黄河鲤胰岛素诱导基因1(INSIG1)序列特征、SNP位点及其与生长性状的关联分析

Sequence characteristics,SNP locus and association with growth traits of insulin-induced gene 1(INSIG1)in Yellow River carp(Cyprinus carpio haematoperus)

为探究胰岛素诱导基因1(insulin-induced gene,INSIG1)在黄河鲤(Cyprinus carpio haematoperus)生长发育过程中的作用,采用候选基因法对黄河鲤生长候选基因INSIG1进行了序列特征分析、组织表达及SNP与生长性状的关联分析。结果表明:黄河鲤INSIG1基因全长为4853 bp,包含5个外显子和4个内含子,其中cDNA全长为1530 bp,蛋白编码区(CDS)全长为756 bp,可编码251个氨基酸;INSIG1在不同物种间序列保守性较高,黄河鲤INSIG1与金线鲃、斑马鱼和鲫等鲤科鱼类聚为一支;预测黄河鲤INSIG1蛋白相对分子质量为27674.25,理论等电点为8.09,属于稳定的疏水性蛋白,该蛋白无信号肽,包含6个跨膜结构,蛋白三级结构包含6个α-螺旋和部分无规则卷曲;qRT-PCR分析显示,INSIG1基因在黄河鲤肝、脾、肾、肠、心脏、鳃、脑、肌肉和皮肤等组织中均有表达,其中在肠和肝组织中表达量较高,在鳃组织中表达量最低;黄河鲤INSIG1基因共鉴定到10个SNP位点,其中g.1854T>C和g.1982A>T 2个SNP位点的不同基因型与黄河鲤肥满度性状显著相关(P<0.05),其他位点的不同基因型与生长性状未表现出显著关联性。研究表明,INSIG1基因参与了黄河鲤的生长发育过程,其突变位点g.1854T>C和g.1982A>T显著影响了黄河鲤的肥满度,该结果为黄河鲤生长性状的分子标记辅助育种提供了可用的分子标记,对于选育具有优良性状的黄河鲤具有重要的实践意义。

In order to explore the function of insulin-induced gene 1(INSIG1)in the growth and development process of Yellow River carp(Cyprinus carpio haematoperus),the sequence characteristics,expression patterns and SNP association with growth traits of INSIG1 in 26 all-sibling lineages of Yellow River carp(Cc-INSIG1)were analyzed based on the candidate gene approach.The results showed that the full-length DNA of Cc-INSIG1 gene was 4853 bp,containing 5 exons and 4 introns,and the full-length cDNA of Cc-INSIG1 gene was 1530 bp,including 756 bp of the coding sequence,encoding 251 amino acids.The amino acid sequence of Cc-INSIG1 showed high homology with INSIG1 of other species,and clustered with other fish species in Cyprinidae like horned golden-line barbel(Sinocyclocheilus rhinocerous),zebrafish(Danio rerio)and crucian carp(Carassius auratus).The Cc-INSIG1 protein had relative molecular mass of 27674.25 and the theoretical isoelectric point(pI)of 8.09,and belonged to the stable hydrophobic protein,without signal peptide.The structure analysis revealed that there were 6 transmembrane structures and the tertiary structure mainly consisted of 6 alpha helices and random coils.Real-time PCR analysis showed that Cc-INSIG1 was ubiquitous expressed in liver,spleen,kidney,intestine,heart,gill,brain,muscle and skin,with extreme predominance in intestine and liver,and the minimum in gill.Ten single-nucleotide polymorphisms(SNP)were identified in DNA sequence of Cc-INSIG1,two,that is,g.1854T>C and g.1982A>T,were significantly associated with Fulton's condition factor(K)in the populations of Yellow River carp(P<0.05),and without association with growth traits in other SNP.It was found that INSIG1 gene was involved in the growth and development of Yellow River carp,and its mutation sites g.1854T>C and g.1982A>T significantly influenced the Fulton's condition factor of Yellow River carp.The findings provided available markers for gene(marker)-assisted selective breeding in Yellow River carp,which has great significance to the breeding of Yellow River carp with excellent economic traits.