西伯利亚鲟CXCR7基因cDNA全长的克隆及其表达分析

采用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE),首次克隆了西伯利亚鲟(Acipenser baerii)的CXCR7a和CXCR7b基因的全长cDNA。CXCR7a的cDNA全长为2 325 bp(登录号为:JQ034508),包括207 bp的5'端非翻译区(UTR)、可编码362个氨基酸的1 086 bp的开放阅读框(ORF)及包括poly(A)尾巴在内的大小为1 032 bp的3'端非翻译区(UTR);CXCR7b的cDNA全长为2 423 bp(登录号为:JQ034509),包括209 bp的5'端非翻译区(UTR)、可编码370个氨基酸的1 110 bp的开放阅读框(ORF)及包括poly(A)尾巴在内的大小为1 104 bp的3'端非翻译区(UTR),并对它们编码的蛋白质序列的分子特征进行了分析。氨基酸序列相似性(similarity)分析表明,CXCR7a与人(Homo sapiens)、小鼠(Mus musculus)、非洲爪蟾(Xenopus laevis)、斑马鱼(Danio rerio)的CXCR7相似性分别为82.6%、81.4%、80.9%、81.0%;CXCR7b与人、小鼠、非洲爪蟾、斑马鱼的CXCR7相似性分别为82.9%、82.3%、80.8%、82.5%;西伯利亚鲟CXCR7a与其CXCR7b的相似性最高为96.5%。系统进化树分析显示,西伯利亚鲟与斑马鱼聚为一簇,两栖类聚为一簇,其它高等脊椎动物再进行聚类。对CXCR7a和CXCR7b在各发育时期胚胎及仔鱼的表达状况分别进行定性分析表明,CXCR7a在除卵裂期、囊胚期的胚胎外,其在原肠胚中期至出膜1 d的仔鱼的各时期中均具有表达,CXCR7b在卵裂期至出膜1 d的仔鱼的各时期中均具有表达。另外,从基因组DNA水平上表明CXCR7a和CXCR7b来自不同的基因拷贝。这些结果为进一步研究CXCR7在西伯利亚鲟侧线系统发育中的作用提供了新的基础资料。

Isolation, sequence and expression analysis of CXCR7 gene from Siberian sturgeon (Acipenser baerii)

The full-length cDNA of CXCR7a and CXCR7b were isolated by performing RT-PCR,3′and 5′RACE.The size of CXCR7a cDNA was 2 325 bp,including a 207-bp 5′untranslated region(5′UTR),a 1086-bp open reading frame(ORF) which encoded a putative protein of 362 amino acids and a 1032-bp 3′untranslated region(3′UTR) with a poly(A) signal sequence.The size of siberian sturgeon CXCR7b cDNA was 2 423 bp,including a 209-bp 5′untranslated region(5′UTR),a 1110-bp open reading frame(ORF) which encoded a putative protein of 370 amino acids and a 1104-bp 3′untranslated region(3′UTR) with a poly(A) signal sequence.The deduced amino acid sequences of CXCR7a and CXCR7b analysis showed that the amino acid sequence of CXCR7a shared 82.6%,81.4%,80.9% and 81.0% similarity with CXCR7 of Homo sapiens,Mus musculus,Xenopus laevis and Danio rerio,respectively,the amino acid sequence of CXCR7b shared 82.9%,82.3%,80.8% and 82.5% similarity with CXCR7 of Homo sapiens,Mus musculus,Xenopus laevis and Danio rerio,respectively,and the amino acid sequence of CXCR7a shared 96.5% similarity with CXCR7b.The phylogenetic tree of CXCR7a and CXCR7b of Siberian sturgeon and CXCR7 of various vertebrate species were constructed by neighbour-joining method and maximum parsimony method respectively,which showed that siberian sturgeon CXCR7 clustered with Danio rerio CXCR7.Also,the expression of CXCR7a and CXCR7b during various stages of embryonic development and one-day larval after hatching were analysed,and it was found that CXCR7a mRNA was detected in other stages from midgastrula stage to 1 day post-hatch except cleavage period,blastula stage and that CXCR7b mRNA was detected in all stages from cleavage period to 1 day post-hatch.Furthermore,it was confirmed that CXCR7a and CXCR7b were from different gene copies by the analysis of genomic DNA.