【作者】 徐永桂；

【导师】 吴益东；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2008， 硕士

【摘要】 二化螟Chilo suppressalis（Walker）是严重威胁我国水稻生产的重要害虫之一。近年来,受耕作制度和种植结构的变化、高产优质水稻品种的大面积种植、气候等原因的影响,二化螟的发生逐年加重,并且由于化学农药的长期大量使用,二化螟的抗药性呈明显上升趋势,给防治工作来了困难。转Bt CryAb基因水稻的出现为防治二化螟提供了一种新的有效方法,但是从生物学和进化角度来看,二化螟对转Bt基因水稻也可以产生抗性是一种胁迫进化现象。因此,研究Bt毒素对二化螟的杀虫机理及二化螟对Bt产生抗性的可能机制,将为制定转基因水稻的抗性治理策略、延长转基因水稻的使用寿命等提供重要的理论依据。本文建立了3种CrylA毒素对二化螟的敏感毒力基线,评估了二化螟及对Cry1Ab的抗性风险,利用配基印迹法研究了Cry1A毒素同二化螟中肠BBMV受体蛋白的结合谱,并通过RT-PCR获得了4个APN基因的cDNA片段。1.二化螟对Cry1Ab的抗性筛选及抗性风险评估用毒蛋白涂表法测定了2002年采自浙江苍南的二化螟室内品系对Cry1Aa、Cry1Ab和Cry1Ac活化毒蛋白的敏感性水平,建立了3种毒素对二化螟的敏感毒力基线。结果表明,Cry1Ab对该品系的毒力最高(LC₅₀为0.14μg/cm²),其次是Cry1Ac(LC₅₀为0.37)和Cry1Aa(LC₅₀为0.49μg/cm²)。用Cry1Ab对2006年采自江苏句容、安徽合肥和宣城、浙江杭州的田间二化螟混合种群（JAZ）进行12代的抗性筛选,其对Cry1Ab的敏感性下降了3.3倍。平均抗性现实遗传力h²为0.068,表明抗性风险较低。根据平均抗性现实遗传力、毒力回归线平均斜率进行估算,在50%-95%的选择压力下,二化螟对Cry1Ab的抗性上升10倍需要26至8代。2.二化螟中肠受体与Cry1A毒素结合分析配基结合分析结果表明二化螟CN品系幼虫中肠刷状缘膜囊泡（BBMV）中有6个Cry1Ac结合蛋白（分子量分别为180,160,120,90,70和50 kDa）,其中180、160和90 kDa结合蛋白的条带明显深于其他结合蛋白的条带,表明这3个受体蛋白结合浓度较高。同源竞争结合结果表明,180和90 kDa结合蛋白为Cry1Ac的低亲合性结合蛋白,其他4个为高亲合性结合蛋白。为了研究Cry1Ac和Cry1Ab受体结合部位的交叉反应,本文还进行了异源竞争结合研究。研究结果表明,Cry1Ab可以与Cry1Ac所有的6个结合蛋白进行竞争性结合,其中与180、120、70和50 kDa结合蛋白具有高亲合性,而与160和90 kDa结合蛋白具有低亲合性.上述结果表明,Cry1Ac与Cry1Ab在二化螟幼虫中肠BBMV上具有多个共享的结合位点,但对每个结合位点的亲合力有差异。基于毒素结合部位的相似性,Cry1Ac和Cry1Ab不宜同时用于转基因Bt水稻来控制二化螟。进一步分离、鉴定二化螟幼虫中肠BBMV中Cry1A毒素的受体蛋白将有助于研究Cry1A毒素的作用机理和二化螟对Cry1A毒素的抗性机理。3.二化螟中肠APN类受体基因片段cDNA的克隆氨肽酶N（aminopeptidase N,APN）受体是昆虫体内Bt毒素的重要作用靶标。本研究利用简并引物,从二化螟中肠中获得4个不同的APN类受体基因cDNA片段。该片段长约600bp,编码约200个氨基酸残基,分别命名为CsAPN1、CsAPN2、CsAPN3和CsAPN4。CsAPN1同家蚕APN（GenBank No.:BAA32140）氨基酸序列相似性高达73%,CsAPN2同二化螟APN（GenBank No.:ABC69855）的相似性高达100%,CsAPN3同棉铃虫APN2（GenBank No.:AAP37951）相似性为68%,CsAPN4同欧洲玉米螟APN3（GenBank No.:ABL01483）相似性为77%。更多还原

