马氏珠母贝CD-MPR基因序列特征及其在耐低温品系的选择分析

Sequence characteristics of CD-MPR gene in Pinctada fucata martensii and its selection analysis in low-temperature resistant strains

  • 摘要: 【目的】明确马氏珠母贝(Pinctada fucata martensii)阳离子依赖性甘露糖-6-磷酸受体(CD-MPR)基因(PmCD-MPR)多态性与其低温耐受能力的相关性,为马氏珠母贝耐低温品系的选育提供理论依据。【方法】采用RACE克隆PmCD-MPR基因序列全长,并运用实时荧光定量PCR检测PmCD-MPR基因在马氏珠母贝各组织及低温胁迫下的表达模式;基于马氏珠母贝耐低温选育系(R群体)和北部湾野生群体(W群体)的全基因组重测序数据筛选出PmCD-MPR基因外显子区的SNP位点,并通过遗传多态性分析、单倍型分析及频率计算获取与马氏珠母贝低温胁迫过程相关的SNP位点。【结果】PmCD-MPR基因序列全长902 bp,其开放阅读框(ORF)为792 bp,共编码263个氨基酸残基; PmCD-MPR基因编码蛋白分子量为30.22 kD,理论等电点(pI)为5.79,属于亲水性蛋白,在第18~263位氨基酸处含有1个典型的甘露糖-6-磷酸受体(Man-6-P_recep)结构域。CD-MPR氨基酸序列在N端的相似性较低,在C端的相似性较高;基于CD-MPR氨基酸相似性构建的系统发育进化树显示,马氏珠母贝与长牡蛎、加利福尼亚海兔等软体动物聚为一支,与经典的动物学分类相吻合。PmCD-MPR基因在马氏珠母贝肝胰腺的相对表达量最高,显著高于在性腺、闭壳肌和外套膜中的相对表达量(P<0.05,下同);低温胁迫组马氏珠母贝鳃组织中的PmCD-MPR基因相对表达量随着胁迫时间的延长整体上呈先上升后下降的变化趋势。从PmCD-MPR基因外显子区筛选获得34个SNPs位点,且这34个SNPs位点构成5个单倍块和15种单倍型,其中GCC、TG、CCCTCT等3种单倍型在R群体中的分布频率显著高于W群体,即与马氏珠母贝耐低温性状显著相关。【结论】PmCD-MPR基因参与马氏珠母贝的低温响应过程,与耐低温性状显著相关的基因型及单倍型可作为马氏珠母贝耐低温品系辅助育种的候选分子标记。

     

    Abstract: 【Objective】To clarify the correlation between the polymorphism of cation-dependent mannose-6-phosphate receptor(CD-MPR)gene(PmCD-MPR)and the low temperature tolerance of Pinctada fucata martensii,and to provide theoretical basis for the breeding of low temperature resistant strains of P. fucata martensii.【Method】The fulllength sequence of PmCD-MPR gene was cloned by rapid-amplification of cDNA ends(RACE),and real-time fluorescence quantitative PCR was used to detect the expression pattern of PmCD-MPR gene in various tissues of P. fucata martensii and under low temperature stress;based on the whole genome resequencing data of P. fucata martensii low temperature resistant line(R group)and Beibu Gulf wild population(W group),we screened for the single nucleotide polymorphism(SNP)loci in exon regions of the PmCD-MPR gene,and obtained the SNP loci related to the low temperature stress process of P. fucata martensii through the analysis of genetic polymorphisms,haplotypes,and the frequency calculations.【Result】The PmCD-MPR gene sequence was 902 bp in length,with an open reading frame(ORF)of 792 bp, encoding a total of 263 amino acid residues;the molecular weight of the protein encoded by the PmCD-MPR gene was 30.22 kD,and the theoretical isoelectric point(pI)was 5.79,which belonged to the hydrophilic proteins,and it contained a typical mannose-6-phosphate receptor(Man-6-P_recep)structural domain at amino acids 18-263. CD-MPR amino acid sequences were less similar at the N-terminal end and more similar at the C-terminal end;the phylogenetic tree constructed on the basis of CD-MPR amino acid similarity showed that P. fucata martensii was clustered with mollusks such as Crassostrea gigas and Aplysia californica,which was in line with the classical zoogeographic classification. The relative expression of PmCD-MPR gene was the highest in the hepatopancreas and significantly higher than that in the gonads,adductor muscle and mantle(P<0.05,the same below);the relative expression of PmCD-MPR gene in the gill tissues of P. fucata martensii in the low temperature stress group showed a tendency to increase first and then decrease with the prolongation of the stress time. A total of 34 SNPs were screened from the exon region of PmCD-MPR gene,and these 34 SNPs constituted 5 haplotype blocks and 15 haplotypes. Among them,the distribution frequency of GCC,TG and CCCTCT haplotypes in R population was significantly higher than that in W population,which was significantly correlated with the low temperature tolerance of P. fucata martensii.【Conclusion】The PmCD-MPR gene is involved in the low temperature response process of P. fucata martensii,and the genotypes and haplotypes that are significantly associated with low temperature tolerance traits can be used as candidate molecular markers for assisted breeding of low temperature tolerant lines of P. fucata martensii.

     

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