一株灰葡萄孢细胞壁完整性缺陷突变株的分子鉴定和表型分析

为探明灰葡萄孢细胞壁相关基因功能,用荧光增白剂Calcofluor White（CFW）从灰葡萄孢T-DNA插入突变体库中筛选细胞壁完整性缺陷突变株,发现一株突变株D-59对CFW的敏感性与野生型相比提高了2.8倍。通过TAIL-PCR扩增突变株的T-DNA插入位点的侧翼序列并对其进行序列分析表明,T-DNA插入于突变株BC1G₀0770.1基因的外显子部位。RT-PCR的结果显示,D-59的BC1G₀0770.1基因不表达。突变株D-59的表型分析表明,突变株的菌丝稀疏,菌落呈土黄色,产孢量明显下降,孢子萌发异常,致病能力减弱,细胞壁的几丁质含量下降了48%。

Molecular identification and phenotypic analysis of a Botrytis cinerea mutant defective in cell wall integrity

To study the cell wall related genes in Botrytis cinerea,T-DNA insertion mutants of Botrytis cinerea were screened by using Calcofluor White（CFW）,and a cell wall integrity defective mutant named D-59 was obtained.Compared with the wild-type strain,sensitivity of the D-59 mutant to CFW increased 2.8 times.The flanking sequence of T-DNA insertion site was amplified by TAIL-PCR and the sequences were analyzed.It revealed that the T-DNA inserted in the extron part of a predicted protein coding gene（BC1G₀0770.1） in B.cinerea genome.As expected,no expression of BC1G₀0770.1 in the mutant was detected by RT-PCR experiment.It was found that D-59 showed sparse hyphae,khaki colonies,decreased spores,abnormal spore germination,decreased pathogenic ability,and the cell wall chitin content decreased by 48%.