新疆南部和田羊、卡拉库尔羊、多浪羊MSTN基因的真核表达  

Eukaryotic Expression of MSTN Gene from Hetian Sheep,Carla Kul Sheep,and Duolang Sheep in the Southern Xinjiang

作　　者：王海涛[1] 金波[1] 李树伟[1] 

机构地区：[1]塔里木大学生命科学学院塔里木盆地生物资源保护利用兵团重点实验室,阿拉尔843300

出　　处：《生物技术通报》2016年第5期82-90,共9页Biotechnology Bulletin

基　　金：新疆生产建设兵团博士资金专项(2010JC08)

摘　　要：旨在构建新疆南部4个地方品种(品系)绵羊,即平原型和田羊(HP)、山区型和田羊(HS)、卡拉库尔羊(KL)和多浪羊(DL)的肌生成抑制素(myostatin,MSTN)基因与增强型绿色荧光蛋白(EGFP)的融合表达质粒并在真核细胞中表达。从4种绵羊骨骼肌中提取总RNA,逆转录获得c DNA,PCR扩增MSTN后与p MD18-T载体连接构建重组质粒并测序分析,然后将MSTN(HP、HS、DL和KL)基因插入到融合表达载体p EGFP-N1中,构建真核融合表达重组质粒p EGFP-N1-MSTN,在细胞铺满80%时用脂质体法转染到CHO中,观察EGFP与MSTN蛋白在CHO细胞内融合表达的绿色荧光。结果显示,平原型和田羊、山区型和田羊、卡拉库尔羊和多浪羊MSTN的ORF序列都是1 125 bp(不含终止密码子),编码375个氨基酸;与Gen Bank中MSTN序列比较,与田羊(HS、HP)没有差异;卡拉库尔羊有2个碱基差异,造成2个氨基酸变异;多浪羊有6个碱基的差异,造成4个氨基酸变异。成功构建EFGP和MSTN共表达重组质粒并在真核细胞CHO中得以表达。This work aims to construct the fusion expression plasmids of myostatin gene（MSTN）from 4 local varieties（lines）sheep,namely plain Hetian sheep（HP）,mountain Hetian sheep（HS）,Carla Kul sheep（KL）,and Duolang sheep（DL）in the southern Xinjiang with enhanced green fluorescent protein（EGFP）,and the expression of MSTN in eukaryotic cells. Total RNA was extracted from the skeletal muscle in 4 varieties of sheep,c DNA was acquired by reverse transcription,and MSTN amplified by PCR was ligated with p MD18-T vector to construct recombinant plasmid and sequenced. Then MSTN（HP,HS,DL,and KL）gene was inserted into the fusion expression vector p EGFP-N1,the recombinant plasmid of p EGFP-N1-MSTN in a eukaryotic fusion expression was constructed. The plasmid with over 80% cells covered was transfected into CHO by liposome,the green fluorescent of fusion protein of EGFP and MSTN in CHO cells was observed. The results showed that the ORF sequence of MSTN gene in any of HP,HS,KL,and DL was 1 125 bp（excluding stop codon）,encoding 375 amino acids. Comparing MSTN sequences in Gen Bank,there was differences in HS and HP;and 2 nucleotide variation in KL caused 2 amino acid mutations;and 6 nucleotide differences in DL resulted in 4 amino acid variations. In conclusion,the recombinant plasmid for EFGP and MSTN co-expression was constructed and expressed in eukaryotic CHO cells.

关 键 词：和田羊 卡拉库尔羊 多浪羊 肌生成抑制素 CHO细胞 绿色荧光蛋白 

分 类 号：Q953[生物学—动物学]