柱花草炭疽菌突变体库致病缺陷转化子筛选及T-DNA插入位点侧翼序列分析

Screening of pathogenicity defect mutant in Colletotrichum gloeosporioides of Stylosanthes guianensias and molecular cloning of T-DNA integration flanking sequence

摘要: 比较了接种病原菌侵染柱花草的几种方法的效果，选定出最佳的初筛和复筛方法，进而对1 230 个转化子进行筛选，得到致病力缺陷菌23 株，其中致病力减弱的菌株18 株，致病力完全丧失的5 株。利用TAIL-PCR 扩增致病力丧失突变子t-2430 的T-DNA 插入位点侧翼序列，得到大小为476 bp 的一段序列，BLAST 比对已测序炭疽菌基因组，发现401nt 和数据库的Contig464的部分序列完全一致，进一步对该段序列的功能进行预测，表明T-DNA 插入在一预测基因的启动子区域，并且这个预测基因与稻瘟病菌(Magnaporthe oryzae）的天冬氨酸转氨酶(XP_003719674.1）同源性为79%。

Abstract: Several inoculation methods were compared to test their effect of pathogenicity to Stylosanthes guianensias. In all 1 230 mutants, 23 had decreased weak pathogenicity, 5 even completely losted its ability to infect S. guianensias. TAIL-PCR was used to identify the T-DNA integration site in the mutant that was coded t-2430 and the sequence of 476 bp was gotten. The flanking sequence of T-DNA was compared with Colletotrichum genome by blast and 401nt of sequence was found in Contig464 of Colletotrichum genome. By predicting the function of flanking sequence, we found T-DNA insertion in the promoter region of putative gene, which had 79% homology to Magnaporthe oryzae aspartate aminotransferase gene (XP_003719674.1).