凡纳滨对虾CCT6基因克隆及SNP位点筛选

Cloning and SNP locus screening of CCT6 gene in Litopenaeus vannamei

  • 摘要: 【目的】克隆凡纳滨对虾CCT6基因并筛选与生长相关的SNP位点,为选育生长快速的凡纳滨对虾分子标记辅助育种提供参考依据。【方法】通过克隆获得凡纳滨对虾CCT6基因的cDNA和DNA序列,应用ExPASy、InterPro、TMHMM、SOPMA及SWISS-MODEL等在线软件进行生物信息学分析,运用实时荧光定量PCR检测CCT6基因在凡纳滨对虾不同组织及2个不同生长速度的凡纳滨对虾群体中的表达情况,利用直接测序法筛选凡纳滨对虾CCT6基因SNP位点并将其与凡纳滨对虾生长性状进行关联分析。【结果】凡纳滨对虾CCT6基因cDNA序列全长为1835 bp,5'和3'非编码区(UTR)分别为55和193 bp,开放阅读框(ORF)为1587 bp,共编码531个氨基酸残基,DNA全长为15873 bp,具有11个外显子和10个内含子。CCT6基因在凡纳滨对虾眼柄、鳃、胃、肝胰腺、心脏、肠道和肌肉组织中均有表达,在肠道组织中相对表达量显著高于其他组织(P<0.05,下同)。A群体凡纳滨对虾最终体质量、最终体长等生长指标显著高于B群体,生长快速的凡纳滨对虾A群体肠道组织中CCT6基因的相对表达量显著高于B群体。于凡纳滨对虾CCT6基因的DNA序列上筛选到6个SNP位点,其中2个SNP位点位于DNA序列的外显子区域,4个位于DNA序列的内含子区域。其中位于内含子区域的C14895C>T位点与凡纳滨对虾的生长性状显著相关,C14895C>T位点CC基因型凡纳滨对虾个体的体长和体质量显著高于TT基因型,且CC基因型在A群体凡纳滨对虾中的数量高于B群体。【结论】CCT6基因在凡纳滨对虾生长发育中具有重要作用,生长快速的A群体趋于纯合的CC基因型,C14895C>T位点可作为选育生长快速的凡纳滨对虾分子标记辅助育种的候选标记。

     

    Abstract: 【Objective】Cloned CCT6 gene of Litopenaeus vannamei and screened SNP loci related to growth to provide reference for screening of fast-growing L. vannamei molecular marker assisted breeding. 【Method】The cDNA and DNA sequences of the CCT6 gene in L. vannamei were obtained through gene cloning,and analyzed them using online bioinformatics softwares such as ExPASy,InterPro,TMHMM,SOPMA and SWISS-MODEL. Real time fluorescence quantitative PCR technology was used to determine the expression of the CCT6 gene in different tissues and two groups of L. vannamei with different growth rates. Using direct sequencing method to screen the SNP loci of CCT6 gene and analyze the correlation between the SNP loci and the growth traits of L. vannamei. 【Result】The total length of CCT6 gene cDNA sequence of L. vannamei was 1835 bp,the 5' and 3' non coding region(UTR) were 55 and 193 bp,the open reading frame(ORF) was 1587 bp,encoding a total of 531 amino acids residues. The length of the DNA sequence was 15873 bp,with 11 exons and 10 introns. The CCT6 gene was expressed in the eye stalks,gills,stomach,hepatopancreas,heart,intestine and muscle tissues of L. vannamei,and the expression level in the intestinal tissue was significantly higher than that in other tissues(P<0.05, the same below). The growth indexes such as final body weight and final body length of A group were significantly higher than that of B group, and the relative expression of CCT6 gene in the intestinal tissue of fast growing L. vannamei A group was significantly higher than that in B group. Six SNP loci were screened from the DNA sequence of CCT6 gene,two SNP loci were located in the exon of DNA sequence,and four SNP loci were located in the intron. The C14895C>T locus located in the intron region was significantly correlated with the growth traits of L. vannamei.The length and weight of L. vannamei with C14895C>T locus CC genotype were significantly higher than those of the TT genotype,and the number of CC genotypes in the A group of L. vannamei was higher than that in the B group. 【Conclusion】CCT6 gene plays an important role in the growth and development of L. vannamei, the fast growing A group tends to be a homozygous CC genotype and the C14895C>T locus can be used as a candidate marker for molecular marker-assisted breeding of the fast growing L. vannamei.

     

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