杏鲍菇SprⅠ基因的克隆、原核表达及其在采后保鲜中的功能研究

Pleurotus eryngii is a newly cultivated fungi with medicinal and edible value, the high activity of serine protease (Spr) which caused the rapid decrease of protein content, is one of the key factors to cause the quality deterioration of Pleurotus eryngii. Our previous studies have shown that the activity of serine protease (Spr) and the expression level of SprⅠ gene were reduced by 2%O2+30%CO2, thus prolonging eryngii shelf. To explore the functional features of Pleurotus eryngii Spr gene, in this project, we will clone the full-length of SprⅠ gene by RACE based on the known sequence fragment. The DNA and promoter sequence will then be determined by PCR. The key domain of SprⅠ gene and the core and regulatory region of promoter sequence were analyzed by bioinformatic, and exploration of its tissue-specific and temporal expression. Moreover, the activity of Spr will be determined by the prokaryotic expression and the expression patterns of SprⅠ gene will be identified by promoter and GUS gene fusion transformed Pleurotus eryngii. The function of SprⅠgene will be verified by overexpression and combining the phenotypic changes during Pleurotus eryngii storability. These results will clarify the biological mechanisms involved in Pleurotus eryngii fresh. Reasearch on the function and expression of SprⅠ gene will help to reveal the molecular mechanism of the postharvest quality change in Pleurotus eryngii, and provide new ways and ideas in regulation the postharvest fresh of Pleurotus eryngii.

杏鲍菇是新培育的一种药食同源的食用菌，而丝氨酸蛋白酶（Spr）高活性导致的蛋白质快速降解是造成其劣变的关键因素之一。我们的前期研究表明：2%O2+30%CO2处理能有效降低菇体中Spr活性和SprⅠ表达量，从而延长杏鲍菇保鲜期。为探明Spr基因的功能，本项目在已获得的杏鲍菇SprⅠ基因片段基础上，通过RACE法克隆SprⅠ基因的全长cDNA、DNA及启动子序列，通过生物信息学分析其关键结构域以及启动子序列中的核心区域和调控区域，并探查其组织特异性和时序表达情况；进一步采用SprⅠ基因的原核表达以及启动子与GUS基因融合转化杏鲍菇来确定Spr的活性和SprⅠ基因的表达模式；利用SprⅠ基因过量表达方法并结合杏鲍菇耐贮表型的变化来验证基因的功能，从而阐明SprⅠ基因介入杏鲍菇保鲜的生物学机理。研究结果不仅有助于揭示杏鲍菇采后品质变化的分子机理，并为调控杏鲍菇采后保鲜提供理论依据。

杏鲍菇是近年来新培育的一种药食同源的食用菌，富含蛋白和人体必须的氨基酸。蛋白质和氨基酸快速降解已成为杏鲍菇采后品质劣变的突出问题。丝氨酸蛋白酶（Spr）的高活性是造成杏鲍菇劣变的关键因素之一，为探明Spr I基因的功能特性并加以利用，本项目以杏鲍菇为研究对象，克隆了Spr I基因的全长序列，明确了其组织特异性和时空表达情况，通过启动子与GUS融合表达确定了基因的表达方式，利用Spr I基因过量表达方法验证了基因的功能，并明确了其作用机制，从而阐明Spr I基因介入杏鲍菇保鲜的生物学机理。研究结果不仅有助于揭示杏鲍菇采后品质变化的分子机理，并为调控杏鲍菇采后保鲜提供理论依据。.本项目于2014年启动，在前期研究基础上经过3年的研究，基本完成了本项目设定的研究内容和考核指标，共发表SCI源论文1篇、EI论文1篇、中文核心期刊2篇，国家授权发明专利1项，培养了1名硕士，在国内学术会议上作大会报告1次。