树舌灵芝免疫调节蛋白基因克隆,生物信息学分析及真核表达载体构建  

Gene Cloning, Bioinformatic Analysis and Eukaryotic Expression Vector Construction of FIP-gap Gene from Ganoderma applanatum

作　　者：林景卫[1,2] 段作文 关山越[2] 韩笑[2] 范文丽[1] 李浩戈[2] 张丽[2] 陈水森[2] 李天来[1] 

机构地区：[1]沈阳农业大学园艺学院,沈阳110161 [2]沈阳农业大学生物科学技术学院,沈阳110161

出　　处：《沈阳农业大学学报》2016年第1期1-7,共7页Journal of Shenyang Agricultural University

基　　金：中国博士后科学基金项目(2013M541251)

摘　　要：树舌灵芝(Ganoderma applanatum)为灵芝属内独特的一类,克隆树舌灵芝免疫调节蛋白(fungal immunomodulatory protein,FIP)基因,对其进行生物信息学分析并构建真核表达载体,将为高效表达重组树舌灵芝FIP和揭示其生物学功能奠定基础。采用染色体步移的方法从树舌灵芝菌丝体基因组DNA中扩增得到1个新型真菌免疫调节蛋白基因—FIP-gap,对其核苷酸序列进行生物信息学分析,结果表明,FIP-gap基因属于灵芝属FIP家族,含有342 bp,编码113个氨基酸。对其氨基酸序列分析表明:树舌灵芝免疫调节蛋白FIP-gap分子量为12.7 k D,理论等电点为4.93,富含丝氨酸,缬氨酸和苏氨酸,不含组氨酸和半胱氨酸。另外将FIP-gap与其他灵芝属FIP进行蛋白质序列比对分析,其具有FIP典型的保守序列,并与灵芝(G.lucidum)、松杉灵芝(G.tsugae)、紫灵芝(G.japoncium)、紫芝(G.sinensis)、小孢子灵芝(G.microsporum)和黑灵芝(G.atrum)具有78%、78%、78%、79%、78%和77%的同源性,为一新型的FIP。构建灵芝属真菌免疫调节蛋白系统进化树,结果表明树舌灵芝FIP-gap与其他灵芝属FIP均不聚类,说明其与其他灵芝属FIP的亲缘性相对较远。同时,构建了毕赤酵母重组表达载体p PIC9-FIP-gap,为进一步研究FIP-gap的生物学功能提供基础,为全面了解真菌免疫调节蛋白提供理论依据。Ganoderma applanatum is a special type of Ganoderma spieces. In this study, a novel gene was cloned from G.applanatum and analyzed through Bioinformatics, which could supply some basis for effectively express of FIP-gap and investigation its biological functions. Using Genome walking method, the noval FIP-gap gene was amplified from the genomic DNA of G.applanatum mycelia. The nucleotide sequence analysis showed that FIP-gap comprises 342 bps and encodes a protein with 113 amino acids. The protein sequence analysis of FIP-gap indicated that molecular mass of FIP-gap is 12.7 k D and pI is 4.93. There are rich in Serines, Valines and Threonines in the peptide of FIP-gap, without Histidines or Cysteines. The homology analysis based on protein sequence blasting indicated that FIP-gap contains typical conserved regions of FIPs, and shares 78%, 78%, 78%, 79%,78% and 77% homology with LZ-8(G. lucidum), FIP-gts(G. tsugae), FIP-gja(G. japoncium), FIP-gsi(G. sinensis), FIP-gmi(G.microsporum) and FIP-gat(G. atrum), respectively. The phylogenetic tree analysis showed that FIP-gap doesn't cluster with any other FIPs from Ganoderma and forms a unique line, which implies its long distance of molecular evolution with the other FIPs. Also, the recombinant expression plasmid pPIC9-FIP-gap was constructed after the FIP-gap gene was inserted in a Pichia expression vector pPIC9. Conclusively, this study will provide helpful basis to further study on the bio-functions of FIP-gap, and full understanding of the FIPs.

关 键 词：树舌灵芝 真菌免疫调节蛋白 生物信息学分析 染色体步移 基因克隆 真核表达 

分 类 号：S567.31[农业科学—中草药栽培]