毛木耳漆酶基因的克隆、序列分析及其鉴定

Cloning, Sequence Analysis and Chacterization of Laccase Gene in Auricularia polytrica

本文利用PCR和RACE技术首次从毛木耳AP4菌株中获得编码漆酶基因的cDNA及其基因组全长序列,基因组大小为2514 bp。通过比较该漆酶基因的cDNA和基因组DNA的全长序列,发现该基因包含14个外显子和13个内含子。cDNA序列的全长为1972 bp,其包含一个完整的ORF,长度为1860 bp,编码619氨基酸,推测的分子量大小为68 kD,等电点pI为5.15。在氨基酸序列的氨基末端存在一个信号肽序列,同时该基因还包括含铜氧化酶的三个功能结构域KOG1263、SufI和pfam00394。氨基酸序列与GenBank中登录的真菌漆酶蛋白序列比对表明:该氨基酸序列与其它真菌漆酶蛋白序列有较高的同源性,氨基酸序列相同性最高达41%,相似性为58%,并且含有真菌漆酶的四个保守的Cu-bind结构域。将获得的漆酶基因lac1与毕赤酵母表达载体pPIC9K连接,构建重组质粒pYH3660,将其转化到毕赤酵母中,经甲醇诱导该基因在第10天产酶高达123 IU/L,并通过Native SDS-PAGE电泳获得预期大小的漆酶蛋白条带。结构分析和功能验证均表明:本研究获得的基因lac1为漆酶基因。

Using PCR and RACE technique,We obtain the cDNA and genomic DNA sequence of the lac1 gene from Auricularia polytrica.The length of genomic DNA is 2514 bp,which contains 14 exons and 13 introns based on the comparison of cDNA and genomic DNA sequence.The length of the lac1 cDNA sequence is 1972 bp,which includes a complete Open Rading Frame（ORF）of 1860 bp,from N0.33 to No.1890,encoding 619 amino acides,the molecular weight is about 68 kD,and isoelectric point is about 5.15.A signal peptide sequence exists in the N-terminal of the deduced amino acids,which also contains three multi-copper oxidase domains： KOG1263,SufI and pfam00394.We blasted the deduced amino acids with the fungal laccases protein sequences on the GenBank and observed that there is higher homologous similarity： the highest identity is 41% and positive is 58% respctively,and it also includes four conserved Cu-bind domains.The full length cDNA sequence of A.polytricha lac1 gene was ligated to the pPIC9K to construct expression vector pYH3660,the recombinant plasmid pYH3660 was transformed into Pichia pastoris,laccase activity was detected from the engineering strain which was induced by methanol with the highest expression level（123 IU/L）.At same time,we get the anticipative protein using the Native SDS-PAGE.Based on the analysis of the sequence structure and expression characterization,we can conclude that the lac1 gene obtained from Auricularia polytrica is laccase gene.