【作者】 俞竹青；

【导师】 杨仲南；

【作者基本信息】 上海师范大学 ， 遗传学， 2006， 硕士

【摘要】 花药及花粉的发育是植物基因功能研究的一个重要研究方向。花药及花粉发育异常会导致雄性不育，雄性不育是生产上杂交制种的基础。因此，对花药及花粉发育的深入研究不仅具有重要的理论研究意义，而且在作物育性的调控方面有着重要的实际应用价值。 目前，在拟南芥中已克隆到二十多个育性相关的花药特异表达基因，而在水稻中只有极少的几个花粉发育关键基因被分离鉴定。本论文利用反向遗传学的方法研究水稻中与拟南芥雄性不育基因相对应的同源基因在花药及花粉发育过程的功能。首先通过Blast检索水稻基因组，通过比较分析选定其中8个与拟南芥雄性不育基因同源的水稻候选基因，构建了这些基因的dsRNA表达载体。然后采用农杆菌介导法转化水稻粳稻品种日本晴(O．sativa japonica．)，得到不同数量的转基因植株。 其中，水稻P0491E01基因与拟南芥茉莉酮酸合成关键基因AtDAD1(DEFECTIVE IN ANTHER DEHISCENCE1)高度同源，同时又是AT4G16820基因的直系同源基因。首先从基因组DNA中克隆P0491E01一段263bp的外显子片段，构建dsRNA表达载体pP0491RNAi。经农杆菌介导法转化水稻，获得了10棵转基因植株。GUS染色和PCR鉴定结果表明，外源DNA已整合进了转基因水稻基因组中。其中3棵转基因植株出现不育性状，其余7棵植株育性均有不同程度下降。细胞学观察发现，不育植株的花药早期发育正常，但是小孢子未能正常发育形成成熟花粉粒。RT-PCR分析显示，转基因植株中P0491E01的表达量明显下降。这些结果表明P0491E01在花药发育后期花粉的成熟过程中起重要的调控作用。更多还原

【Abstract】 The development of the anther and pollen is an important aspect in the plant genomic research. The defection of anther and pollen development may result in male sterility. Male-sterile is the basis to produce hybrid seeds in agriculture. Thus, the research of anther and pollen development has importance for both basic research and agricultural application.At present, over twenty anther specific genes related to fertility have been cloned in arabidopsis. In rice, however, only a few genes essential for pollen development has been identified. In this paper, we investigated the function of rice genes homologous to Arabidopsis male sterile genes in the anther development using reverse genetics strategy. At first, a total of eight rice candidate genes were chosen via Blast search of Arabidopsis genes in rice genome. After the vectors for expressing dsRNA of these genes were constructed, they were transformed into O.sativa japonica by Agrobacterium-mediated transformation. Transgenic plants were obtained for all of these constructs.The rice P0491E01 gene shares high similarity in amino acid sequence with Arabidopsis genes AtDAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) which plays a key role in the biosynthesis of jasmonic acid. And it was found to be orthologous with AT4G16820 gene. At first, dsRNA expressing vector pP0491RNAi with 263bp-longed exon fragment of P0491E01 was constructed .Ten transgenic plants were obtained after Agrobacterium-mediated transformatioa GUS-staining and PCR analysis confirmed that T-DNA had integrated into rice genome. Three of the transgenic plants were male sterile, and the other transgenic plants showed reduced fertility. Cytological observation indicated that anther development in the early stage of male sterile transgenic plants was normal, however, the microspores could not develop into mature pollen grains. Further investigations of the expression of P0491E01 gene in these transgenic lines by RT-PCR revealed that its transcript was significantly reduced. Our results suggest that P0491E01 may play an important role during the late stage of anther development.更多还原