【作者】 葛小佳；

【导师】 王石平；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 白叶枯病（Bacterial Blight，BB）是世界各地水稻生产中危害十分严重的病害之一，由黄单胞杆菌水稻变种（Xanthomonas oryzae pv．Oryzae，Xoo）引起。借助于转基因技术的抗病育种被认为是有效而直接的方式越来越受到人们的重视。目前，至少有8个水稻白叶枯抗性基因（Xa1，Xa2，Xa3，Xa4，xa5，Xa10，xa13和Xa21）被转入籼稻品种IR24中，产生了一系列以IR24为遗传背景的抗白叶枯近等基因系。本室在水稻白叶枯病抗性基因的克隆和相关基因的鉴定上开展了大量的工作，已经将Xa4和xa13定位于来自近等基因系品种的DNA片段中，另外，从这些近等基因系中也获得了大量的抗病相关基因。然而，目前对该基因型品种的组织培养体系还不成功，大大限制了这些抗病基因和抗病相关基因的功能验证。因此，本研究立足服务于本室的白叶枯病抗病基因克隆和转基因的需要，通过对籼稻组织培养体系的优化，摸索一套适合于以IR24为代表的白叶枯病近等基因系的转基因组培体系，从而可以推动抗白叶枯病研究的进程。同时，由于IR24也是国际上，尤其是东南亚、南亚国家主栽品种的亲本来源之一，以该基因型为背景的有关水稻各性状的研究开展得非常广泛，因此，该转基因体系的建立可以更好地推动基因工程育种。 通过调整培养基中植物生长调节因子（plant growth regulators，PGRs）、有机成分和无机盐的水平，分别筛选出适合籼稻的愈伤诱导以及继代的培养基。实验表明：IRBB13、IRBB10、IRBB4、IR24、明恢63、珍汕97和93-11共7个籼稻品种的成熟种子通过优化培养基的诱导以及继代过程，能够产生大量胚性愈伤；相对于MS基本培养基，愈伤组织的生长率和再生率都得到了提高，能够作为农杆菌介导的遗传转化的理想受体。另外，参考本室林拥军老师建立的籼稻遗传转化体系，在这两种培养基的基础上，进一步确立了转化过程中预培养、共培养以及筛选的培养基，并完成了IRBB13的高效遗传转化。 另外，本室储昭晖利用同源序列法在明恢63中分离了基因22E6EI14，表达分析表明该基因在抗病品种IRBB13中受白叶枯病原菌PXO99接种诱导表达，而在感病品种IR24中不表达，由此，储昭晖推测其可能参与xa13基因介导的抗病反应途径。为了验证抗病相关基因22E6EI14的功能，本研究利用双链RNA表达抑制（double-strand RNA interference,dsRNAi）技术对该基因进行表达抑制的遗传转化，观察转化植株的抗病表型变化。 通过3’-RACE（3’-rapid amplification of cDNA end）和RT-PCR（reverse transcription-polymerase chain reaction）分析，确定了22E6EI14的转录终止位点及基因内含子的剪切位点，序列分析显示该基因编码NBS类蛋白质。构建22E6EI14的双链RNA表达抑制载体，通过农杆菌介导的遗传转化方法转入抗病品种IRBB13，抑制该基因的表达。对获得的39株T₀代转化植株进行PCR阳性检测，其中有23更多还原

【Abstract】 Rice bacterial blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo), is one of the most devastating diseases in rice production worldwide. Gene transfer technology, as a powerful and direct tool to minimize the loss caused by plant diseases, has been getting more and more attention in resent years. Up to now, at least eight rice resistance genes, Xa1, Xa2, Xa3, Xa4, xa5, Xa10, xa13, and Xa21, for bacterial blight resistance have been introduced into rice variety IR24 to form a set of indica near-isogenic lines. In this lab, two bacterial blight resistance genes, Xa4 and xa13, have been targeted to DNA fragments from two near-isogenic lines with the genetic background of IR24 and a great number of resistance-responsive genes have also been identified from the same sets of rice lines. However, the lack of a successful tissue culture system based on these indica lines is still a major obstacle to functional complementary test of these candidate genes. Therefore, the establishment of effecient tissue culture system for the near-isogenic lines with an IR24 background will greatly facilitate molecular characterization of the R genes for bacterial blight resistance and clarification of the interaction between the R genes. On the other hand, IR24 is also one parent of some widely cultivated varieties worldwide, especially in Southeast Asia or South Asia. Genetic improvement of important rice qualities by way of gene transfer technology will also be benefited by this tissue culture system.By manipulating plant growth regulators (PGRs), organic components and salts within the culture media, we established two media for callus induction and subculture, respectively, in tissue culture of indica rice. The modified media could guarantee the production and proliferation of a great number of embryogenic calli with high regeneration capacity from mature seeds of different indica rice varieties, IRBB13, IRBB10, IRBB4, IR24, Minghui63, Zhenshan97 and 93-11. The calli obtained from this system should be ideal material for Agrobacterium-mediated transformation. Futhermore, on the basis of the composition of the induction and subculture media optimized in this study, the media for pre-culture, co-culture, and selection that were subsequent steps in Agrobacterium-mediated transformation of rice were also developed. With these media and the transformation protocols developed by Lin yongjun, we have succeeded in the efficient production of transgenic plants from rice line IRBB13.On the other hand, Chu zhaohui in this lab isolated a gene 22E6EI14 from Minhui 63. Expression of the 22E6EI14 was induced in IRBB13 after inoculation with an incompatible Xoo strain PXO99, but not in the susceptible line IR24. With the results above, Chu zhaohui supposed that 22E6EI14, as a defense-related gene, may be involved更多还原