【作者】 林静；

【导师】 王才林；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2006， 硕士

【摘要】 抗除草剂转基因水稻在快速鉴定和提高杂交稻纯度方面表现出明显的优越性。通过基因枪法、农杆菌介导法和花粉管通道法等途径已获得多例抗除草剂转基因水稻。而通过杂交、回交将抗Basta除草剂的bar基因转育到其它品种,是获得抗除草剂转bar基因水稻新品系的一种简便有效的方法。因此有必要对外源基因bar在回交转育后代中的遗传表现进行研究。本研究通过对转bar基因水稻人工杂交与回交后代除草剂抗性的连续跟踪鉴定,研究了外源基因bar在水稻杂种后代的遗传表现,并着重对抗除草剂水稻9311HR中bar基因异常分离的遗传机制作了进一步分析,主要结果如下:1、以从中国水稻研究所引进的基因枪转化粳稻京引119获得的抗除草剂转bar基因材料HR为供体亲本,与扬稻6号(9311)、E32、扬稻4号、2403、苏御糯、宁恢1号和镇香粳等7个目标亲本进行杂交并通过连续回交与自交,获得的F1、F2、F3、BC1F1、BC1F2、BC1F3、BC2F1、BC2F2和BC2F3等世代为材料,对不同分离世代bar基因的遗传分析表明,2403、苏御糯、宁恢1号和镇香粳等粳稻材料与HR杂交后代的除草剂抗性分离符合一对显性基因的遗传,而9311等籼稻材料与HR杂交后代的bar基因遗传表现异常,抗性单株自交后代始终表现1:1分离。2、以9311回交转育获得的高世代回交自交群体9311HR为材料,研究了外源基因bar的导入对其主要农艺性状、花粉育性、花粉萌发率的影响,结果表明抗除草剂基因bar的导入对目标亲本的主要农艺性状无明显影响,9311HR抗性植株正常染色花粉率及花粉离体萌发率均在90%左右,其结实率正常,与轮回亲本9311均无显著差异。花粉DNA的PCR分析结果显示,9311HR抗性植株形成了携带外源bar基因的成熟花粉。这些结果表明9311HR抗性植株不存在雌雄配子败育。3、以9311HR抗性单株作母本,常规非转化品种为父本杂交获得的F1,除草剂抗性植株和非抗性植株比例为1:1;而以9311HR抗性单株作父本,与常规非转化不育系测交的后代均不抗除草剂。PCR分析结果显示,以9311HR抗性单株作母本获得的F1抗性植株PCR扩增呈阳性,非抗性单株PCR扩增呈阴性;而以9311HR抗性单株作父本获得的F1后代PCR扩增均呈阴性。表明9311HR中外源基因bar仅能通过雌配子传递给后代,而携带bar基因的粳型雄配子未参与授精。4、以9311与HR杂交构建的368个F2单株,为作图群体,利用公共图谱的分子标记对外源基因bar进行定位的结果表明,bar基因插入位置位于第3染色体上SSR标记RM5551和RM6080之间,遗传距离分别为4.4cM和1.2cM。推测外源基因bar与位于第3染色体的配子体基因ga2紧密连锁,导致携带bar基因的粳型雄配子败育,从而引起9311HR除草剂抗性的异常分离。更多还原

【Abstract】 Herbicide-resistant transgenic rice has obvious superiority on improving and fast checkup seed purity in hybrid rice. Some herbicide-resistant transgenic rice varieties were obtained by particle bombardment, Agrobacterium tumefaciens and pollen-tube pathway method. However, transfer herbicide-resistant gene bar into other variety by cross and backcross is a simple and effective approach to getting new transgenic rice material resistant to herbicide“Basta”. So, it is necessary to study the inheritance of foreign gene bar in the backcrossing progeny. In present experiment, the inheritance of foreign gene bar was studied by the tracking investigation of the herbicide resistance of generations crossed or backcrossed with bar transgenic rice, and more analysis have been made for the abnormal segregation of foreign gene bar in herbicide-resistant rice 9311HR. The main results obtained were as follows.1. By cross breeding, the herbicide-resistant gene bar from the donor, a genetically modified organism (GMO) HR obtained from introduction of bar into japonica rice Jingyin119 by biolistic method, was transferred into the breeding lines Yangdao 6(9311), E32, Yangdao 4, 2403, Suyunuo, Ninghui 1 and Zhenxiangjing. Some lines of bar transgenic rice F1 , F2, F3, BC1F1, BC1F2, BC1F3, BC2F1, BC2F2 and BC2F3 have been developed by continuous backcrossing and self-crossing. The inheritance of bar gene in different separate population was studied. The results showed that the segregation of herbicide resistance was accorded with the law of Mendelian heredity of a single dominant gene in japonica varieties such as 2403, Suyunuo, Ninghui 1 and Zhenxiangjing. However, abnormal segregation was observed in indica variety 9311. The herbicide resistance would segregate again with a ratio of 1:1 in selfcrossing progenies from herbicide-resistant plants.2. The effects of foreign gene bar on main agronomic traits, pollen fertility and pollen germination were studied in 9311HR which developed by backcrossing from 9311 and HR. Results indicated that the herbicide-resistant gene bar had no significant effect on the main agronomic traits of herbicide-resistant plants of 9311HR. The pollen fertility and in vitro pollen germination rate for herbicide-resistant plants of 9311HR were about 90%. Also, the herbicide-resistant plants of 9311HR showed normal seed fertility. There existed no siginificant difference in pollen fertility, in vitro pollen germination rate and seed fertility between 9311HR and 9311. Moreove, PCR analysis of pollen DNA indicated that mature pollen which carrying foreign gene bar were formed in herbicide-resistant plants of 9311HR. Above results sugested that there was no gametal sterility in herbicide resistant plants of 9311HR.3. In order to know the heredity transmission of bar gene in the progeny, the herbicide-resistant plant segregated from 9311HR was used as female or male to cross with conventional rice variety Zhenxian 96 or (HR-2×881)F9 or CMS line, and the segregation ratio of herbicide-resistant to sensitive plants was investigated in the test F1 poppulation. When the resistant plant was used as female parent, the segregation ratio of resistant to sensitive plants was 1:1. However, when it was used as male parent, all the progenies were sensitive to herbicide. Result of PCR analysis indicated that bar gene amplification of the herbicide resistant plants were positive and the herbicide sensitive plants were negative. The biological and genetic basis of distorted segregation of bar gene was dicussed and deduced on the basis of above results that foreign gene bar only could be transferred to progenies by pistils. The authors are inclined to ascribe these phenomena to the absent impregnation of japonica pollen which carried herbicide-resistant gene bar.4. To genetically map the foreign bar , 368 F2 plants developed by crossing 9311 with HR were used as mapping population. Genomic DNA from F2 plants of 9311×HR was extracted and analyzed for co-segregation, using available molecular markers. Mapping results showed that the foreign gene bar was located between SSR marker RM5551 and RM6080 at a distance of 4.4cM from RM5551 and 1.2cM from RM6080 on chromosome 3. We presumed that foreign gene bar was tightly linked with gametophyte gene of ga2 on chromosome 3. It conduced gametophytic sterility of japonica male gametes which carried foreign gene bar. Thereby, it caused abnormal segregation of herbicide resistance in 9311HR.更多还原