【作者】 李付广；

【导师】 郭三堆；

【作者基本信息】 中国农业科学院 ， 分子生物学， 2003， 博士

【摘要】 2001年我国抗虫棉种植面积已达160万公顷，但几乎全部是单价Bt基因抗虫棉。研究表明，单价Bt基因抗虫棉在大面积推广应用后，棉铃虫可能会对其产生抗性。为此，将双价基因(GFMcry IA+CpTI)导入优良棉花品种，获得新类型抗虫种质，加速双价转基因抗虫棉新品种培育，并对其遗传规律和抗虫性表达等进行全面研究具有重要意义。 本研究通过农杆菌介导法和花粉管通道法转化获得双价转基因抗虫棉；通过卡那霉素筛选、ELISA检测、生物鉴定以及PCR扩增、Southern杂交等方法进行筛选鉴定、时空表达研究、逆境的影响以及分子验证等；根据其后代分离情况，研究其遗传行为；根据相应酶活性、激素、叶绿素等的测定研究其生化特征。 在前期工作的基础上，完善了农杆菌介导法和花粉管通道法遗传转化技术，优化了主要技术参数，拓宽了棉花基因型范围，实现了部分优良受体材料高效转化，初步建立了规模化转基因技术体系，并获得了大量双价转基因抗虫棉新材料。研究了部分双价转基因抗虫棉抗性分离情况，对双价转基因抗虫棉不同发育时期的抗虫性、相同时期不同器官的抗虫性、不同龄期棉铃虫幼虫对其敏感性以及不同世代棉铃虫的敏感性等进行了分析。仿照我国主要棉区的某些特定或易变气候特点、环境条件，研究了温度变化、干旱处理、盐碱条件等对双价转基因抗虫棉抗性的影响。在不同生育期取样，研究了双价转基因抗虫棉超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)等抗氧化系统酶活性，研究了丙二醛(MDA)、脯氨酸(pro)等生化物质、吲哚乙酸(IAA)与脱落酸(ABA)等内源激素以及叶绿素等的含量变化规律。 通过本项研究发现，采用嫁接技术，可使转基因再生植株成活率达到90%以上，可有效提高转化效率。分子检测结果显示，双价基因已整合到棉花染色体基因组中。遗传分析数据表明，花粉管通道法转化所获得的转基因抗虫材料多拷贝比例可能偏高，多数后代材料不能按照孟德尔遗传规律分离。生物鉴定结果说明，双价转基因抗虫棉同样具有时空表达性，在3代棉铃虫发生期间其抗虫性也有所下降，在4代棉铃虫发生期间又有所回升，形成一个波谷，但双价基因抗虫棉的抗虫性下降幅度小于单价Bt基因抗虫棉，其抗虫稳定性优于单价Bt基因抗虫棉；在棉花生长中后期，双价基因抗虫棉生殖器官如花、棉蕾等组织仍具有较强抗虫性。试验证明，在实际生产中，短时间环境条件的突然变化如温度等不影响双价转基因抗虫棉的抗虫性表达。双价转基因抗虫棉叶片ABA含量低于常规对照品种，IAA含量高于常规品种，表明双价转基因抗虫棉叶片中期生长旺盛，有利于提高光合强度，增加光合产物，提高生物产量。 研究结果对双价转基因抗虫棉在生产中的推广应用及其综合防治等具有一定的指导意义。更多还原

【Abstract】 In 2001, insect-resistant transgenic cotton were grew in 1.6 million ha, and it was about 40% of the planting area of Chinese cotton, but they were almost Bt transgenic cotton. Studies showed bollworm possibly produced some resistance to Bt transgenic cotton after massive planting. Therefore, it was very important of the introduction the synthesized double-gene, GFM CryIA gene and modified CpTI gene, to the elite cotton (G. hirsutum L.) and the production of new style resources of insect-resistance were obtained, cultivation of new style insect-resistant transgenic cotton and rules of inheritance and their insect-resistance and main characters related to physiology and biochemistry were studied.Insect-resistant transgenic cotton plants harbouring double-gene were obtained by the pollen-tube pathway transformation system and the agrobacterium tumefaciens-mediated transformation system. Their selection and identification, studies on space-time expression of the target-gene, influences from adversities, molecular analysis, etc. were proceeded by kanamycin selection, ELISA detection, bio-identification, PCR amplification, Southern Blotting; their inheritance rules was resulted from the separation results of their offspring, their physiological and biochemical characters was studied by activities of enzymes, contents of endogenous hormones, the trends of contents variations of MDA, proline, chlorophyll ,etc.Based on my previous works, the techniques of the pollen tube pathway transformation system and agrobacterium tumefaciens-mediated transformation system were improved, main parameters of technical were optimized, the genotypes of the cotton acceptors were widen, a highefficiency of transformation was achieved, a technical system of transformation on a large-scale was established and a lot of transgenic cotton plants harboring double-gene were acquired. In my experiment, I studied the separation results of the insect-resistance to bollworm of part of the transgenic cotton plants, analyzed and compared their insect-resistance at different growth stages and their insect-resistance of different organs at same growth stages, analyzed and compared the sensitivities of bollworm of different instar of larvae, sensitivities of different generationsbollworms to the transgenic cotton. Leaves. Imitated some special and variable characters of climate and condition of environment, I studied the influences of variations of temperature and the treatments by drought and NaCl, etc to the transgenic cottons. I Studied the activities of SOD, CAT, POD and the trends of contents variations of MDA, proline and chlorophyll and endogenous ABA,IAA of the transgenic cottons.The results showed the surviving rates of the regeneration transgenic plants of different varieties exceed 90% by grafting. PCR and Southern blotting detection results showed the existence of the target gene and indicated the Bt and CpTI genes were inserted into cotton genome. The results of inheritance indicated a lot of transgenic cottons harboring double-gene by the pollen tube pathway transformation did not fit to Mendel Rules that resulted from many copies of insert-gene in the transgenic cotton plants. Same as Bt transgenic cotton, the space-time expression of insect-resistance of the transgenic cotton harboring double-gene to bollworm was different in different times and organs, their insect-resistance had a descending trend during 3rd generation, and afterward, it began to rise during 4rd generation, but the descending extent of the transgenic cottons was less than that of Bt transgenic cotton, the stability of their insect-resistance to bollworm was more than that of Bt transgenic cotton. In the middle-last days of growth, the insect-resistances of the flowers and buds of the transgenic cottons were more than that of its leaves. The research results showed the insect-resistance of the transgenic cottons did not affected by the changes of temperature in the process of growth, and sudden-variations of environment did not influenced the expression更多还原