Constitutive expression of a cowpea trypsin inhibitor gene,CpTi, in transgenic rice plants confers resistance to two major rice insect pests

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.     

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.     

豇豆胰蛋白酶抑制剂基因转化芥菜及抗虫鉴定

用农杆菌介导将豇豆胰蛋白酶抑制剂 (CpTI)基因导入芥菜 ,获得了Kan抗性植株 .经PCR扩增、PCR Southern印迹和Northern印迹分析 ,转化再生植株大部分呈阳性 ,而非转化的再生植株均为阴性 ,证明CpTI基因已存在于芥菜基因组中 .在室内进行了喂虫试验 ,结果表明转基因芥菜抗虫性明显高于对照 ,转基因植株之间存在抗虫性差异     

Protein and cDNA sequences of Bowman-Birk protease inhibitors from the cowpea (Vigna unguiculata Walp.)

The protein and gene sequences of the cowpea Bowman-Birk type trypsin inhibitor which confers enhanced insect resistance to transgenic tobacco plants, and of cowpea trypsin/chymotrypsin inhibitors are presented. There are regions of high conservation and high divergence within the 5 leader, mature protein and 3 non-coding regions of the Bowman-Birk inhibitors and in the genes which encode them in different members of this family within the Leguminosae. The practical implications of this finding for studies on the evolution of plants and the utilization of these genes for enhancing insect resistance is discussed.     

Transgenic rice plants expressing a trypsin inhibitor are resistant against rice stem borers, Chilo suppressalis

A synthetic gene, mwti1b, coding for a winged bean trypsin inhibitor WTI-1B, has been introduced and expressed in rice plants, Oryza sativa. Protein extracts from transgenic rice plants expressing the trypsin inhibitor inhibited the gut proteases of larvae of the serious insect pest, the rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) in vitro. The growth of larvae reared on transgenic rice plants expressing WTI-1B at more than 1 ng/10 g total protein was significantly retarded compared to that on non-transgenic control plants.     

Use of cowpea trypsin inhibitor (CpTI) to protect plants against insect predation

Transgenic tobacco plants expressing a cowpea trypsin inhibitor gene have enhanced levels of insect resistance to a variety of insect pests. Furthermore, insect bioassay has shown the cowpea trypsin inhibitor to have anti-metabolic activity to insect pests of the orders Lepidoptera, Coleoptera and Orthoptera. The advantages and disadvantages of this approach to developing insect resistant crops is discussed in relationship to other methods, including conventional plant breeding and chemical control. Eventually it is hoped that African farmers will benefit from this industrially sponsored research.     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

Inhibition of trypsin by cowpea thionin: characterization,molecular modeling,and docking

Higher plants produce several families of proteins with toxic properties, which act as defense compounds against pests and pathogens. The thionin family represents one family and comprises low molecular mass cysteine-rich proteins, usually basic and distributed in different plant tissues. Here, we report the purification and characterization of a new thionin from cowpea (Vigna unguiculata) with proteinase inhibitory activity. Cowpea thionin inhibits trypsin, but not chymotrypsin, binding with a stoichiometry of 1:1 as shown with the use of mass spectrometry. Previous annotations of thionins as proteinase inhibitors were based on their erroneous identification as homologues of Bowman-Birk family inhibitors. Molecular modeling experiments were used to propose a mode of docking of cowpea thionin with trypsin. Consideration of the dynamic properties of the cowpea thionin was essential to arrive at a model with favorable interface characteristics comparable with structures of trypsin-inhibitor complexes determined by X-ray crystallography. In the final model, Lys11 occupies the S1 specificity pocket of trypsin as part of a canonical style interaction.     

豇豆胰蛋白酶抑制剂(CpTI)基因转化欧美杨的研究

本研究建立了欧美107杨的高频再生体系,用改良的根癌农杆菌介导法将豇豆胰蛋白酶抑制剂(CpTI)基因导入107杨中。经过严格的卡那霉素(Km)筛选,得到了Km抗性(Kmr)植株。对部分Kmr植株进行PCR及PCR-Southern杂交鉴定,证实CpTI基因已整合进杨树基因组中。转基因植株的饲虫实验表明,其中部分株系的叶片可在一定程度上抑制扁刺蛾幼虫的生长。     

The promoter of the gene Itr1 from barley confers a different tissue specificity in transgenic tobacco

Tissue-specific expression of the gene coding for trypsin inhibitor BTI-CMe in barley (Itr1) occurs during the first half of endosperm development. In transgenic tobacco, theItr1 promoter drives expression of the β-glucuronidase reporter gene not only in developing endosperm but also in embryo, cotyledons and the meristematic intercotyledonary zone of germinating seedlings. A promoter fragment extending 343 bp upstream of the translation initiation ATG codon was sufficient for full transgene expression, whereas, the proximal 83 bp segment of the promoter was inactive. Possible reasons for the differences in expression patterns are discussed. These authors have contributed equally to this work     

Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene

Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were recovered as a result of Agrobacterium tumefaciens-mediated transformation performed with EHA105 pBI-KUN strain. Plasmid pBI-KUN contains a 650 bp insert derived from the soybean (Glycine max L.) KTi₃, gene, coding for a Kunitz trypsin proteinase inhibitor. A total of 58 independent transgenic lines were obtained from 200 co-cultivated leaf explants. Southern blot hybridization analysis demonstrated the presence of KTi₃ gene in the poplar genome. Northern blot analysis of different kanamycin-resistant plantlets confirmed the accumulation of KTi₃ mRNA and revealed different levels of expression. The trypsin inhibitory activity was determined in poplar transgenic tissues by means of specific assay. Moreover, the trypsin-like digestive proteinases of the polyphagous moth Lymantria dispar (Lepidoptera, Lymantriidae) and Clostera anastomosis (Lepidoptera, Notodontidae) were detected and inhibited in vitro by Kunitz proteinase inhibitor from selected transgenic plants. Two insect bioassays were performed on P. nigra transgenic plant lines, using larvae of the above mentioned insects. In both cases larval mortality and growth as well as pupal weight were not significantly affected when the insects were fed on transgenic leaves and control leaves, respectively.     

