灰葡萄孢犬尿氨酸单加氧酶基因BcKMO与病菌cAMP信号途径的关系

【目的】明确灰葡萄孢（Botrytis cinerea）犬尿氨酸单加氧酶基因BcKMO与病菌cAMP信号途径之间的关系，为进一步阐明BcKMO调控灰葡萄孢生长、发育和致病力的分子机制打下基础。【方法】利用cAMP信号途径特异性抑制剂SQ22536，检测灰葡萄孢野生型BC22、BcKMO的T-DNA插入突变体BCG183和回复菌株BCG183/BcKMO对cAMP信号途径特异性抑制剂的敏感性；分别提取灰葡萄孢野生型BC22、突变体BCG183和回复菌株BCG183/BcKMO中的cAMP，利用HPLC检测各菌株中cAMP的含量；利用real-time PCR技术检测BcKMO与cAMP信号途径中cAMP依赖的蛋白激酶（PKA）的催化亚基基因pka1和pka2、调节亚基基因pkaR、编码异源三聚体G-蛋白Gα亚基基因bcg2和bcg3在病菌不同发育阶段、组织部位以及培养条件下的表达规律；利用real-time PCR技术检测BcKMO突变体中cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的表达水平；利用real-time PCR技术检测cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的RNAi突变中BcKMO的表达水平。【结果】灰葡萄孢BcKMO的T-DNA插入突变体BCG183对cAMP信号途径特异性抑制剂SQ22536不敏感，受抑制的程度明显低于野生型BC22和回复菌株BCG183/BcKMO。BcKMO的T-DNA插入突变体BCG183中cAMP含量明显低于野生型菌株BC22和回复菌株BCG183/BcKMO。BcKMO与cAMP信号途径中cAMP依赖的蛋白激酶（PKA）的催化亚基基因pka1、编码异源三聚体G-蛋白Gα亚基基因bcg2和bcg3的表达规律基本一致，均为在7 d的菌丝和菌核中表达水平较高；此外，BcKMO和cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3均为在含果糖培养基上培养的病菌中表达水平较高。BcKMO的T-DNA插入突变体BCG183中cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的表达水平均明显高于野生型BC22和回复菌株BCG183/BcKMO。pka1、bcg2的RNAi突变体中BcKMO表达水平明显高于野生型，而pka2、pkaR、bcg3的RNAi突变体中BcKMO表达水平明显低于野生型。【结论】BcKMO负调控cAMP信号途径关键基因pka1、pka2、pkaR、bcg2、bcg3的表达；cAMP信号途径关键基因pka1、bcg2负调控BcKMO的表达，而cAMP信号途径关键基因pka2、pkaR、bcg3正调控BcKMO的表达。

Relationship between kynurenine 3-monooxygenase gene BcKMO and cAMP signaling pathway in Botrytis cinerea

【Objective】The objective of this study is to analyze the relationship between BcKMO and cAMP signaling pathway in Botrytis cinerea, and to lay a foundation for clarifying the molecular mechanism of the BcKMO in growth, development and pathogenicity in B. cinerea. 【Method】 A specific inhibitor SQ22536 of cAMP signaling pathway was used to detect the sensitivity of the wild-type strain BC22, the BcKMO T-DNA insertion mutant BCG183, and the BcKMO complementing mutant BCG183/BcKMO. The cAMP was extracted from the wild-type strain BC22, the BcKMO T-DNA insertion mutant BCG183, and the BcKMO complementing mutant BCG183/BcKMO and detected by using HPLC assay, respectively. Real-time PCR technology was used to detect expression pattern of the BcKMO,the PKA catalytic subunit gene pka1 and pka2, the PKA regulatory subunit gene pkaR, G-protein Gα subunits gene bcg2 and bcg3 in different developmental stages, tissues and culture conditions of BC22. The expression level of cAMP signaling pathway key genes pka1, pka2, pkaR, bcg2, and bcg3 in mutants BCG183 and BCG183/BcKMO was detected by using real-time PCR technology. The expression level of BcKMO in RNAi mutantsof cAMP signaling pathway key genes pka1, pka2, pkaR, bcg2, and bcg3 was detected by using real-time PCR technology.【Result】The BcKMO T-DNA insertion mutant BCG183 was insensitive to the cAMP signaling pathway specific inhibitor SQ22536. The inhibition rate of the cAMP signaling pathway specific inhibitor SQ22536 to mutant BCG183 was significantly lower than the wild-type strain BC22 and the BcKMO complementing mutant BCG183/BcKMO. The cAMP content of the mutant BCG183 was significantly lower than that of the wild-type strain BC22 and the mutant BCG183/BcKMO. The expression pattern of BcKMO, the PKA catalytic subunit gene pka1, G-protein Gα subunits gene bcg2 and bcg3 was basically the same, and the expression level of BcKMO, pka1, bcg2, and bcg3 was higher in 7th day of mycelia and sclerotia of BC22. In addition,the expression level of BcKMO and the cAMP signaling pathway key genes pka1, bcg2, pkaR, bcg2, and bcg3 was higher in BC22 cultured on medium with fructose. The expression level of cAMP signaling pathway key genes pka1, pka2, pkaR, bcg2, bcg3 in mutant BCG183 was significantly higher than that of strains BC22 and BCG183/BcKMO. The BcKMO expression level in the RNAi mutants of pka1and bcg2 was obviously higher than that of BC22, the BcKMO expression level in the RNAi mutants of pka2, pkaR, and bcg2 was obviously lower than that of BC22.【Conclusion】The BcKMO negatively regulated the expression of pka1, pka2, pkaR, bcg2, and bcg3. The pka1 and bcg2 negatively regulated the BcKMO expression, and the pka2, pkaR, and bcg3 positively regulated the BcKMO expression.