玉木耳漆酶基因Aclac的克隆及原核表达

【目的】对玉木耳漆酶基因Aclac进行克隆及原核表达分析，为深入研究漆酶基因在玉木耳子实体发育中的生物学功能提供理论依据。【方法】克隆Aclac基因的cDNA和DNA全长序列，对其进行生物信息学分析，并将目的基因连接至pET-28a质粒上以构建原核表达载体pET28a-Aclac，将其转化大肠杆菌BL21（DE3）中诱导表达。【结果】克隆获得的Aclac基因cDNA序列长度为1734 bp，编码577个氨基酸残基；Aclac基因DNA序列长度为2521 bp，含14个内含子。AcLAC蛋白理论分子量为64.75 kD，理论等电点为5.70，不稳定指数为34.01，无跨膜区域，存在1个信号肽，在4个铜离子结合区高度保守，且含有Cupredoxin超家族蛋白保守功能域。AcLAC蛋白与真菌的漆酶蛋白聚为一支，其中，AcLAC蛋白与木耳属真菌的漆酶蛋白亲缘关系最近。AcLAC融合蛋白在大肠杆菌BL21（DE3）表达宿主中成功表达，分子量为67 kD。【结论】克隆获得的Aclac基因属于Cupredoxin超家族基因，可在原核表达系统中异源表达，推测其与其他真菌漆酶基因在生物学功能上具有一致性，丰富了真菌漆酶资源。

Cloning and prokaryotic expression analysis of laccase gene Aclac from Auricularia cornea

【Objective】 The cloning and prokaryotic expression analysis of the laccase gene(Aclac) of the Auricularia cornea provided a basis for in-depth study of the biological function of the laccase gene in the development of the fruit body of A.cornea.【Method】 Aclac gene of A.cornea was cloned, and the full-length cDNA sequence of the gene was obtained and analyzed by bioinformatics. The target gene was also ligated to the pET-28a plasmid to construct the prokaryotic expression vector pET28a-Aclac, and the recombinant plasmid was induced to express in Escherichia coli BL21 (DE3). 【Result】 The cloned Aclac gene cDNA sequence was 1734 bp long, encoded 577 amino acids, DNA sequence was 2521 bp, contained 14 introns, the theoretical AcLAC protein molecular weight was 64.75 kD, the isoelectric point was 5.70, the instability index was 34.01, there was no transmembrane region, and it contained a signal peptide.it was highly conserved in 4 copper ion binding regions, and contained the conservative functional domains of the Cupredoxin superfamily protein. The results of phylogenetic analysis showed that AcLAC protein and fungal laccase protein were clustered together. Among them, AcLAC protein had the closest relationship with the Auricularia. AcLAC fusion protein was successfully expressed in E. coli BL21(DE3), and its molecular weight was 67 kD.【Conclusion】 The cloned Aclac gene belongs to the Cupredoxin superfamily gene and can be expressed heterologously in a prokaryotic expression system. It is speculated that it has the same biological function as other fungal laccase genes, and enriches fungal laccase resources.