypsin inhibitor genes, PtTI2. was over-expressed in Escherichia coli and its product shown to inhibit bovine trypsin in vitro. Both classes of PtTI genes are induced by wounding and herbivory, permitting rapid adaptive responses to herbivore pressure. The response appears to be mediated by an octadecanoid-based signaling pathway, as methyl jasmonate treatments induced the trypsin inhibitors. Wound-induced accumulation of trypsin inhibitor protein was also observed by western blot analysis. The pattern of expression, the apparent rapid evolution of TI genes, and the in vitro trypsin inhibitory activity are consistent with a role in herbivore defense. This work establishes the presence of a functional protein-based inducible defense system in trembling aspen.     

Enzyme inhibitors from plants. Isolation and characterization of a protease inhibitor from arrow root (Maranta arundinaceae) tuber

A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a Mr of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0–12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a two-fold increase in inhibitor potency     

Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Frankliniella occidentalis) in greenhouse trials

Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), cause very large economic damage on a variety of field and greenhouse crops. In this study, plant resistance against thrips was introduced into transgenic potato plants through the expression of novel, custom-made, multidomain protease inhibitors. Representative classes of inhibitors of cysteine and aspartic proteases [kininogen domain 3 (K), stefin A (A), cystatin C (C), potato cystatin (P) and equistatin (EIM)] were fused into reading frames consisting of four (K-A-C-P) to five (EIM-K-A-C-P) proteins, and were shown to fold into functional inhibitors in the yeast Pichia pastoris. The multidomain proteins were expressed in potato and found to be more resistant to degradation by plant proteases than the individual domains. In a time span of 14-16 days, transgenic potato plants expressing EIMKACP and KACP at a similar concentration reduced the number of larvae and adults to less than 20% of the control. Leaf damage on protected plants was minimal. Engineered multidomain cysteine protease inhibitors thus provide a novel way of controlling western flower thrips in greenhouse and field crops, and open up possibilities for novel insect resistance applications in transgenic crops.     

斜纹夜蛾幼虫诱导的油菜抗虫性及其与茉莉酸信号途径的关系

植物在受植食性昆虫为害时能产生防御反应,并且植物的茉莉酸信号转导途径在这一过程中发挥着重要作用。然而, 迄今为止对于油菜Brassica campestris的诱导防御反应很少有研究报道。为此, 本实验通过测定油菜内茉莉酸和胰蛋白酶抑制剂含量研究了油菜在斜纹夜蛾Spodoptera litura幼虫为害后的抗虫性和胰蛋白酶抑制剂含量的变化,并分析这些变化在油菜诱导抗虫性与茉莉酸信号转导途径中的关系。结果表明：斜纹夜蛾幼虫取食能导致油菜体内茉莉酸和胰蛋白酶抑制剂含量系统性上升,外用茉莉酸甲酯处理也能系统性增加油菜的胰蛋白酶抑制剂含量,并且取食茉莉酸甲酯处理或斜纹夜蛾幼虫取食过的叶片能显著降低斜纹夜蛾幼虫的体重,两者的体重分别为对照植株上的67.5%和60.2%。机械损伤加斜纹夜蛾幼虫口腔分泌物处理能引起处理叶中茉莉酸和胰蛋白酶抑制剂含量的增加,但其诱导效果与机械损伤加水没有显著差异,并且两者明显低于虫害的诱导效果,两种处理的茉莉酸和胰蛋白酶抑制剂含量分别为虫害诱导的68.4%和24.4%及62.9%和36.9%;多次连续机械损伤的诱导效果与一次损伤的没有明显差异。结果说明斜纹夜蛾幼虫诱导的油菜抗虫性与茉莉酸信号转导途径有关,而其激活油菜抗虫反应的机理则可能与其特定的取食行为相关。     

入侵植物乌桕防御策略的适应性进化研究

生长于不同昆虫群落胁迫下的植物地理种群可能进化出不同的防御策略。入侵植物在原产地同时受到专食性昆虫和广食性昆虫的取食危害, 而在入侵地“逃逸”了专食性昆虫的取食危害。入侵植物对不同类型昆虫防御策略的演化可能在其成功入侵的过程中起着至关重要的作用。该文主要以原产中国入侵北美的木本植物乌桕(Triadica sebifera)为例, 并结合其他入侵植物防御策略演化的研究, 从抗性和耐受性、直接抗性和间接抗性、组成抗性和诱导抗性三个方面系统分析不同昆虫选择压力下入侵植物防御策略的演化, 同时探讨入侵植物防御策略演化对生物防治效果的影响, 指出未来的重点研究方向。