论文标题:融合杀虫基因的分子设计、构建及表达研究
Molecular Design, Construction and Expression of A Fused Insecticidal Gene
论文作者 武东亮
论文导师 郭三堆,论文学位 博士,论文专业 作物遗传育种
论文单位 中国农业科学院,点击次数 72,论文页数 102页File Size5000k
2000-07-01论文网 http://www.lw23.com/lunwen_64179927/ Cry1A;CpTI;融合基因;抗虫性;酵母
crylA, CpTI, Fused gene, Insect resistance, Yeast
虫害严重影响着农业生产，转基因抗虫植物是控制害虫的最有效手段之一。但是随着转基因抗虫植物的大面积推广，昆虫会逐渐对其产生抗性。构建融合杀虫基因可能是延缓昆虫对转基因抗虫植物产生抗性的一种有效策略。 根据Bt杀虫蛋白Cry1A和豇豆胰蛋白酶抑制剂（CpTI）的高级结构、杀虫机理及昆虫体内肠激酶的作用机理，本着尽量不影响二者功能的原则，设计出了构建Cry1A和CpTI融合杀虫基因CryCI的方案，即将CpTI基因的编码序列连接到GFM Cry1A基因编码区3’端，并在其间用一段编码肠激酶切割位点的序列相连。 利用设计的CpTI引物扩增出282bp的CpTI基因片段，将其克隆到pUC19载体上，然后将含有肠激酶切割位点编码序列的接头连接到CpTI基因的5’端，进而把此带有接头的CpTI基因片段克隆到质粒pG4AB上GFM Cry1A基因编码区的3端，构建成携带融合杀虫基因CryCI的载体pGF4ABC。将pGF4ABC上的融合杀虫基因CryCI分别克隆到酵母表达载体pPIC9K和植物表达载体pBI121.1上，构建成融合杀虫基因的分泌型酵母表达载体pPIC9KBC和高效植物表达载体pGBIF4ABC。 应用电激法将酵母表达载体pPIC9KBC导入甲醇酵母（Pichia pastoris）菌株KM71，共获得52个His~+Mut~s转化子。以Bt杀虫基因Cry1A的引物进行PCR筛选，得到5个酵母重组菌株，重组率为9.6％。在以甲醇为唯一碳源的培养基中，进行了重组酵母菌株的诱导表达，在培养物上清中检测到了约75kD的融合蛋白。酵母中表达的融合蛋白具有杀虫活性，并且可以被肠激酶进行体外切割。 通过农杆菌介导法，获得了34株转融合杀虫基因的烟草植株。经PCR、Southern blot、Western blot等分子生物学方法分析，结果证明融合杀虫 中国农业科学院博士学位论文2基因Ci－－－x＊x己整合到烟草基因组中，并能表达出75kD的融合蛋白。以Bt杀虫晶体蛋白抗体作为一抗的 EL SA分析和蛋白酶抑制活性测定结果表明，融合基因同时使Bt杀虫晶体蛋白和更豆胰蛋臼酶抑制剂（CPTI）两个构成成分得以表达。 用棉铃虫幼虫对34株转基因烟草进行生物杀虫试验，3株O．8％）表现高抗，14株O．2％）表现中抗，这一结果证明融合杀虫基因 CrW在高等植物中表达的融合蛋白具有杀虫生物学特性。 用对单价Bt抗虫棉产生抗性的棉铃虫幼虫进行转基因烟草杀虫试验，结果表明转融合杀虫基因烟草对抗性棉铃虫仍具杀虫活性，即融合杀虫基因可能会延缓害虫对抗虫转基因作物产生抗性的进程。
Pests seriouly threaten agricultural production. The genetic engineering of insect-resistant plants has been proved very effective in the Integrated Pest Management Program. On the other hand, pests will develop resistance to insect- resistant transgenic plants with the wide planting of these plants. The fused insecticidal protein may delay the process of insects? developing resistance. After investigation of the secondary structure and action mode of Bt insecticidal protein (CryIA) and Cowpea trypsin proteinase inhibitor (CpTI), as well as the action mechanism of insect enterokinase, we devised the fusion gene on the base of maintaining their functions respectively. The method follows: CpTI gene is linked to 3? -end of GFM crylA gene by a nucleotide sequence encoding cleavage site of insect enterokinase. The 282bp of CpTJ gene obtained by PCR amplification was cloned into the plasmid pUC 19 and then the adaptor, which contains a nucleotide sequence encoding cleavage site of insect enterokinase, was linked to 5? -end of CpTJ gene. The CpTI gene with the adaptor was cloned into pG4AB at the 3? -end of the encoding region of GFM cry]A gene and therefore we obtained the plasmid pGF4ABC harboring the fused insecticidal gene CryCI. The CryCI gene and essential regulation elements cut from the plasmid pGF4ABC were inserted into yeast expression vector pPIC9K and plant expression vector pBI 121.1, so we got the secretive yeast expression plasmid pPIC9KBC and plant high-efficiency expression plasmid pGBIF4ABC, Fifty-two His+Muts transformants were obtained after the expression vector pPIC9KBC was introduced into Pichia pasoris, KM7I strain, by electroporation. Five yeast clones were verified to be recombinant clones carrying the fused gene CryCI by PCR amplification with the primers of lit Cry IA gene. The recombinant 96 rate was 9.6 percent. The induction expression of the recombinant yeast clones was carried out by the medium which had the methanol as the only carbon resource and the fused protein of 75kD was expressed. The fused protein from pPIC9KBC/KM71 yeast strain had the insecticidal activity and could be digested by enterokinase in vifro. The tobacco leave discs were transformed with Agrobacterium tumefaciens carrying the plant expression plasmid pGBIF4ABC. We got thiity-four transgenic tobacco plants containing the fused gene CryCI after kanamycin screening, PCR detection and Southern blot analysis. The expression of 75kD fused protein in transgenic plants was proved by western blot. ELISA analysis with the antibody of Bt crystal protein as the first antibody and the inhibition activity analysis of the trypsin proteinase demonstrated that both of the two constitutive parts (Cry 1 A and CpTI) of the fused protein could be expressed effectively in transgenic tobacco plants with the fused gene cryCi Leaf disc bioassay of the total 34 transgenic plants resistant to cotton bollworm (H Armigera) larvae showed that three of them (8.8 percent) had high resistance and 14 of them(4 1.2 percent) had moderate resistance. The result proves that the fused protein has the insecticidal activity. Leaf disc bioassay of the transgenic plants resistant to cotton bollworm (H Armigera) larvae which had developed resistance to monovalent Bt transgenic cotton plants
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