【作者】 李宏宇；

【导师】 王宗华；

【作者基本信息】 福建农林大学 ， 植物病理学， 2003， 博士

【摘要】 稻瘟病菌(Magnaporthe grisea)既是经济重要的病原真菌，也是研究植物与微生物相互作用的重要生物，从全基因组水平分析研究该菌关键基因的功能，可以加速对稻瘟病菌生物学及其致病分子机制的认识，为培育持久抗病品种和制定稻瘟病菌持续管理的策略提供理论依据。为此，本研究利用农杆菌介导转化T—DNA插入技术，大规模创建稻瘟病菌T-DNA插入突变体，并对部分突变体进行了分析，取得了如下结果： 在Mullins等建立的农杆菌转化Fusarium方法基础上，进一步优化了农杆菌介导转化稻瘟病菌获得T-DNA插入突变的条件，包括选择转化子的潮霉素B用量、抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比、不同转化阶段培养基的选择等。 多次转化结果表明农杆菌转化稻瘟病菌分生孢子的效率为每转化1*10~6孢子可以得到约385个插入转化子。分子检测表明所有转化子均含hph基因插入，但是只有约85％含T-DNA插入。Southern杂交结果表明突变体中含1—3个拷贝的T—DNA插入，其中50％为单插入。 随机取120个突变体进行形态发育观察，发现了13个颜色突变体、5个生长速度减慢突变体、14个孢子形态和产孢突变体、2个孢子萌发和10个附着胞形态和形成突变体。 在C101LACPi-1(t)、C101A51Pi-2(t)、C104PKTPi-3(t)、C101PKTPi-4a、C105TTP-4L-23Pi-4b、BL22(Pi-9)、多系1号、CO39和LTH等9个品种上，对160个突变体进行毒性分析，结果发现15个致病性减弱突变体，40个致病性增强突变体。在致病性增强突变体中，有17个突变体致病性由无毒变为有毒。 TAIL-PCR可以有效地扩增T-DNA插入侧翼稻瘟病菌基因组序列，AD9是最佳随机引物。对10个TAIL-PCR特异产物测序分析，结果表明6个T-DNA插入在基因上，4个则插入在基因间隔区中，并将10个侧翼序列登陆在GeneBank上，登陆号为AY295779-AY295788。 推测蛋白MG03076.1、MG09558.1、MG08181.1被标签后，其对应的突变体T940001901、T940003102和T940002001分别表现为对Pi-1(t)致病性增强；对CO39的致病性显著减弱；对Pi-1(t)和Pi-4a的致病性增强，可能表明这些基因与稻瘟病菌致病性有关。福建农林大学博士学位论文 突变体T940000302对尸i一I(t)和pi-甲b致病性增强，T94000一001对尸1-1(t)从无毒变为有毒，T940001 604对Pi-I(t)和Pi一夕的致病性增强，但是分析其T-DNA插入却发现是分ZIJ插在MG02674.1与MG02675.1、MG09558.1与MG09559.l、MG08092.1与MG08093.l之间隔区中，说明T-DNA插在与致病性有关的基因调控区。 在突变体T940004402中，T-DNA插在稻瘟病菌推导蛋白NCU03 146.1上，该蛋白在Saccharo袱lyces cerevisia。和Schizosaccharomycespombe中也有同源蛋白，但未观察到任何表型发生变化。 本文利用农杆菌转化方法成功地将T-DNA插入稻瘟病菌基因组，获得了一批基因被标签的突变体，并发现了若干关键性状对应的基因，是稻瘟病菌功能基因组学的良好开端，具有重要的科学意义和应用前景。更多还原

【Abstract】 The rice blast fungus (Magnaporthe grisea Barr.) is important both economically and in the study of plant-microbe interaction. Dissection of gene functions at the whole genome level will speed up understanding of the fungal biology and molecular mechanisms regulating the fungal pathogenicity and facilitate the rice breeding for durable resistance and the disease integrated mangament. Therefore, we studied on large scale T-DNA insertional mutagenesis of the fungus by Agrobacterium fumefaciens-mediated transformation (ATMT) and achieved the following results.The conditions for ATMT in the rice blast fungus was further optimized based on the basic procedures established in Fusarium by Mullins and co-workers, including the optimal concentration of hydromycin B in transformant selection, the ratio of cefotaxime to carbenicillin for Agrobacterium inhibition, the efficient medium in the different stage of transformation,etc.In average, about 385 transformants could be achieved by transforming 1*106 conidia of the fungus. Polymerase chain reaction (PCR) and Southern blot analysis showed that all transformants had hph gene insertion but only 85% had T-DNA insertion, among which there were one to three copies of T-DNA insertion with 50% having single-copy insertion.Morphology and development were observed with 120 randonly selected mutants. Thirteen mutants were found mutated in colony color, 5 in growth rate, 14 in conidium morphology and sporulation, 2 in spore germination and 10 in appressorium development.Virulence analysis of 160 mutants was conducted on rice variety C101LACPi-1(t), C101A51Pi-20, C104PKTPi-3(t), C101PKTPi-4a, C105TTP-4L-23Pi*46, BL22Pi-9, Duoxi . No.1, CO39, and LTH. Fifteen mutants were weakened and 40 were enhanced in pathogenicity. Seventeen out of 40 pathogenicity-enhanced mutants changed from avirulent to virulent.Thermal asymmetric interlaced PCR (TAIL-PCR) was efficiently used to amplify the T-DNA flanking regions. AD9 was the most suitable arbitrary primer among 9 primers used.Among 10 specific tertiary TAIL-PCR products sequenced, 6 mutants were found with T-DNA insertion within gene regions and 4 with T-DNA insertion in the region between two genes. The sequences of T-DNA flanking regions were submitted to GenBank (Accession numbers AY295779-AY295788).It was found that the hypothetical protein MG03076.1, MG09558.1 and MG08181.1 were tagged respectively in mutant T940001001 T940003102 and T940002001 which showed pathogenicity-enhanced on Pi-l(t), pathogenicity-decreased on CO39 and pathogenicity-enhanced both on Pi-1(t) and Pi-4b, which suggested that these be pathogenicity related.genes.It was also found that mutant T940000302 was enhanced in pathogenicity on Pi-1(t) and Pi-4b, T940001001 changed from avirulent to virulent on Pi-1(t), and T940001604 was enhanced in pathogenicity on Pi-1(t) and Pi-9. However, the T-DNA was found to insert respectively in between hypothetical proteins MG02674.1 and MG02675.1.MG09558.1 and MG09559.1, MG08092.1 and MG08093.1, which suggested that T-DNA insert at the promoting regions regulating the pathogenicity.Mutant T940004402 had T-DNA inertion at the hypothetical protein NCU03146.1 which homologs were found both in Saccharomyces cerevisiae and in Schizosaccharomyces pombe, but no known functions and mutated phenotypes were observed.In summary, T-DNA was successfully inserted into genome of the rice blast fungus by ATMT technique, a large scale of T-DNA insertional mutants were generated, and several genes related to important biological functions were found to be tagged by T-DNA in this study. This is a good start for functional genomics of the fungus and will have important impact in science and future application.更多还原