希金斯刺盘孢T-DNA插入突变体表型筛选及其特性分析
Screening and characterization of T-DNA insertion mutants of Colletotrichum higginsianum

以希金斯刺盘孢菌株Ch-1为供试野生型菌株，通过农杆菌介导转化方法获得含2 000个转化子的T-DNA插入突变体库，筛选菌落生长异常或致病缺陷突变体，分析致病缺陷突变体的T-DNA 插入拷贝数和位点。结果显示，筛选到14株菌落异常突变体，包括2株生长缓慢突变体Ch-1-E393和Ch-1-C135、12株菌落形态异常突变体（包括色素异常、菌落扇变或菌丝坍塌现象），另外筛选到1株致病缺陷突变体Ch-1-G090。致病性测定结果表明，致病缺陷突变体Ch-1-G090接种拟南芥后，叶片发病明显减弱，且未产生水渍状病斑。显微观察发现该突变体分生孢子在叶片上仅能产生少量初生菌丝，不能正常形成次生菌丝。Southern 杂交显示，致病缺陷突变体Ch-1-G090为T-DNA双拷贝插入；通过Inverse-PCR法获得插入T-DNA的侧翼序列，明确该突变体T-DNA插入位点分别为假定蛋白（Ch063_10682）和RNA加工蛋白FCF1（CH063_10671）编码区。
In this study,insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) was used to build a T-DNA insertion library of Colletotrichum higginsianum containing a collection of 2 000 insertion mutants. From the library,development and pathogenicity deficient mutants were screened and T-DNA insertion copies and sites were analyzed. As a result,we isolated 2 growth-deficient mutants Ch-1-E393 and Ch-1-C135 with significant reduced growth on PDA medium,12 mutants with abnormal colonies and one pathogenicity deficient mutant Ch-1-G090. Pathogenicity assays showed that after inoculation of the pathogenic defective mutant Ch-1-G090 in Arabidopsis,the incidence of leaf diseases was significantly reduced. Under microscope,few primary hyphae were found in the leaves and no secondary hyphae was observed in Ch-1-G090. Southern blot analysis indicated that the mutant Ch-1-G090 harbored two T-DNA insertions. Border flanking sequences of T-DNAs from these mutants were recovered by Inverse PCR. Sequence analyses revealed that the two T-DNA insertion sites in the mutant Ch-1-G090 were coding genes for a hypothetical protein (Ch063_10682) and an RNA processing enzyme (CH063_10671)