柱花草炭疽菌致病力丧失突变菌株1869的T-DNA插入位点侧翼序列的克隆

通过对实验室已构建的柱花草炭疽菌T-DNA突变体库中各转化子致病力的测定,获得致病力丧失突变菌株1869。对其进行PCR检测以验证T-DNA在1869基因组中插入情况,并测定观察其菌落直径、菌落形态及产孢量等生物学特性,利用TAIL-PCR克隆标记基因侧翼序列,采用生物信息学方法比对基因信息。结果表明,与柱花草炭疽菌野生型菌株CH008相比,突变菌株1869表现致病力丧失,菌落生长速率也显著低于野生型;然而,在分生孢子形态、产孢能力、孢子萌发率等方面与野生型并无明显差异。TAIL-PCR扩增得到T-DNA插入位点RB端序列467bp,LB端侧翼序列388bp,经两侧序列拼接比对,所得序列与Pac1同源性达到92%~96%,推测可能由于基因表达产物调节菌株对pH值的敏感性,从而影响了其致病过程。

Molecular characterization of the flanking gene of T-DNA insertional,pathogenicity-defective Colletotrichum gloeosporioides mutant strain 1869

Colletotrichum gloeosporioides is a fungal pathogen of Stylosanthes guianensis causing anthracnose disease.Pathogenicity defective mutant strains of C.gloeosporioides were screened in the laboratory and strain 1869 selected for study.The mutant strain was compared with wild type strain CH008 for phenotypic charac-teristics,including colony diameter,colony morphology,sporulation ability and spore germination rate.Colo-
ny diameter of CH008 and strain 1869 differed,but the other traits did not.DNA of the mutant strain was ex-tracted,and using thermal asymmetric interlaced PCR (TAIL-PCR)a T-DNA insertion mutation site be-lieved responsible for loss of pathogenicity was identified.The RB and LB flanking sequences at the insertion site were cloned,also using TAIL-PCR.The RB flanking sequence was 467 bp in length,while the LB flan-king sequence was 388 bp.The BLAST result showed that the sequence had 92% -96% homology to gene Pac1.By predicting the function of a protein coded by the flanking sequence,we inferred that the gene in ques-tion regulated the sensitivity to pH,and in this way affected the pathogenic potential of C.gloeosporioides .