甘蔗梢腐病菌t与-dna插入突变体库的构建和分析.docxVIP

甘蔗梢腐病菌T-DNA插入突变体库的构建和分析 甘蔗梢腐病菌T-DNA插入突变体库的构建和分析 摘要 甘蔗梢腐病是世界范围内重要的经济作物真菌性病害，其病原真菌为 轮枝镰孢菌(F．verticillioides)和层出镰刀菌(F．proliferatum)。通过农杆 菌介导的遗传转化分别构建了这两个镰刀菌种(菌株CNO．1和YN41)的 突变体库，并利用高效TAIL．PCR和菌落形态对突变体库进行鉴定。结果 分别构建了库容量为195的YN41突变体库和库容量为956的CNO一1突变 体库。从两个突变体库中分别随机挑选6个转化子，进行PCR鉴定，结果 均为阳性。通过菌落特征和致病性筛选YN41和CNO。1突变体库，鉴定出 生长速度减慢且致病力下降突变体1个(A5)，生长速度减慢且致病力不变 突变体1个(C8)，生长速度加快且致病力增强突变体2个(A1、A2)，菌 丝变浓密且菌丝疏水性变弱突变体1个(A3)；采用高效TAIL．PCR对这些 转化子的T-DNA侧翼序列进行扩增，获得了A1、A2、A3突变体的T-DNA 侧翼序列，通过BLAST鉴定到其插入位点(下游)基因，这些基因分别与 色氨酸加氧酶和苯丙氨酸解氨酶等有关。CNO一1突变体库中，筛选出生长 速度减慢突变体23个，色素变异突变体1个；对47株突变体进行高效 TAIL．PCR，扩增插入位点侧翼序列，得到有效序列22条，BLAST结果显 示，T-DNA的22个插入位点分布在CNO．1的15条基因组scaffold上；对 插入位点(下游)基因进行分析，结果显示，生长速度减慢突变体的插入 失活基因分别与细胞分裂、DNA修复、△12脂肪酸脱氢酶、E3泛素连接 酶等有关。 关键词：遗传转化，插入突变体库，表型变异，侧翼序列 万方数据 CONSTRUCTION CONSTRUCTION AND ANALYSIS OF T-DNA INSERTIONAL Ⅳ叮TANT LIBRARIES 0F FUSARIUM SPECIES CAUSED SUGARCANE POKKAH BOENG SUGARCANE AB STRACT Sugarcane pokkah boeng is an economically important fungal disease worldwide，which are caused by two species of Fusarium，F．verticillioides and F．proliferatum．Two T-DNA insertional mutant libraries of these two Fusarium species(isolate CNO一1 and YN4 1)were generated by Agrobacterium tumerfaciens mediated transformation(ATMT)and analyzed by Hi TAIL—PCR and fungal characterizations．The results indicated that two library of 1 95 and 956 insertional mutants were generated from isolate YN4 1 and CNO一1， respectively．Six mutants were randomly selected from each library and identified to be positive by PCR amplification．From YN4 1 library,five mutants were identified by fungal characterization and pathogenicity,including one mutant(A5)with slower growth phenotype and lower pathogenicity；one mutant(C8)with slower growth but less different pathogenicity；two mutants (A1 and A2)with more growth and higher pathogenicity；one mutant(A3)with denser mycelium and weaker hydrophobic ability．The flanking sequences of ll 万方数据 T-DNA T-DNA from the mutants A 1，A2 and A3 were amplified and sequenced by Hi