【作者】 杨慧；

【导师】 杨世湖；

【作者基本信息】 南京农业大学 ， 遗传学， 2006， 硕士

【摘要】 Pib是利用图位克隆技术从水稻克隆出来的第一个抗稻瘟病主效基因。由于Pib基因来自基因组文库，其DNA序列保持了该基因在原基因组中的自然状态。这为从分子水平研究稻瘟病抗性基因分子结构与抗稻瘟病功能之间的相互关系提供了材料。 首先采用DNAman软件对全长Pib基因序列进行酶切位点分析。 其次根据Pib基因的结构特点，将Pib结构基因克隆进双元载体构建了5个植物表达载体pNAR501、pNAR502、pNAR503等，这5个载体分别由35S驱动。pNAR501携带Pib结构基因的外显子2-外显子3，pNAR502携带5’端部分缺失外显子1，pNAR503携带5’端部分缺失的外显子1-外显子2-外显子3的3个不同基因组片段等。 再次用农杆菌介导法获得了上述表达载体的水稻品种日本晴转基因植株30株。 随后经PCR、Southern和后代潮霉素抗性分离检测确证外源基因已整合进水稻基因组。 最后进行T₀代和T₁代转基因植株的稻瘟病接种鉴定。T₀代转基因植株分蘖期大苗离体叶片抗稻瘟病鉴定结果表明三个构建的转基因植株的抗稻瘟性都显著高于对照日本晴，其中又以pNAR501为最高，对E、F、G三个小种抗性均为2级。T₁代3-4叶种子苗活体接种鉴定结果与T₀代离体叶片鉴定结果不同，三个构建的转基因植株后代对E、F、G三个小种抗性均为8级，低于对照日本晴种苗（7级）。更多还原

【Abstract】 The Pib gene is the first major resistant rice blast gene that was cloned by map-based clone technique. This Pib gene was isolated from rice genome, its sequence of DNA kept the original state. It is a good material for studying the correlation of the structure function of resistance gene in the molecular level.First, the sequence of Pib was analyzed using DNAman.Second, five plant binary expression vectors pNAR501, pNAR502 and pNAR503 et al driven by CaMV35S constitute promoter were constructed respectively according to the structure characteristics of open reading frame of Pib gene. Exon2-exon3 fragment of Pib gene was constructed into pNAR501. 5’ partial deletion exonl fragment of Pib was constructed into pNAR502. 5’ partial deletion exonl- exon2-exon3 fragment of Pib was constructed into pNAR503 et al.Third, the vectors were transformed into calli from mature embryos of Nipponbare by Agrobacterium mediated transformation, and 30 independent transgenic plants were obtained.Forth, transgenic plants were analyzed by PCR, Southern and Hyt resistance.Last, T₀ and T₁ transgenic plants were inoculated rice blast pathogen.Rice blast resistance test in vitro for T₀ transgenic plants to test their blast resistance showed higher blast resistance than that of control Nipponbare and transgenic plants of pNAR501 were highest resistance in three constructions which was class 2 to El, F1 and G1 races. But the results of rice blast resistance test for T₁ transgenic plants in 3-4 leaves stage showed different: all of the transgenic progeny were worse than that of control and all were class 8 to E1, F1, G1 races of rice blast while resistance of control Nipponbare was only class 7.更多还原