Transformation of Populus tomentosa with Insecticidal Cowpea Proteinase Inhibitor Gene

Cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene, was introduced into poplar ( Populus tomentosa Carr. ) by gene transformation mediated by Agrobacterium tumefac/ens (Smith et Townsend) Conn. The influences on regeneration and transformation frequency of poplar by the concentration and addition of kanamycin were compared. Kanamycin resistant (Kmr) plantlets were obtained by 3 -4 cycles screening in selective condition. The ability of leaf regeneration and shoot subculture and rooting from the transformed and non-transformed plants in the presence of 50 mg/L kanamycin was examined. The presence of CpTI gene in the transgenic plants were confirmed by PCR and PCR-Southern blot. Assay on proteinase inhibition activity demonstrated that leaf protein extracts of the transgenic poplar showed higher inhibition activity against trypsin than that of control plants.     

An Insect-Resistant Transgenic Cabbage Plant with Cowpea Trypsin Inhibitor(CpTI) Gene

Cowpea trypsin inhibitor (CpTI) gene was transformed into Brassica oleracea var. capitara variety "Yingchun" and "Jingfeng" mediated by the Agrobacterium tumefaciens LBA4404(pRCL27). Transgenic plants were obtained from transformed calli or explants. It was shown from the ELISA assay that NPT Ⅱgene was expressed in the transgenic cabbage cells. The integration of the CpTI gene into cabbage genome DNA was confirmed by Southem blotting. Insect-tolerance of the transgenic plants to Pieris rapae L. was observed by bioassays on the transgenic plants in the laboratory.     

通过细胞内的靶向定位大幅度提高外源蛋白在转基因植株的积累水平

通过体外操作,对豇豆胰蛋白酶抑制剂(cpti)基因进行修饰,获得了一个融合蛋白基因(sck)。该基因是在cpti基因的基础上,在其5’端添加了信号肽编码序列,在3’端添加了内质网滞留信号编码序列,旨在引导基因转译产物进入细胞内质网,并最终滞留在内质网及其衍生的蛋白体内。用sck基因转化烟草(Nicotiana tabacum L.),对获得的转基因植株进行ELISA检测。结果表明,含有修饰基因的转基因烟草CpTI蛋白含量有明显提高,比转末修饰cpti基因烟草平均高出2倍,最高单株可达4倍以上,同时转基因植株的抗虫性也有了显著的提高。结果表明,采用外源蛋白靶向定位的策略,可大幅度提高外源蛋白在转基因植物细胞内的积累量,在植物基因工程研究中具有广泛的借鉴意义。     

通过细胞内的靶向定位大幅度提高外源蛋白在转基因植株的积累水平

通过体外操作,对豇豆胰蛋白酶抑制剂(cpti)基因进行修饰,获得了一个融合蛋白基因(sck).该基因是在cpti基因的基础上,在其5'端添加了信号肽编码序列,在3'端添加了内质网滞留信号编码序列,旨在引导基因转译产物进入细胞内质网,并最终滞留在内质网及其衍生的蛋白体内.用sck基因转化烟草(Nicotiana tabacum L.),对获得的转基因植株进行ELISA检测.结果表明,含有修饰基因的转基因烟草CpTI蛋白含量有明显提高,比转未修饰cpti基因烟草平均高出2倍,最高单株可达4倍以上,同时转基因植株的抗虫性也有了显著的提高.结果表明,采用外源蛋白靶向定位的策略,可大幅度提高外源蛋白在转基因植物细胞内的积累量,在植物基因工程研究中具有广泛的借鉴意义.     

胰蛋白酶抑制剂基因siRNA表达体系的构建

以大豆基因组DNA为模板,利用聚合酶链式反应(PCR)技术克隆了大豆胰蛋白酶抑制剂基因KSTI3的全长DNA片段,并将其构建到pMD18-T vector上。核苷酸序列测定结果表明:该基因片段全长654bp,与已发表的KSTI3基因序列同源性达99%。将反义 正义基因片段插入到pBI121 35S启动子下,构建重组质粒pBIKSTI3。通过冻融法将该重组质粒转入农杆菌EHA105中,获得了siRNA表达体系。利用农杆菌介导法将带有pBIKSTI3的菌株转化大豆,从2棵再生植株中得到2 100bp的特异性扩增条带,而未转化的植株中无该片段的产生。     

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     

Transgenic cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>) seeds expressing a bean α-amylase inhibitor 1 confer resistance to storage pests,bruchid beetles

Cowpea is one of the important grain legumes. Storage pests, Callosobruchus maculatus and C. chinensis cause severe damage to the cowpea seeds during storage. We employ a highly efficient Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) α-amylase inhibitor-1 (αAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants. The use of constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404, thiol compounds during cocultivation and a geneticin based selection system resulted in twofold increase in stable transformation frequency. Expression of αAI-1 gene under bean phytohemagglutinin promoter results in accumulation of αAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine α-amylase in vitro. Transgenic cowpeas expressing αAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays.     

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.