暗纹东方鲀sox9基因的克隆和组织表达分析

Molecular cloning and tissue expression analysis of sox9 gene in Takifugu obscurus

摘要:

为初步探究暗纹东方鲀sox9基因在性腺发育和性别分化过程中的作用，通过设计兼并引物、RACE及荧光定量PCR技术，成功克隆出暗纹东方鲀sox9基因的cDNA全长序列，并分析其相应的生物信息学特征及其在雌雄个体的组织表达水平。结果表明：sox9a基因cDNA全长为1 248 bp （NCBI登录号：MH218818），包括684 bp的ORF，编码227个氨基酸，297 bp的5'UTR和267 bp的3'UTR。sox9b基因全长为1 941 bp （NCBI登录号：MH218819），包括1 470 bp的ORF，编码489个氨基酸，306 bp的5'UTR和165 bp的3'UTR。同源性和系统发育分析结果表明暗纹东方鲀sox9基因与红鳍东方鲀同源性最高，亲缘关系最近。氨基酸多重序列比对结果显示两个Sox9氨基酸序列的HMG box结构域在哺乳动物和鱼类中均高度保守。荧光定量PCR分析显示两个sox9基因普遍存在于雌鱼和雄鱼的各个组织中，且均在雌鱼下丘脑中表达量最高，在精巢中有少量表达，卵巢中几乎不表达。总体来说，除少数组织外，两个sox9基因在雌鱼组织中的表达量普遍高于雄鱼。本研究为了解暗纹东方鲀sox9的遗传特性及相关的生理功能，探索性别分化与性腺发育的分子调控机制提供理论依据。

Abstract:

The purpose of this study was to preliminarily investigate the role of sox9 gene in gonad development and sex differentiation in Takifugu obscurus. The full-length cDNA sequence of sox9 gene in Takifugu obscurus is successfully cloned through the design of degenerate primer amplification, RACE and Real-time PCR technology, and subsequently its corresponding bioinformatics characteristics and the level of tissue and expression were also analyzed. The results showed that the full-length of Takifugu obscurus sox9a gene is 1 248 bp (NCBI accession number:MH218818), including a 684 bp ORF, encoding 227 amino acids; the 5'UTR is 297 bp and the 3'UTR is 267 bp. The full-length of Takifugu obscurus sox9b gene is 1 941 bp (NCBI accession number:MH218819), including a 1 470 bp ORF, encoding 489 amino acids; the 5'UTR is 306 bp and the 3'UTR is 165 bp. The results of homology and phylogenetic analysis showed that the two sox9 sequences shared the highest homology and closest relationship with Takifugu rubripes. The results of multiple amino acid sequence alignment showed that the HMG box domains of the two Sox9 amino acid sequences were highly conserved in mammals and fish. Quantitative fluorescence PCR analysis showed that the two sox9 genes are ubiquitous in all tissues of female and male fish, and they were most highly expressed in the hypothalamus of female fish, a little in the testis, and very little in the ovary. Overall, except for a few tissues, the expression of two sox9 genes in tissues of females was generally higher than that of males. The purpose of this study was to understand the genetic characteristics and physiological functions of sox9 in Takifugu obscurus, and to explore the molecular regulation mechanism of sex differentiation and gonadal development.