【Abstract】 The rice stem borer C.suppressalis(Walker) is one of the most serious pests threaterning rice production in China.Recently,C.suppressalis has become more serious because of cropping system and farming pattern changes,extensive planting of high yield and high quality rice,and suitable weather conditions.Resistance of C.suppressalis to chemical insecticides has obviously increased and let it more difficult to be controlled.Bt transgenic rice offers a new effective and safe weapon to control the rice stem borer.But from the evolutionary biology view,the target pests will inevitably evolve resistance to Bt toxins expressed in the Bt rice after extensive planting of Bt rice cultivars.Therefore, researching the mode of action of Bt toxins to C suppressalis and the potential resistance mechanisms will be important for developing resistance manangement strategy and prolonging the using longevity of Bt rice.In this study,we assessed resistance risk to Cry1Ab,analysized binding of Cry1A toxins to the midgut BBMV of C.suppressalis.The cDNA fragments of four APN genes from the midgut of C.suppressalis were cloned and sequenced.1.Laboratory selection for Cry1Ab resistance and resistance risk assessmentSusceptibility base-lines of activated Cry1Aa,Cry1Ab and Cry1Ac against a laboratory strain(CN) of C.suppressalis were determined using a surface contamination assay.Cry1Ab was more toxic than Cry1Ac,and Cry1Ac was more toxic than Cry1Aa against the third instar larvae of the CN strain.A mixed field population(JAZ) of C. suppressalis collected from Anhui,Jiangsu and Zhejiang provinces during 2006 was selected with activated Cry1Ab for 12 generations,and its susceptibility to Cry1Ab decreased 3.3-fold.The realized heritability(h~2) of Cry1Ab resistance was estimated as 0.068,indicating resistance development potential of Cry1Ab was low.According to the mean realized heritability and the mean slope of LD-P lines,10-fold resistance to Cry1Ab will be developed after 26 generations under selection pressure of 50%,and 8 generations under selection pressure of 95%.2.Ligand blot analysis of Cry1A toxins to midgut BBMV proteinsLigand blot analysis showed that there were 6 major binding proteins(50,70,90,120, 160 and 180 kDa) of Cry1Ac in the midgut brush border membrane vesicles(BBMV) of the CL strain.Binding protein bands of 180,160 and 90 kDa were much darker than the others,indicating their high binding concentration.Homologous competition binding results indicated that 180 kDa and 90 kDa protein bands were of low binding affinity and the other four protein bands(160,120,70 and 50 kDa) were of high binding affinity. Heterologous competition binding assays were conducted to study the binding site cross-reactivity between Cry1Ab and Cry1Ac.Cry1Ab competed for all binding sites recognized by Cry1Ac,with high affinity to 180,120,70 and 50 kDa proteins and low affinity to 160 and 90 kDa proteins.These data suggested that Cry1Ac and Cry1Ab shared several common binding sites in the midgut BBMV of C.suppressalis,but they had different binding affinity for each binding site.Considering the similarity in binding sites, Cry1Ab and Cry1Ac should not be used together in the transgenic Bt rice to control the target pest C.suppressalis.Isolation and identification of the binding proteins of Cry1A toxins in the midgut BBMV of C.suppressalis will facilitate our understanding of mode of action and resistance mechanism of Cry1A toxins.3.Cloning of cDNA fragments of APN genes of C.suppressalisAminopeptidase N is one kind of the important receptors of Bt toxins.A 600kb cDNA fragment of APN genes of C.suppressalis was amplified by RT-PCR with degenerate primers.The fragment was cloned and sequenced,and four cDNA sequences(named as CsAPN1,CsAPN2,CsAPN3 and CsAPN4) of about 600kb bases encoding APN genes were obtained.CsAPN1,CsAPN2,CsAPN3 and CsAPN4 shared 73%,100%,68%and 77% amino acid sequence similarity with Bombyx mori APN(GenBank accession No. BAA32140),C.suppressalis APN(GenBank accession No.ABC69855),Helicoverpa armigera APN2(GenBank accession No.AAP37951),and Ostrinia furnacalis APN (GenBank accession No.ABL01483).更多还原