中华大蟾蜍皮肤galectin-3 cDNA分子多样性及氨基酸变异分析

 以中华大蟾蜍Bufo gargarizans皮肤总核糖核酸（RNA）反转录第1链互补脱氧核糖核酸（cDNA）为模板，以聚合酶链式反应（PCR）扩增出galectin-3基因片段并通过TA克隆将其插入到pGM-T载体，通过DNA测序确定该基因片段序列，然后利用美国国家生物技术信息中心（NCBI）中ORF finder查找基因的全长开放阅读框（ORF）。筛选到9个编码galectin-3的cDNA序列，其中2个序列编码N-端截短型galectin-3（galectin-3C）。Clustal X2.0多序列比对和DnaSP 4.0分析cDNA序列的多样性。结果发现：中华大蟾蜍galectin-3C区域具有高密度的单核苷酸多态性（SNP），频率为每20 bp为1个单核苷酸多态性。推导的氨基酸序列比对显示这9个基因在N端和糖识别结构域（CRD）有较高同源性，包括磷酸化位点、糖基结合位点和相关基序，而在串联重复区则存在较大差异。galectin-3基因及其编码蛋白的多样性很可能赋予中华大蟾蜍很强的环境适应能力。图3表1参24

The galectin-3 cDNA diversity and amino acid variation of Bufo gargarizans skin tissue

To obtain the cDNA clone of galectin-3,a first-strand cDNA library was synthesized from the total RNA of Bufo gargarizans skin and then used as a template for subsequent polymerase chain reaction(PCR) amplification.The PCR product was successfully cloned into pGM-T vector.After sequencing and using the National Center for Biotechnology Information(NCBI),an open reading frame(ORF) finder was utilized to determine the cDNA.Then,Clustal X 2.0 and DnaSP 4 software were used to analyze cDNA diversity and the deduced amino acid variation.Results of the ORF finder showed nine cDNA clones of galectin-3,two of which were estimated to encode truncated protein.Clustal X 2.0 and DnaSP 4.0 revealed a high density of single nucleotide polymorphism(SNP) with an average of 1 SNP per 20 bp.For deduced amino acid residues,the N-terminal and carbohydrate recognition domain(CRD) were highly consistent with a phosphorylation site,a CRD,and two related motifs.Meanwhile,the tandem repeated domain showed strong differences.Thus,this study indicated a molecular basis for bio-adaptation of B.gargarizans in certain habitat.[Ch,3 fig.1 tab.24 ref.]