摘要
根据棉花纤维特异表达cDNA文库得到的咖啡酰辅酶A-O-甲基转移酶基因EST序列设计引物,采用RT-PCR技术从棉花中克隆了一个CCoAOMT基因,命名为GhCCoAOMT2。GhCCoAOMT2基因cDNA(GenBank登录号为FJ376606)具有一个747 bp的开放阅读框,5′非编码区为12 bp,3′非编码区为243 bp,编码248个氨基酸,预测分子量约为28.023 kD,等电点为5.39。GhCCoAOMT2基因组序列长度为1 442 bp,包含4个外显子和3个内含子。氨基酸同源分析发现,GhCCoAOMT2与来自毛白杨、烟草和苎麻的CCoAOMT同源性较高。半定量RT-PCR检测表明,GhCCoAOMT2基因在棉花各个组织中都有表达,其中茎部的表达量最高。原核表达分析表明,最佳诱导表达条件为0.2 mmol/LIPTG在37℃下诱导6 h。
According to results of cotton fibre preferentially expressed cDNA library analysis,we designed primers from EST sequences.RT-PCR method was used to clone CCoAOMT gene.A gene coding for CCoAOMT,designated as GhCCoAOMT2（GenBank accession no.FJ376606） was isolated from cotton（Gossypium hirsutum L.）.The length GhCCoAOMT2 cDNA is 1 002 bp,including a 12 bp 5′-UTR,an ORF of 747 bp,and a 243 bp 3′-UTR.This cDNA sequence encoded a polypepide of 248 amino acid residues with a predicted molecular mass of 28.023 kDa and a basic isoelectric point of 5.39.Furthermore,a length of 1 442 bp sequence from genomic DNA of GhCCoAOMT2 was also cloned by PCR.The genomic DNA of GhCCoAOMT2 contains four exons and three introns.The deduced amino acid sequence of GhCCoAOMT2 had a high homology with PtCCoAOMT,NtCCoAOMT-3,and BnCCoAOMT-1.Semi-quantitative RT-PCR analysis revealed that GhCCoAOMT2 could be expressed in different kinds of cotton tissues,and its mRNA accumulated most abundantly in stem.To investigate the function of the GhCCoAOMT2 gene,the full-length open reading frame was fused into a prokaryotic expression vector pET-28a.SDS-PAGE indicated that the most high expression quantity was induced by 0.2 mmol/L IPTG treatment for 6 h at 37℃.
出处
《西北植物学报》
CAS
CSCD
北大核心
2010年第6期1083-1091,共9页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(30660088)
国家"863"项目(2006AA10Z184)
新疆自治区高技术研究发展计划(200611101)
农业部转基因重大专项(2009ZX08005-011B)
关键词
棉花
咖啡酰辅酶A-O-甲基转移酶
基因克隆
表达分析
原核表达
Gossypium hirsutum L.
caffeoyl-CoA O-methyltransferase
gene cloning
expression analysis
prokaryotic expression
