灰葡萄孢犬尿氨酸单氧酶基因BcKMO调控病菌致病力的机制分析

摘要

【目的】揭示灰葡萄孢犬尿氨酸单氧酶基因BcKMO（kynurenine 3-monooxygenase）调控病菌致病力的机制，阐明灰葡萄孢致病的分子机理。【方法】利用DNS和Hoffman方法，对BcKMO的T-DNA插入突变体BCG183和回复突变体（BCG183/BcKMO）的多聚半乳糖醛酸酶（PMG）、果胶酶（PG）、纤维素酶（Cx）、多聚半乳糖醛酸反式消除酶（PGTE）和果胶甲基反式消除酶（PMTE）的活性进行分析；提取灰葡萄孢野生型和BCG183、BCG183/BcKMO突变体的粗毒素并进行生物活性测定；利用溴百里酚蓝显色试验，对突变体BCG183和BCG183/BcKMO的产酸能力进行分析；利用洋葱表皮穿透试验，检测突变体BCG183和BCG183/BcKMO的穿透能力；利用real-time PCR技术，检测突变体BCG183和BCG183/BcKMO中已知致病相关基因腺苷酸环化酶编码基因Bac、异源三聚体G-蛋白Gα亚基基因Bcg2和Bcg3、蛋白激酶调节亚基基因PkaR、MAP激酶编码基因Bmp1和Sak1、MAP激酶Bmp3和BcSak1下游的靶基因Bcreg1、TCHK-MAPK信号途径基因Bos1、亲环蛋白A编码基因Bcp1、小G蛋白基因Ras2、多聚半乳糖醛酸酶基因Bcpg1和超氧化物歧化酶基因Sod1的表达水平。【结果】突变体BCG183中的PMG和PG的酶活性较野生型和回复突变体明显增强，CX、PGTE和PMTE的酶活性与野生型和回复突变体没有明显差别；突变体BCG183的毒素活性较野生型和回复突变体明显增强；突变体BCG183的产酸能力较野生型和回复突变体明显减弱；突变体BCG183能够穿透洋葱表皮，在培养基上形成菌落，与野生型和回复突变体没有明显差别；突变体BCG183中已知致病相关基因Bac、Bcg2、Bcg3、PkaR、Bmp1、Sak1、Bcreg1、Bos1、Bcp1、Ras2、Bcpg1和Sod1的表达水平明显强于野生型和回复突变体。【结论】灰葡萄孢BcKMO通过调控病菌的胞壁降解酶活性、毒素活性、产酸能力、致病相关基因的表达而影响病菌的致病力。%[Objective]The objective of this study is to reveal the molecular mechanism of kynurenine 3-monooxygenase gene BcKMO in regulating pathogenicity ofBotrytis cinerea and lay a foundation for clarifing the pathogenic mechanism ofB. cinereain the future.[Method]The activities of polymethylgalacturonase (PMG), endopolygalacturonase (PG), cellulase (CX), polygalacturonic acid transeliminase (PGTE) and pectin methyl transelimination enzyme (PMTE) were analyzed in the wild-type strain (WT), the BcKMO gene ATMT mutant (BCG183) and gene complement mutant (BCG183/BcKMO) by DNS and Hoffman methods. The toxin was isolated from WT, BCG183 and BCG183/BcKMO and the activity of toxin was analyzed. Acid production assays was performed on PDA medium with bromothymol blue. Penetrability of WT, BCG183 and BCG183/BcKMO was detected with onion epidermis spread on PDA medium. Real-time PCR was used to measure the transcription levels of pathogenicity-related genes, e.g.,Bac, Bcg2,Bcg3, PkaR,Bmp1,Sak1,Bcreg1,Bos1,Bcp1,Ras2,Bcpg1, andSod1 in WT, BCG183 and BCG183/BcKMO.[Result]The activity of PMG and PG in mutant BCG183 were significantly higher than that of WT and BCG183/BcKMO. The CX, PGTE and PMTE activities were no significant difference while compared with WT and BCG183/BcKMO. The toxin activity of mutant BCG183 was significantly higher than that of WT and BCG183/BcKMO. The acid production of the mutant BCG183 significantly decreased. The penetrability of mutant BCG183 appeared no significant difference compared to WT and BCG183/BcKMO. The expression level of pathogenicity-related genes, i.e.,Bac,Bcg2,Bcg3, PkaR,Bmp1,Sak1,Bcreg1,Bos1,Bcp1,Ras2,Bcpg1, and Sod1, was obviously up-regulated in mutant BCG183.[Conclusion]TheBcKMO gene is involved in regulating cell wall degradation enzyme activity, toxin activity, acid production, penetrability, and the expression of pathogenicity- related genes inB. cinerea.