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

Mechanism Analysis of Kynurenine 3-monooxygenase Gene BcKMO in Regulation of Pathogenicity in Botrytis cinerea

【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G?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 of Botrytis cinerea and lay a foundation for clarifing the pathogenic mechanism of B. cinerea in 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, and Sod1 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】The BcKMO gene is involved in regulating cell wall degradation enzyme activity, toxin activity, acid production, penetrability, and the expression of pathogenicity- related genes in B. cinerea.