马氏珠母贝Perlucin基因序列特征及其SNP与耐低温性状的关系

Sequence Characteristics of Perlucin Gene in Pinctada fucata martensii and Relationship between Its SNPs and Low Temperature Tolerance

【目的】克隆马氏珠母贝(Pinctada fucata martensii)新的凝集素分子基因,命名为Perlucin,研究在低温胁迫下马氏珠母贝Perlucin的表达以及与抗低温性状相关的单核苷酸多态性(SNP)位点。【方法】根据马氏珠母贝基因组中Perlucin基因序列设计引物,克隆Perlucin基因全长;用生物信息学方法分析Perlucin的结构和理化特征;设计17和22℃(对照)2个温度组,对马氏珠母贝进行低温胁迫实验,用实时荧光定量PCR检测低温胁迫下Perlucin表达量的变化;筛选和比较分析马氏珠母贝耐低温选育系(R)F3和北部湾野生群体(W)的Perlucin外显子区的SNP位点和单倍型。【结果】马氏珠母贝Perlucin全长631 bp,编码152个氨基酸;包含1个信号肽和1个C型凝集素结构域。同源分析表明,马氏珠母贝Perlucin与紫贻贝(Mytilus galloprovincialis)Perlucin的亲缘性最近。Perlucin在鳃中表达量最高,其次为足和肝胰腺。低温胁迫时,鳃组织中Perlucin基因在17℃低温组的表达量呈先升高后降低的趋势,在12 h时达到最高并显著高于22℃对照组(P<0.05),表明Perlucin可能参与马氏珠母贝的低温响应过程。对Perlucin外显子区的SNP进行分析,共得到30个SNP,其中13个SNP位点的基因型频率在R和W群体间差异显著(P<0.05)。【结论】Perlucin是参与调节马氏珠母贝低温适应过程中的候选基因,筛选出两个SNP位点g.40078856、g.40078945。

【Objective】The aim of this study is to clone a new lectin molecular gene,named Perlucin,from Pinctada fucata martensii,and to study its expression under low-temperature stress and its SNPs associated with low-temperature resistance traits.【Method】According to the sequence of Perlucin in the genome of P.f.martensii,primers were designed to clone the full length of Perlucin.The structure and physicochemical characteristics of Perlucin was analyzed by bioinformatics methods.The low temperature stress(17℃)experiment(22℃as the control)was conducted on P.f.martensii,and the expression of Perlucin under low-temperature stress was detected by Quantitative-PCR.SNP loci and haplotypes in the Perlucin exon region of the low temperature resistant line(R)and Beibu Gulf wild population(W)were screened and compared.【Result】Sequence analysis revealed that the full length of Perlucin is 631bp that encoded 152 amino acids and contained 1 signal peptide and 1 C-type lectin domain.Homologous analysis showed that Perlucin was most closely related to Mytilus galloprovincialis.Perlucin showed highest expression in the gill,followed by the foot and hepatopancrea.Under the low temperature stress(17℃),the expression of Perlucin firstly increased and then decreased.The highest expression level at 12 h,significantly higher than that in the control group(22℃),indicated that Perlucin may participate in the cryogenic response process of P.f.martensii.The SNPs of the exon region of perlucin were analyzed,and a total of 30 SNPs were obtained,among which the genotype frequencies of 13 SNP loci varied significantly between R and W populations(P<0.05).【Conclusion】Perlucin is a candidate gene involved in the low-temperature adaptation of P.f.martensii.Two SNP loci were screened(g.40078856 and g.40078945).