中华大蟾蜍gal-3基因多样性的原因分析

Analysis of gal-3 gene diversity of Bufo gargarizans

使用TAKARA的SMARTer^(TM)RACE cDNA Amplification Kit和改进的Quantscript RT kit方法制备了3组源于同一中华大蟾蜍(Bufo gargarizans)个体皮肤总核糖核酸(RNA)的第一链互补脱氧核糖核酸(cDNA),并以此为模板通过聚合酶链式反应(PCR,polymerase chain reaction)扩增半乳糖凝聚素3(gal-3)的全长开放阅读框(ORF,open reading frame),然后进行PCR产物的克隆、测序,再通过多种形式的序列比对分析该基因多样性产生的原因.从3组实验得到31个克隆,去除组内和组间重复出现的序列,共获得16个不同的cDNA.相同的序列,相同的单核苷酸多态性位点(single nucleotide polymorphism,SNP)以及相同位置相同数目的碱基缺失在3组独立的实验中重复出现,表明gal-3基因多样性.实际上,gal-3基因的多样性导致其编码蛋白的多样性.尽管如此,推导的氨基酸序列比对显示半乳糖凝聚素3糖基结合位点、磷酸化位点以及与其生物学功能关系密切的相关基序均高度保守,暗示中华大蟾蜍皮肤gal-3基因多样性的客观存在.

Three independent groups of first-strand cDNAs were synthesized from the total RNA extracted from the skin of a single individual of Bufo gargarizans using different kits including SMARTerTM RACE cDNA Amplification Kit and modified Quantscript RT kit,which were then used as templates for the subsequent polymerase chain reaction （PCR）.The PCR products were cloned into the pGM-T vector and sequenced.As a result,16 different uni-cDNAs were singled out of the total 31 cDNA clones.Same sequences,single nucleotide polymorphism sites （SNPs） and base deletions were detected in two or three independent experiments/groups,indicating gal-3 diversity,which led to diversity of proteins encoded.But alignment of the amino acid residues deduced form these gal-3 cDNAs revealed high conservation of glycosyl-binding sites,phosphorelation sites and the motifs highly associated with biological functions,which suggested the existence of galectin-3 diversity in the skin of B.gargarizans.