香菇草酰乙酸水解酶基因LeOAH1克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2019-1246

摘要/Abstract

摘要： 草酰乙酸水解酶（Oxaloacetate acetylhydrolase/Oxaloacetate hydrolase）是香菇（Lentinula edodes）中草酸合成的关键性酶,克隆香菇LeOAH1,并分析其在香菇体内与体外的表达情况,旨为研究香菇LeOAH1的功能奠定基础。从香菇L808中克隆LeOAH1,并进行生物信息学分析,采用 RT-PCR 和高效液相技术检测 LeOAH1在不同pH条件下香菇菌丝内的表达情况和草酸含量。此外,构建了重组原核表达载体 pET28a-oah1,在大肠杆菌中进行体外表达。基因克隆结果表明,该基因的gDNA长度为1 906 bp,cDNA长度为 1 356 bp,编码氨基酸数量为451个,蛋白分子质量约为48.9 kD,等电点为 6.80。生物信息学分析表明：该酶为不稳定的疏水性蛋白,具有α-螺旋（40.08%）、无规则卷曲（34.59%）、延伸链（17.52%）、β-折叠（7.10%）4种二级结构;亚细胞定位在细胞质中;序列比对的结果显示该酶有保守结构域;系统发育树分析该蛋白,与日本香菇OAH的相似度最高,同源性为82%;其次是灰光柄菇,同源性为64%。RT-PCR、HPLC的结果显示：在不同pH条件下,随着pH增加,LeOAH1基因表达量增加,香菇草酸分泌量也随之增加。而大肠杆菌中诱导表达的蛋白分子量符合预测结果。初步明确了香菇草酰乙酸水解酶的基因特性以及表达规律,以及与草酸分泌的关系。

关键词: 香菇, 草酰乙酸水解酶, 基因克隆, 生物信息学分析, 原核表达

Abstract: Oxaloacetate acetylhydrolase/Oxaloacetate hydrolase is one of the key enzymes for oxalate synthesis in Lentinula edodes. The LeOAH1 was cloned and its expression in vivo and in vitro was analyzed to lay the foundation for the functional research of LeOAH1. LeOAH1 was cloned from L. edodes L808 and bioinformatics analysis was conducted. The expression of LeOAH1 and the content of oxalic acid secreted by L. edodes under different pH conditions were respectively detected by RT-PCR and HPLC during the vegetative phase. In addition,a recombinant prokaryotic expression vector pET28a-oah1 was constructed and successfully expressed in Escherichia coli. The results showed that the length of gDNA of the LeOAH1 was 1 906 bp,the length of cDNA was 1 356 bp,encoding 451 amino acids,the protein molecular weight was about 48.9 kD,and the isoelectric point was 6.80. Bioinformatics analysis revealed that the enzyme was an unstable hydrophobic protein with four secondary structures of α-helix（40.08%）,random coil（34.59%）,extended chain（17.52%）,and β-sheet（7.10%）level structure;it was in the cytoplasm by subcellular localization. The sequence alignment results demonstrated that the enzyme had a conserved domain. The phylogenetic tree analysis suggested that the protein had the highest similarity with OAH from Japanese mushroom with 82% homology,followed by Pluteus cervinus with 64% homology. The RT-PCR and HPLC results showed that the expressions of LeOAH1 gene and the amount of oxalic acid secreted by L. edodes both increased with the increase of pH. The molecular weight of the induced protein in E. coli was in line with the predicted results. The gene characteristics and expression pattern of oxaloacetate hydrolase,as well as its relationship with oxalic acid secretion are preliminarily clarified.

Key words: Lentinula edodes, oxaloacetate hydrolase, gene cloning, bioinformatics analysis, prokaryotic expression

段应策, 胡姿仪, 杨帆, 李金涛, 邬向丽, 张瑞颖. 香菇草酰乙酸水解酶基因LeOAH1克隆及表达分析[J]. 生物技术通报, 2020, 36(9): 227-234.

DUAN Ying-ce, HU Zi-yi, YANG Fan, LI Jin-tao, WU Xiang-li, ZHANG Rui-ying. Cloning and Expression Analysis of Oxaloacetate Hydrolase（LeOAH1）Gene from Lentinula edodes[J]. Biotechnology Bulletin, 2020, 36(9): 227-234.

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