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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。
Through the determination of the pathogenicity of each transformant in the T-DNA mutant library of Colletotrichum which has been constructed in the laboratory, a virulence mutating strain 1869 was obtained. PCR was carried out to verify the insertion of T-DNA in the genome of 1869. The biological characteristics such as colony diameter, colony morphology and sporulation were observed. TAIL-PCR was used to clone the flanking sequence of the gene and bioinformatics methods Compare gene information. The results showed that compared with the wild type strain CH008 of Stylosanthes anthracnose, the mutant strain 1869 showed a loss of virulence and a colony growth rate significantly lower than that of the wild type. However, in conidial morphology, sporulation ability, germination rate of spores There is no significant difference between the wild type and. TAIL-PCR amplified 467bp of the RB end sequence of the T-DNA insertion site and 388bp of the LB-terminal flanking sequence. The sequences flanked by the two flanking sequences have a homology of 92% -96% with Pac1, which may be due to the gene expression The product regulates the pH sensitivity of the strain, which affects its pathogenicity.