辣椒疫霉菌果胶裂解酶PL101基因真核表达及致病性分析

Eukaryotic Expression and Pathogenicity Analysis of Pectate Lyase PL101Gene from Phytophthora capsici

辣椒疫霉菌(Photophthora capsici)常引起辣椒等多种作物的毁灭性病害,对该病原菌重要功能基因尤其是致病基因的研究具有重要的经济学意义。果胶裂解酶(pectate lyase,PL)为植物病原菌关键致病因子,通过降解寄主细胞壁引起寄主发病。本研究以辣椒疫霉菌PL关键成员(PL101)为材料,通过真核表达系统进行诱导表达重组蛋白。真核表达载体选用pPIC9K,测序验证后的重组质粒pPIC9K/PL101经电击法转化毕赤酵母GS115表达菌株,对阳性转化子进行甲醇诱导型(Mut^(+))、不同浓度抗生素G418即高拷贝转化子筛选获得表达量可观的重组蛋白表达菌株。重组酵母菌经1%甲醇诱导7 d获得重组蛋白,经硫酸铵沉淀法、Ni-NTA亲和层析纯化后获得纯度较高的PL101纯蛋白。经聚半乳糖醛酸反应法测得的PL101酶活达到970 U/mg,接种健康辣椒叶片测试结果表明PL101具较高致病性,表明PL101是辣椒疫霉菌重要致病因子。本研究为进一步阐明PL作用机理提供科学依据,进而为辣椒的抗病育种提供基因资源。

Phytophthora capsici often causes destructive diseases of many plants,such as pepper.It is of great economic significance to study the important functional genes,especially the pathogenic genes of P.capsici.Pectate lyase(PL) is one of the key pathogenic factors of plant pathogens,causing disease by degrading the host cell wall.In this study,one of the key PL members(PL101) of P.capsici was used to express the recombinant protein through eukaryotic expression system.The recombinant plasmid pPIC9 K/PL101,which was confirmed by sequencing,was transformed into Pichia pastoris GS115 by electric shock.The positive transformants were screened by Mut+and antibiotics of G418 with different concentrations,obtaining a recombinant expressing strain with considerable amount of expression.The recombinant protein was induced by 1% methanol for 7 days and purified by ammonium sulfate precipitation and Ni-NTA affinity chromatography.PL101 enzyme activity measured by polygalacturonic acid reaction method reached 970 U/mg,and the test results of inoculating healthy pepper leaves show that PL101 has high pathogenicity,showing PL101 is an important pathogenic factor of P.capsici.This study will provide a scientific basis to clarify the mechanism of PL and genetic resources for disease resistance breeding of pepper.