【作者】 周晓梅；

【导师】 沈晋良；

【作者基本信息】 南京农业大学 ， 农药学， 2004， 博士

【摘要】 转Bt基因棉的推广为控制我国棉铃虫猖獗为害提供了一项新途径。但棉铃虫对其具有产生抗性的风险将严重威胁转Bt基因棉在生产上的应用寿命。本文采用AFLP分子标记技术，作抗性基因连锁图，确定抗性基因位点，从分子遗传学角度阐明棉铃虫对转Bt基因棉的抗性机理，是转Bt基因棉安全性评价的重要组成部分之一。研究结果为制定抗性治理方案、延缓抗性发展及促进分子技术的进展具有重要的现实和理论价值。 本论文第一章对转Bt基因作物的抗虫性、害虫对转Bt基因作物的抗性发展、抗性治理策略以及AFLP的研究进展等四大方面进行了阐述，这为后续的实验研究提供了理论依据。 第二章为棉铃虫对转Bt基因棉高抗品系的纯化及保持研究。叶片喂饲法进行抗性棉铃虫对转Bt基因棉的筛选结果表明，棉铃虫初孵幼虫取食转Bt基因棉NUCOTN33^B苗期叶片3d的平均死亡率为31.6％～62.1％，5d的平均死亡率为69.5％～82.3％。经过12代的筛选，棉铃虫对Bt毒素CrylAc的抗性由第49代的329.7倍上升到第59代的2824.0倍，使其重新获得极高水平抗性，抗性种群得到进一步纯化，为进一步研究棉铃虫对Bt棉抗性的分子遗传机理提供保证。 提取高质量的棉铃虫基因组DNA是进行遗传分析的重要前提条件，本论文的第三章即为CTAB法提取棉铃虫基因组DNA的研究。CTAB法主要包括组织消化、CTAB去多糖、氯仿：异戊醇（24：1，Ⅴ：Ⅴ）除蛋白、PEG沉淀DNA、TE重溶DNA沉淀以及RNA酶消化RNA等过程；结果表明该方法提取的棉铃虫基因组DNA的A₂₆₀/A₂₈₀的比值绝大部分在1.7～1.9之间，EcoR I／Mse I双酶切的产物大小主要为50～1000bp，符合AFLP分析的要求。 第四章为棉铃虫对转Bt基因棉的抗性遗传及AFLP标记构建的研究。饲料感染法测定结果表明，棉铃虫回交后代（BC）初孵幼虫用含区分剂量40μg／ml CrylAc的饲料饲喂5d后，存活虫数：死亡虫数的比例为1：1，这与该虫对转Bt基因棉的抗性遗传方式为常染色体上不完全隐性的研究结果相一致：AFLP技术是一种多态性检出效率棉铃虫对转Bt基因棉抗性的分子遗传机理较高的分子标记技术，本文在前人研究的基础上，将此技术用于棉铃虫对转Bt基因棉抗性基因（刀次）分子标记的构建，结果表明BtR与AFLP标记EaaMcta02连锁，位于第2条连锁群上EaaMcta02和Eaal讨cta03两个标记之间，遗传图距分别为ro.40cM和13 .90cM;对标记EaaMcta02回收、克隆、测序，仅在68lbp处获得一条特异性条带。本研究结果是抗性分子机理的重要组成部分，并将为制定棉铃虫对转Bt基因棉的抗性监测技术及抗性治理方案提供一个依据。 第五章为两种转Bt基因棉杀虫蛋白Cry1A。表达量的检测研究。用EnviroLogixcry1Ab/cry1A。平板试剂盒检测了两个转cry1Ac基因的棉花品种NucoTN 33“、NucoTN 99B以及常规对照品种苏棉12不同生育期主茎嫩叶、侧枝嫩叶、蕾及蕾的苞叶中杀虫蛋白cry1Ac的含量。结果表明，两种转Bt基因棉Nuco翎33”和99”主茎嫩叶生长前期杀虫蛋白c叮IAc的含量显著高于生长后期，花铃期（33B和99”分别为4.43和2.93林留g）和吐絮期（3 .87和2.86拌留g）主茎嫩叶中Cry1Ac的含量分别降至苗期第6叶（7.64和8.38林留g）的58%、35%和51%、34%;相同时期的主茎嫩叶和侧枝嫩叶中Cry1Ac的含量均显著高于蕾及蕾的苞叶，前两者Cry1A。的含量均为后两者的两倍以上。关键词:棉铃虫;转Bt基因棉:抗性机理;分子遗传;AFLP;杀虫蛋白CrylAc更多还原

