猕猴桃溃疡病菌phaseolotoxin毒素合成相关基因的克隆与功能分析

Kiwifruit bacterial canker is destructive disease affected safety of kiwifruit industry, and has become the main limiting factor of kiwifruit production in China and other country. It was showed that phaseolotoxin is the major causal agent for pathogenicity in Pseudomonas syringae pv. actinidiae. However, the molecular mechanism of action of P. syringae pv. actinidiae phaseolotoxin in the pathogenic process is still unclear. In previous study, we constructed the Tn5-like transposon insertional mutant library by transposon mutagenesis based on the strain of P. syringae pv. actinidiae. On the basis, phaseolotoxin biosynthetic-deficient mutants are screened in the research. Genomic fragments flanking the insertion site of the mutants are amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and sequenced, and the genes required for phaseolotoxin biosynthesis are identified by combining genetic complementation technology. It will be analyzed for the affect of phaseolotoxin biosynthetic genes on the pathogenicity and biological character of P. syringae pv. actinidiae, based on the results of testing for the phaseolotoxin biosynthetic-deficient mutants and wild-type strain. It will be further analyzed for the relationship between the effector and phaseolotoxin biosynthetic gene regulation by RT-PCR. The expectation results of studies will have important significance to reveal pathogenic mechanism of P. syringae pv. actinidiae. It will also establish foundation for searching for novel bactericide, and provide theoretical basis for breeding of resistant cultivars.

猕猴桃溃疡病是影响猕猴桃产业安全的毁灭性病害，已成为制约我国及其它国家猕猴桃生产的主要因素。猕猴桃溃疡病菌产生的phaseolotoxin毒素是引起病原致病的重要因子，但关于该毒素在病菌致病过程中的作用分子机制尚不明确。本项目拟在前期构建猕猴桃溃疡病菌转座子突变体库的基础上，筛选毒素合成丧失突变体；利用TAIL-PCR等技术克隆毒素合成丧失突变体中转座子插入位点的侧翼序列，并获得其全长基因序列，结合功能互补技术鉴定毒素合成相关基因；通过突变体致病性和生物学性状的测定，明确毒素合成相关基因与病菌致病力间的关系及对病菌生物学特性的影响；通过QRT-PCR方法进一步分析毒素合成相关基因与效应因子间的表达调控关系。预期研究结果对揭示猕猴桃溃疡病菌致病机制具有重要的意义，为探索新型杀细菌剂奠定基础，并可为抗病品种选育提供可靠的理论依据。

猕猴桃溃疡病是影响猕猴桃产业安全的毁灭性病害，已成为制约我国及其它国家猕猴桃生产的主要因素。引起猕猴桃溃疡病的病原菌是丁香假单胞猕猴桃致病变种，其中phaseolotoxin毒素是猕猴桃溃疡病菌致病的重要因子。本项目利用转座子诱变方法获得猕猴桃溃疡病菌的转座子插入突变体库，利用室内平板E.coli生长抑制试验快速筛选毒素合成丧失突变株，并结合TLC层析技术对突变株鉴定和验证，获得47株猕猴桃溃疡病菌毒素合成丧失或减弱的突变体。利用TAIL-PCR等技术克隆毒素合成丧失突变体中转座子插入位点的侧翼序列，并获得其全长基因序列，确定了phaseolotoxin毒素密切相关的glmS基因，该基因的突变引起病菌毒素合成和致病性的丧失。利用同源重组和功能互补试验，明确了gacA基因的缺失不仅降低了病菌的致病性，同时引致病菌毒素和胞外酶合成的丧失。研究结果为揭示phaseolotoxin毒素的致病机制奠定了基础，对揭示猕猴桃溃疡病菌致病机制具有重要的意义，并为抗病品种选育提供了可靠理论依据。