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     

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.     

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.     

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。     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests

A member of the potato proteinase inhibitor II (PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco (Nicotiana tabacum) usingAgrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R₀) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R₀ plants (designated R₁) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae ofChrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper),Spodoptera litura (F.) (Lepidoptera: Noctuidae) andThysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently,C. eriosoma larvae fed leaf tissue from transgenic plants expressing thePPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectablePPI II protein accumulation. However, larvae of bothS. litura andT. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured inC. eriosoma digestive tract extracts, but not in extracts fromS. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants.     

Transgenic Expression of Trypsin Inhibitor CMe from Barley in Indica and Japonica Rice,Confers Resistance to the Rice Weevil Sitophilus Oryzae

Indica and japonica rice (Oryza sativa L.) plants were transformed by particle bombardment with the Itr1 gene encoding the barley trypsin inhibitor BTI-CMe, under the control of its own promoter that confers endosperm specificity, and the maize ubiquitin promoter. From 38 independent transgenic lines of indica (breeding line IR58) and 15 of the japonica (cv Senia) selected, 22 and 11, respectively, expressed the barley inhibitor at detectable levels. The transgene was correctly translated as indicated by western blot analysis with a level of expression in R₃ seeds up to 0.31% (IR58) and 0.43% (Senia) of the total extracted protein. The functional integrity of BTI-CMe was confirmed by trypsin activity assays in liquid media and by activity staining gels, performed with seed extracts. The significant reduction of the survival rate of the rice weevil (Sitophilus oryzae, Coleoptera: Curculionidae) reared on homozygous transgenic indica and japonica rice seeds expressing the BTI-CMe, compared to non-transformed controls, and the decrease in the trypsin-like activity of insect crude midgut extracts, confirmed the utility of this proteinase inhibitor gene for the control of important storage pests.     

Introduction and Expression of Cowpea Trypsin Inhibitor (CpTI) Gene in Transgenic Tobacco

A trypsin/chymotrypsin inhibitor (CpTI) was purified from mature cowpea seeds. A full length cDNA clone encoding the trypsin/chymotrypsin inhibitor protein was isolated from a cDNA library that was constructed using poly(A⁺) RNA from developing seeds. Tobacco protoplasts were transformed with a construct composed of CaMV 35S promoter, NPTII gene, CpTI gene, and NOS terminator, using polyethylene glycol (PEG)-mediated direct gene transfer. The CpTI gene has been integrated into the plant genome, and the expressed CpTI protein from transgenic plants was catalytically active.     

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 -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.Resistance of the transgenic plants to insect attack was assayed by exposing the plants to larvae of the tomato moth, Lacanobia oleracea. All the plants tested which were expressing GNA showed an enhanced level of resistance. Leaf damage was reduced by more than 50% compared to controls; total insect biomass per plant was reduced by 45-65%, but larval survival was only slightly reduced (20%). These results support the hypothesis that GNA has a significant antifeedant effect on insects. Expression of BCH had no protective effect against this insect. Expression of CpTI in transgenic potatoes had similar effects to expression of GNA on total insect biomass and survival, but did not afford protection against insect damage to the plant.     

Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthopper (Nilaparvata lugens Stål) in transgenic rice

Proteinase inhibitors are widely distributed in animals, plants and microorganisms and their roles in plants are associated with defense against pests. The utilization of proteinase inhibitors for crop protection has been actively investigated with a variety of proteinase inhibitors. Soybean Kunitz trypsin inhibitor (SKTI), one of the major seed storage protein, is synthesized for a short period during seed development. To investigate the role of SKTI in a plant's defense system against insect predation, a recombinant plasmid containing the full-length cDNA of SKTI under control of the CaMV 35S promoter was introduced into rice protoplasts by using the PEG direct gene transfer method and a large number of transgenic rice plants were regenerated. The integration, expression, and inheritance of this gene was demonstrated in R1 and R2 generations by Southern, northern, and western analyses. Accumulation levels (0.05–2.5% of soluble proteins) of SKTI protein were detected in R1 and R2 plants. Bioassay with R1 and R2 transgenic plants revealed that transgenic plants are more resistant to destructive insect pest of rice, brown planthopper (Nilaparvata lugens Stål), than the control plants. Thus, introduction of SKTI into rice plants can be used to control insect pests.     

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.     

Phenotypic cost to plants of an extra gene

In lines of transgenic tobacco plants containing cowpea trypsin inhibitor gene constructs, the cost to various phenotypic characteristics has been measured in plants which express the gene at a high level and in plants which possess, but do not express, the cowpea sequences. Small, but in some cases significant, differences between transgenic and untransformed control plants were found in various parameters. There was no additional difference between transgenic plants which expressed cowpea trypsin inhibitor and those which did not. Thus, although the processes of transformation/regeneration may have some small effects on non-targeted phenotypic characteristics, the expression at high levels of this ‘foreign’ protein imposed no additional yield penalty on the plants.     

Cloning and Characterization of Trypsin Inhibitor Gene from Vigna unguiculata

A trypsin inhibitor, member of Bowman-Birk family, was isolated and purified from cowpea (Vigna unguiculata]). Polyclonal antibodies were raised against cowpea trypsin inhibitor (CpTI) protein in rabbits. The gene for CpTI was amplified by polymerase chain reaction and cloned in a bacterial expression vector pVCATFR18416. The expression of CpTI protein in BL21 (DE3) strain of Escherichia coli was confirmed by western blot studies. The CpTI gene was also sequenced and found to exhibit 100% homology with already published sequence of CpTI gene.     

转双基因烟草对棉铃虫的杀虫活性评价

以含Ｂｔ杀虫蛋白基因（单基因）烟草和常规烟草为对照,系统测定了含Ｂｔ与豇豆胰蛋白酶抑制剂蛋白基因（双基因）的抗虫烟草对棉铃虫不同龄期幼虫的杀虫活性。结果表明：１￣３龄幼虫取食转双基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食转基因烟草３ｄ后死亡率为８０．５％￣９９．３％,取食６ｄ后死亡率达１００％,均显著高于转单基因烟草。２龄幼虫取食     

抗虫植物基因工程研究进展

虫害是造成农业减产的主要原因之一。据不完全统计,全世界每年因虫害引起的作物减产达总产量的15％,损失高达数千亿美元。在我国,因虫害水稻减产在lO％以上;小麦减产近20％;棉花减产在     

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.