草鱼诱导型一氧化氮合酶cDNA和启动子的克隆及分析  

Cloning and Sequence Analysis of the cDNA and Promoter of Grass Carp Inducible Nitric Oxide Synthase

作　　者：汪新艳[1] 赵太强[1] 郭嘉聪[1] 杨牧[1] 周红[1] 

机构地区：[1]电子科技大学生命科学与技术学院,成都610054

出　　处：《四川动物》2011年第4期510-516,共7页Sichuan Journal of Zoology

基　　金：电子科技大学青年科学基金项目资助(L08010901JX0856);国家自然科学基金(30972280)

摘　　要：应用PCR和RACE技术,以细菌脂多糖(LPS)刺激的草鱼头肾白细胞cDNA为模板,获得草鱼iNOS(In-ducible nitric oxide synthase)全长cDNA序列。该序列共4286 bp,编码含1080个氨基酸残基的蛋白。氨基酸序列比对分析发现草鱼iNOS与金鱼iNOS a和b、斑马鱼iNOS 2b以及鲤鱼iNOS高度保守,序列一致性均大于80%;它们的血红素、四氢生物蝶呤、钙调蛋白、FMN、FAD和NADH等辅因子结合区域也十分保守。用NJ法对新获得的序列和其它脊椎动物NOS的编码序列进行进化分析证实它属于诱导型NOS。在此基础上,运用基因步移技术分离草鱼iNOS基因的启动子序列,共有1978 bp。生物信息学分析发现该序列含有包括GR、AP1、C/EBP、ER、YY1、IRF1和IL-6 REBP在内的多个转录因子的潜在结合位点。The full length cDNA of grass carp iNOS was isolated from LPS-stimulated grass carp head kidney leucocytes by using PCR amplification coupling to 5＇-and 3＇-RACE.This cDNA was 4286 bp in length which encodes a protein with 1080 amino acid residues.Amino acid alignment showed that the identities between grass carp iNOS and goldfish iNOS,zebrafish iNOS2b and common carp iNOS were all more than 80%.The cofactor-binding sites for heme,tetrahydrobiopterin,calmodulin,FMN,FAD pyrophosphate,FAD isoalloxazine,NADPH ribose,NADPH adenine and NADPH are also highly conserved in these sequences.Phylogenetic analysis confirmed that this newly cloned iNOS is a member of inducible NOS.The promoter sequence of grass carp iNOS with 1978 bp was also obtained by using the Genome Walker Universal kit.Multiple transcription factors including GR,AP1,C/EBP,ER,YY1,IRF1 and IL-6 REBP binding sites were revealed by using transcription element search system in this study.

关 键 词：INOS CDNA序列 启动子序列 序列分析 

分 类 号：S917.4[农业科学—水产科学] Q78[生物学—分子生物学]