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白灵菇漆酶基因的克隆及其序列分析Cloning and Sequence Analysis of Laccase Gene from Pleurotus nebrodensis
薛丹, 刘腾, 那日, 王志永, 郭九峰
白灵菇, 漆酶基因, 克隆, 序列分析Pleurotus nebrodensis, Laccase Gene, Cloning, Sequence Analysis
《Advances in Microbiology》, Vol.3 No.2, 2014-06-18
白灵菇作为一种珍稀食用菌，提高其产量及质量具有重要的现实意义，而漆酶对真菌的生长发育具有重要影响。本文根据侧耳科漆酶基因共同保守序列，设计引物，利用RT-PCR的方法，克隆白灵菇漆酶序列，经测序获得其cDNA序列。为进一步获得该漆酶基因序列，以白灵菇基因组DNA为模板，PCR扩增出长2606 bp的漆酶基因DNA片段，DNA经纯化后克隆到pGM-T载体上，经筛选、PCR鉴定、序列分析，证明该片段为完整的白灵菇漆酶基因(GenBank注册号KC789845)。对基因序列的内含子/外显子进行分析，分析结果与经反转录测序获得的cDNA序列一致。该基因的开放阅读框由1596个核苷酸组成，编码一个由531个氨基酸组成的多肽(GenBank登录号AGO64758.1)，Protein Blast 分析结果表明，该基因含有3个铜氧化酶(Cu-oxidase)保守结构域，并具有高度保守的漆酶特征序列L3；又进一步对白灵菇漆酶二级结构进行分析，并通过 SWISS-MODE预测了该酶的三维结构。
Pleurotus nebrodensis as a rare mushroom, it is necessary to improve its yield and quality; laccase has an important influence on the growth of fungi. A pair of specific primers was designed by conservative sequence of pleurotaceae laccase gene, and the laccase cDNA sequence of Pleurotus nebrodensis was amplified by RT-PCR. In order to obtain the laccase gene, taking genomic DNA as test material, 2606-bp-long laccase gene fragment was cloned by PCR. Through purification, the DNA is cloned to the pGM-T; after screening, PCR identification and the sequence analysis, it is proved that the fragment is complete laccase DNA (GenBank KC789845). Through the intron and exon analysis, the analysis results consistent with the cDNA sequence obtained from the reverse transcription of sequencing. It contains one open reading frame (ORF), which encodes for a polypeptide containing 519 amino acids (Genbank AGO64758.1). The result of protein blast showed that the gene had three Cu-oxidase conserved domains, and the highly conserved laccase signature sequence L3 was also found from the gene sequence. The secondary structure and three-dimensional structure of the enzyme were predicted by bioinformatics software.