灰葡萄孢犬尿氨酸途径关键酶基因的鉴定

Identification of key enzyme genes of kynurenine pathway in Botrytis cinerea

摘要:

【背景】灰葡萄孢是一种重要的植物病原真菌，实验室前期明确了灰葡萄孢犬尿氨酸单加氧酶(kynurenine 3-monooxygenase，KMO)基因BcKMO参与调控病菌的生长发育和致病力。犬尿氨酸单加氧酶(KMO)是犬尿氨酸途径的关键酶，但灰葡萄孢是否存在犬尿氨酸途径及其在病菌生长、发育和致病过程中的功能尚未见相关报道。【目的】鉴定灰葡萄孢犬尿氨酸途径中的关键酶基因，确定灰葡萄孢犬尿氨酸途径的存在，为阐明灰葡萄孢生长发育和致病力的分子机理奠定基础。【方法】利用生物信息学方法，对灰葡萄孢犬尿氨酸途径中犬尿氨酸酶(kynureninase，KYN)、吲哚-2,3-双加氧酶(indoleamine-2,3-dioxygenase，IDO)、犬尿氨酸氨基转移酶(kynurenine amino transferase，KAT)的编码基因进行分析；利用Real-time PCR技术，检测灰葡萄孢野生型BC22、BcKMO基因T-DNA插入突变体BCG183、恢复菌株BCG183/BcKMO中犬尿氨酸途径关键酶基因的表达水平；利用真菌犬尿氨酸酶KYN检测试剂盒，测定BcKMO突变体中犬尿氨酸酶(KYN)的含量。【结果】灰葡萄孢中含有2个犬尿氨酸氨基转移酶(KAT)的编码基因、3个吲哚-2,3-双加氧酶(IDO)的编码基因、10个犬尿氨酸氨基转移酶(KAT)的编码基因。灰葡萄孢KYN编码基因、IDO编码基因、KAT编码基因在突变体BCG183中的表达水平显著高于或低于在野生型和恢复菌株。突变体BCG183中犬尿氨酸酶(KYN)的含量显著低于野生型BC22和恢复菌株。【结论】灰葡萄孢中存在犬尿氨酸途径，灰葡萄孢BcKMO基因突变影响KYN、IDO和KAT编码基因的表达以及犬尿氨酸酶(KYN)的含量。

Abstract:

[Background] Botrytis cinerea is an important phytopathogenic fungus. In previous studies, it was confirmed that the BcKMO gene encoding kynurenine 3-monooxygenase (KMO) is involved in the regulation of the growth and pathogenicity of Botrytis cinerea. KMO is a key enzyme in the kynurenine pathway, but the presence of the kynurenine pathway in Botrytis cinerea and its function in the growth, development and pathogenesis of pathogens have not yet been related reported. [Objective] To identify the key enzyme genes in the kynurenine pathway of Botrytis cinerea, and to determine the presence of kynurenine pathway in Botrytis cinerea, lay the foundation for elucidating the molecular mechanism of growth and pathogenicity of Botrytis cinerea. [Methods] Bioinformatics methods were used to analyze the coding genes of key enzyme, kynureninase (KYN), indoleamine-2,3-dioxygenase (IDO), and kynurenine amino transferase (KAT), in the kynurenine pathway of Botrytis cinerea. Real-time PCR was used to detect the expression level of key enzyme genes of the kynurenine pathway in Botrytis cinerea wild-type BC22, BcKMO mutant BCG183, and the BcKMO complementing strain BCG183/BcKMO. The content of kynureninase in the BcKMO mutant was determined by using a fungal kynureninase assay kit. [Results] Botrytis cinerea contains 2 genes coding KYN, 3 genes coding IDO, and 10 genes coding KAT. The expression levels of Botrytis cinerea KYN, IDO, KAT coding genes in mutant BCG183 were significantly higher or lower than those in BC22 and BCG183/BcKMO. The content of kynureninase (KYN) in the mutant BCG183 was significantly lower than that of BC22 and BCG183/BcKMO. [Conclusion] Kynurenine pathway is present in Botrytis cinerea. BcKMO mutation affects the expression of KYN, IDO and KAT encoding genes and the content of kynureninase (KYN) in Botrytis cinerea.