【作者】 王秋华；

【导师】 王政逸；

【作者基本信息】 浙江大学 ， 植物病理， 2004， 硕士

【摘要】 尖孢镰刀菌(Fusarium oxysporum)是一种世界性分布的重要植物病原真菌，寄主范围广泛，可引起100多种植物的维管束萎蔫病害，了解它的致病分子机理对植物维管束萎蔫病害的有效防治具有重要意义。显然，通过致病相关基因的鉴定是达到这一目标的关键所在。许多致病相关基因是通过DNA插入突变产生致病缺陷突变而分离得到的。限制酶介导整合转化(restriction enzyme-mediated integration，REMI)正被越来越多地应用于丝状真菌致病相关基因的标记与克隆。另外，近年来农杆菌介导的DNA转化(Agrobacterium tumefaciens mediated transformation，ATMT)也开始应用于一些丝状真菌的转化。 本研究较系统地对尖孢镰刀菌的REMI转化体系进行了优化，并对部分转化子的表型进行了初步的分析。研究结果表明尖孢镰刀菌菌体的培养时间、胞壁降解酶的种类、酶解时间和温度、再生培养基等一系列因素对原生质体释放及再生有明显的影响。根据研究结果总结出一套有效的尖孢镰刀菌原生质体制备的方法：从PDA固体培养基上培养8天的菌落上洗下尖孢镰刀菌孢子，将孢子按浓度7.4×10~7～1.0×10~8个／100ml接种到PDB液体培养基中，27℃下振荡培养11～13个小时，过滤洗涤收集菌丝，然后用12mg／ml溶壁酶和4mg／ml蜗牛酶组成的混合酶在33℃、80rpm的条件下酶解4～5个小时，过滤、洗涤、收集原生质体。一般300ml菌丝培养液可以得到3ml左右、浓度为5.0×10~7～1.44×10~8个／ml的原生质体。通过对比实验表明，不同的质粒和限制性内切酶的种类对尖孢镰刀菌的REMI转化效率也有较明显的影响。在相同实验条件下，pCB1004质粒的REMI转化效率比pCB1003的高；用内切酶BamH Ⅰ介导的转化效率也比Hind Ⅲ的高。实验结果还表明，REMI的转化效率是一般质粒转化效率的19.12～22.72倍。 本文利用限制酶介导DNA插入突变(REMI)转化尖孢镰刀菌黄瓜专化型代表菌株ATCC16416，共获得了123个尖孢镰刀菌转化子。对以上所得的123个转化子进行生长速度及产孢量的初步测定，发现产孢量比ATCC16416降低了1～4.5倍的有13个转化子，产孢量升高了1.35～3.59倍的有12个转化子。对所 浙江大学硕士学位论文有转化子在PDA上生长速度的测定结果显示，其中10个转化子的生长速度比ATCCI 6416有不同程度的减慢。对以上生长速度变慢的10个转化子做了初步的致病性分析，发现H60和52两个转化子的致病性变弱。 本文还构建了1个适用于丝状真菌农杆菌介导转化(ATMT)和基因突变的双元载体AfMTI。该质粒是在pCAMBIA1300质粒的基础上构建的，其关键之处是用仰C启动子代替了355(CaMN35S)启动子，使之适合丝状真菌的转化。更多还原

【Abstract】 Fusarium oxysporum causes plant vacicular wilt diseases worldwide and is one of the most economically important phytopathogens. A better understanding of the molecular basis involved in the pathogenesis of the fungus would be invaluable in obtaining targets for fungicide fungicide development and therefore control of the disease. Obviously, the identification of genes required for pathogenicity is a key step in achieving this goal. REMI(restriction enzyme-mediated integration), a widely applied gene tagging technique, is an efficient way to identify genes required for pathogenicity in some filamentous fungi. Recently ATMT (Agrobacterium tumefaciens mediated transformation) has also shown to be potentially useful for the transformation of filamentous fungi.In this paper, REMI transformation technique of Fusarium oxysporum f. sp. cucumerinum was established and some transfomation parmeters were optimized. The results showed REMI tranformation efficiency of the fungus was significantly increased by the improvement of protoplast preparation and regeneration. Meanwhile an optimized protocol of the protoplast preparation and regeneration of Fusarium oxysporum was developed by analyzing key factors which could influence protoplast release and vigor, such as fungal cultures, cell wall degradation enzymes, digesting conditions and regeneration media. Normally we could isolate 3ml protoplasts of Fusarium oxysporum with 5.0 X 107~1.44X 108 cell/ml by digesting fungal culture in osmotic buffer containing 12mg/ml lywallzyme and 4mg/ml Snailase for 4-5h. In REMI transformation of Fusarium oxysporum, we also found that plasmids and restriction enzymes used in the transformation can effect on the transformation efficiency. The results showed that pCB1004’s REMI transformation efficiency is higher than pCB1003’s and the BamH I’s REMI transformation efficiency is higher than Hind Ill’s. REMI transformation efficiency was 19.12~22.72 times higher than normal plasmid transformation without restriction enzyme.Based on REMI transformation techniques, we obtained 123 REMI transformants of Fusarium oxysporum f. sp. cucumerinum ATCC16416. We had analysed some phenotypes of these transformants, such as growth, conidiation and pathogenicity. Thirteen transfonnants were reduced in conidiation by 1-4.5 times that of the wild strain. Twelve transformants were 1.35-3.59 times more in conidiation. Ten transfonnants grew slower on PDA than wild strain. Meanwhile, the results of pathogenicity assay showed two of the transformants were of reduced virulence.In this study, a binary vector ATMT1 was also constructed based on pCAMBIA1300. The key point in this construction was 35S (CaMV35S) promoter replaced by trpC promoter to make it suitable for the transformation of filamentous fungi.更多还原