灰葡萄孢BcPDR1基因与PKA编码基因的关系研究

A cross-regulation between BcPDR1 and PKA coding genes in Botrytis cinerea

为明确灰葡萄孢BcPDR1基因与病菌cAMP信号途径中PKA编码基因BcPKA1、BcPKA2、BcPKAR之间的关系,利用Real-time PCR技术分析了BcPDR1基因突变对PKA编码基因BcPKA1、BcPKA2、BcPKAR表达的影响,以及PKA编码基因突变对BcPDR1基因表达的影响。结果发现,BcPDR1基因突变体中BcPKA1、BcPKA2、BcPKAR的表达水平均显著高于野生型和回复菌株,BcPKA1、BcPKA2、BcPKAR基因的RNAi突变体中BcPDR1基因表达水平显著低于野生型。我们分别构建了PKA编码基因BcPKA1、BcPKA2、BcPKAR在敲除突变体ΔBcpdr1中过表达的菌株ΔBcpdr1/BcPKA1-OE、ΔBcpdr1/BcPKA2-OE、ΔBcpdr1/BcPKAR-OE;对过表达菌株的表型和致病力进行分析发现,过表达菌株的菌落形态、菌核形成、菌丝形态、生长速率、产孢量和致病力均与突变体ΔBcpdr1表现显著的差异,而更接近野生型BC22的表型和致病力。结果表明,灰葡萄孢BcPDR1基因与PKA亚基基因BcPKA1、BcPKA2和BcPKAR的表达水平密切相关,BcPDR1基因负调控这3个基因的表达,而这3个基因对BcPDR1基因表达有正调控作用。本研究为进一步阐明灰葡萄孢BcPDR1基因调控病菌生长发育和致病力的分子机制奠定了基础。

To clarify the relationship between BcPDR1 gene and PKA coding genes of the cAMP signaling pathway in Botrytis cinerea. Real-time PCR was used to analyze the expression levels of BcPKA1, BcPKA2 and BcPKAR in BcPDR1 gene mutants and the expression levels of BcPDR1 in mutants of BcPKA1, BcPKA2 and BcPKAR. It was found that the expression levels of PKA coding genes BcPKA1, BcPKA2 and BcPKAR in BcPDR1 gene mutations were significantly higher than those in wild-type BC22 and the BcPDR1 complementing strain. The expression levels of BcPDR1 gene in RNAi mutations of BcPKA1, BcPKA2 and BcPKAR genes were significantly lower than those in wild-type BC22. We constructed the over-expression isolates of BcPKA1, BcPKA2 and BcPKAR in ΔBcpdr1 background, respectively. The colony morphology, sclerotium forming, mycelial morphology, growth rate, conidiation, and pathogenicity of the over-expression isolates significantly different from ΔBcpdr1, and closer to the wild-type BC22. These results suggest that the expression of BcPDR1 was closely related to BcPKA1, BcPKA2 and BcPKAR in B. cinerea. The BcPDR1 gene negatively regulates the expression of BcPKA1, BcPKA2 and BcPKAR, while BcPKA1, BcPKA2 and BcPKAR genes positively regulate the expression of the BcPDR1. In a summary, our findings would increase understanding the molecular mechanism of the BcPDR1 gene in growth, development and pathogenicity in B. cinerea.