【Abstract】 The extension of Bt transgenic cotton offers a new method on controlling Helicoverpa armigera which is one of the most important pests of cotton in China. But the resistance risk of H. armigera to transgenic Bt cotton will badly impact the longevity of its application in the cotton production. In this thesis, we used one kind of molecular technique, amplified fragment length polymorphism (AFLP), to map the gene conferring resistance to Bt cotton in H. armigera, decide the sites of the resistance gene. Understanding the resistant mechanism of H. armigera to transgenic Bt cotton based on molecular genetics is an important part of safety evaluation system of transgenic Bt cotton. The results will provide some practical and theoretical value for establishing the resistance management strategies, staying the development of resistance, and improving the progress of the molecular technique.In chapter 1 of this thesis, we summarized the progress of resistance of transgenic crops to pests, resistance of pests to transgenic Bt crops, strategies of resistance management and AFLP, which provide theoretical basis for the successive research.Chapter 2 involves the purification and maintenance of resistant population of H. armigera to transgenic Bt cotton. Selection experiment of purification and maintenance for high resistance to transgenic Bt cotton expressing Cry 1 Ac toxin in population of H. armigera were conducted using a leaf-feeding method. The results showed that the mean survival rate of the neonate feeding on the leaves for 3d were 31.6%~62.1%, and that for 5d were 69.5%~82.3%. By 12 generation of selection, the resistant level of H. armigera toCrylAc was enhanced from 329.7-fold to 2824-fold. The very high level of resistance of//. armigera to Bt toxin, CrylAc, was recovered. It will provide a guarantee for the research work about molecular mechanism of H, armigera to transgenic Bt cotton.The extraction of complete DNA from H. armigera (HUbner) is a key precondition of genetic analysis, which were studied in chapter 3. The analysis of amplified fragment length polymorphism (AFLP) has the potential to become a powerful new DNA fingerprinting technique for studying genetic relationship and genetic diversity in insects. The CTAB based DNA extraction protocol provided DNA suitable for AFLP assay. Its process mainly contained six parts, including digesting of tissue by lysis buffer, precipitation of polysaccharides by CTAB, removal of proteins by chloroform : isoamylalcohol (24:1, V:V), precipitation of DNA by PEG, dissolving DNA by TE buffer and consuming of RNA by RNase A. The ratios of A260/A280 of genomic DNAs gotten by the method were almost between 1.7 and 1.9, and the sizes of products digested by EcoR I / Mse I were mainly 50~1000bp.In chapter 4, we studied the inheritance and AFLP markers of resistance in H. armigera to transgenic Bt cotton. The results of infected diet method showed that the ratio between the survival and the dead in the backcross population (BC) of H. armigera was 1:1 after feeding diet with 40ng/ml CrylAc, a determining dose between resistant colony and heterozygote colony for 5d, which was consistent with that of its resistance genetic model-incompletely recessive autosomal heredity. AFLP is a reliable and powerful approach in detecting polymorphic loci. In this paper, AFLP method was applied to map the resistant gene marker in H. armigera to Bt cotton on the basis of previous studies. The results showed that BtR gene flanked with two AFLP marker, EaaMctaO2 and EaaMctaO3, on the 2nd linkage group in H. armigera, with 10.40 cM and 13.90 cM linkage distance. The AFLP marker, EaaMcta, was collected, cloned, and sequenced, and a band with 681bp length was gotten. The results provide a guidance in understanding, monitoring, and managing resistance to Bt cotton in H. armigera.In chapter 5, the usefulness of EnviroLogix CrylAb/CrylAc plate kits for quantification of the Cry1Ac content was determined with two Bt transgenic cotton varieties, NUCOTN 33B and NUCOTN 99B, and a non-Bt variety, Sumian 12更多还原