【作者】 潘初沂；

【导师】 王宗华；

【作者基本信息】 福建农林大学 ， 植物病理学， 2004， 硕士

【摘要】 由稻瘟病菌(Magnaporthe grisea(Hebert)Barr．，无性世代为Pyricularia grisea(Cooke)Sacc．)引起的稻瘟病是全球水稻生产上最主要的病害之一(Ou，1985；Zeigler et al．，1994)。由于稻瘟病菌的致病性复杂多样、生理小种多而且变异大，高抗水稻品种大面积种植往往3-5年便丧失抗病性。因此只有深入了解稻瘟病菌的致病性及其变异机制，特别是要研究无毒基因在稻瘟病菌致病演化中所起的作用，加深稻瘟病菌与水稻互作机制的了解，才能培育出抗瘟持久的水稻品种，为稻瘟病防治提供更加有效的策略。 本研究利用农杆菌介导的稻瘟病菌遗传转化，构建T-DNA插入突变体库，已得到6000个转化子，转化每1~*10~6个分生孢子可得到约300个稻瘟病菌T-DNA突变体，随机抽取80个转化子于含潮霉素的JM液体培养基中筛选，有72个具有潮霉素抗性，从中再抽取14个转化子进行PCR验证，综合结果推测可能得到5400个真正的T-DNA插入突变体。 随机选取的300个突变体分别接种在C101LACPi-1(t)、C101A51Pi-2(t)、C104PKTPi-3(t)、C101PKTPi-4a、C101TTP-4L-23Pi-4b、75-1-127Pi-9(t)及感病对照CO39上，进行致病性分析。所有接种的突变体中有32个致病性发生了变异。其中在CO39、Pi-4b和Pi-3(t)上致病性减弱(感病的4-5型反应变为3型以下的抗病反应)的突变体分别有3个、19个和1个。19个C101LACPi-1(t)致病性增强突变体中有17个由抗病变为感病；11个C101A51Pi-2(t)致病性增强突变体中8个由抗变为感；6个由抗变为感的C101PKTPi-4a致病性增强突变体；8个对75-1-127Pi-9(t)致病性增强突变体中3个由抗变为感。将无毒转变为有毒(4-5级致病类型)的菌株接种产生的感病病斑进行单孢分离，重新接种，筛选到11个强毒力(整体为感病反应)的单孢突变体菌株。 用分子方法对这些致病性变异的突变体进行了分析。普通PCR验证表明，它们都含有潮霉素磷酸转移基因，Southern blot杂交结果，4个致病性变异的突变体都只有T-DNA单拷贝，这有利于这几个突变体的后续分析。用TAIL-PCR扩增这些致病性变异的突变体，其中6个突变体已扩增到特异的片断，其中T940009401已测序，与稻瘟病菌contig2.1003中的福建农林大学硕_卜学位论文26462一27034bp相同，正好反向插在克隆末端OgD12上，离编码未知蛋白MG05451 .4有1 .2kb距离，如果此菌的T-DNA整合是通过双交换同源重组的，那么T-DNA可能交换掉蛋白MGO5451.4的启动子区，使该蛋白无法表达。对Pi一夕(t)表现为强致病力的PiZ一T940009701一1一2的不DNA左边界的侧翼序列与稻瘟病菌coniig2.1354中的11630一12304bp相同，正好反向插在mgns007xL21f.b 28 561:(12825一13461)BLATeeSPAN和mgns007xN17f.b28 504:(1 3049一13460)BLAT一PAN的两个ESTs序列区前，同样可能导致这两个ESTs表达受阻。更多还原

【Abstract】 The rice blast disease ,caused by the rice blast fungus (Magnaporthe grisea (Hebert) Barr. , Pyricularia gmea(Cooke)Sacc.) ,is the dominant rice disease worldwide. Due to the varieties of pathogenicity, the varieties of races and volatility of the races, the resistant rice variety will decrease in the resistance after application about 3 to 5 years. In order to cultivate the persistent resistant rice varieties and make out more powerful control strategy, we should understand the pathogenicity of rice blast fungus and the mechanism of variance deeply, especially we should find out the function of avirulent genes in the pathogenicity of blast.In the study, we constructed the T-DNA-inserted mutant library of the rice blast fungus by Agrobacterium tumefaciens-mediated transformation (ATMT), and got 6,000 transformants. In average, about 300 transformants could be achieved by transforming 1*106 conidia of the fungus. 80 transformants selected randomly were cultured them in the JM liquid media containing hygromycin, and 72 transformants could grow well. 14 of these transformants were positive transformant after being tested by PCR. So we confer that we can get 5400 positive T-DNA-inserted mutants.We selected 300 mutants randomly for virulence analysis on rice variety C101LACPi-1(t), C101A51Pi-2(t), C104PKTPi-3(t) , ClOlPKTPi-4a, C101TTP-4L-23Pi-4b , 75-1-127Pi-9(t) and CO39. Among them, 32 pathogenicity mutants were screened, 3 mutants were decreased in pathogenicity on CO39,19 mutants decreased on Pi-4b, and 1 was decreased on Pi-3(t). 19 mutants enhanced pathogenicity on C101LACPi-1 (t) ,and 17 mutants were compatible with C101LACPi-1 (t); 11 mutants’ pathogenicity were enhanced on C101A51Pi-2(t), and 8 mutants were compatible to C101A51Pi-2(t); 6 mutants were compatible with C101PKTPi-4a; 8 mutants’ pathogenicity were enhanced on 75-1-127Pi-9(t), and 3 mutants were compatible with 75-1-127Pi-9(t).The PCR results showed that pathogenecity mutants all had hph gene. Thesouthern-blot result showed that 4 pathogenecity mutants were only inserted by single copy of T-DNA, which is in favour of further analysis. Moreover, we analyzed the pathogenecity mutants by TAIL-PCR. We amplified certain fragment among 6 mutants. One in six mutants, T940009401, was sequenced, and its sequence was the same as contig 2.1003 of rice blast fungus from 26462 to 27034 bp; T-DNA was inserted in the end of clone 09D12, it was 1.2 kb away from the unknown protein MG05451.1; if T-DNA in mutant was integrated by double exchange, the promoter region of Protein MG05451.4 might be exchanged by T-DNA, and the protein would not express. The T-DNA-franking sequence of mutant Pi2-T940009701-l-2 was also sequenced, and its sequence is the same as contig 2.1354 of rice blast fungus from 11630 to 12304 bp; the T-DNA inserted site was in front of two close ESTs’ sequence, mgns007xL21f. b 28 561: (12825 - 13461) BLAT_SPAN and mgns007xN17f. b 28 504: (13049 - 13460) BLAT_SPAN, so may lead both ESTs not to express.更多还原