血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

Cloning and Sequence Analysis of Laccase Gene from Pycnoporus sanguineus

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～

To clone laccase gene from Pycnoporus sanguineus, a pair of degenerate primers was designed according to the conservation sequence at both ends of amino acid residues of laccase protein family from known fungus. A 1227 bp fragment of laccase gene was amplified using genomic DNA from Pycnoporus sanguineus as template. And then the full-length cDNA of laccase gene was obtained by 5′/3′ RACF. The full-length sequence was 1902 bp,in which the unencoded regions at 5′ and 3′ ends were 51 bp and 297 bp,respectively,and an open reading frame was 1554 bp,encoding a protein of 518 amino acids. The protein had four catalytic cupric ion domains,with 56313.2 of relative molecular weight and predicted pI value of 559. The putative N-glycosylation sites showed the highest homology of 96% with laccase(lcc3-2) from Pycnoporus cinnabariuns. Using the terminal sequence of encoding region as primers,genomic clone of 2154 bp has been isolated and showed exon-intron structure with 10 intron of 52～70 bp.