大黄鱼sox9a/b基因的克隆与表达分析

Cloning and expression of sox9a/b gene in the large yellow croaker(Larimichthys crocea)

为探究sox9基因在大黄鱼性别决定与分化过程中的作用,利用RACE方法克隆得到大黄鱼sox9a和sox9b 2个基因,采用实时荧光定量PCR(qRT-PCR)分析其在雌雄个体不同组织和不同发育阶段的表达规律,并检测了其在17β-雌二醇处理后的遗传雄性和17α-甲基睾酮诱导处理后遗传雌性幼鱼中的表达变化。结果显示,大黄鱼sox9a c DNA全长2 442 bp(NCBI登录号:MH996431),包含476 bp 5′UTR、466 bp 3′UTR、1 500 bp ORF,编码499个氨基酸。大黄鱼sox9b cDNA全长为2 199 bp(NCBI登录号:MH996432),包含335 bp5′UTR、415 bp 3′UTR、1 449 bp ORF,编码482个氨基酸。qRT-PCR分析显示,sox9a基因主要在性腺、眼、脑、肝脏中表达,精巢中的表达量显著高于卵巢;sox9b在大黄鱼各组织中广泛表达,但以精巢中表达量最高,而卵巢中只有痕量表达。在鱼苗性腺发育初期,sox9a/b的表达量都维持在较低水平;随着鱼苗长大,sox9a/b的表达量在84 dph、123 dph 2次达到高峰,随后开始下降,10 mph后又逐渐上升。17β-雌二醇处理能够显著下调遗传雄性大黄鱼sox9a和sox9b基因在性腺中的表达,17α-甲基睾酮处理则显著上调遗传雌性大黄鱼sox9a和sox9b基因在性腺中的表达。研究表明,sox9a/b基因与大黄鱼性别发育和分化过程密切相关,对大黄鱼精巢的发育与维持起重要的调控作用,而且两个基因的功能可能具有一定程度的分化。

To elucidate the role of sox9in sex determination and differentiation of the large yellow croaker(Larimichthys crocea),the full length of sox9a and sox9b were cloned using reverse transcription-polymerase chain reaction(qRT-PCR)and rapid amplification of cDNA ends(RACE).The difference of the gene expression in various tissues and development stages was analyzed through quantitative real-time PCR.Expression profiles of sox9a/b after 17β-estradiol or 17α-methyl testosterone treatments were also examined.The full-length cDNA of sox9a gene is 2 442 bp(NCBI:MH996431),including a 476 bp 5′UTR,a 466 bp 3′UTR and a 1 500 bp ORF coding a polypeptide of 499 amino acids.The full-length cDNA of sox9b gene is 2 199 bp(NCBI:MH996432),including a 335 bp 5′UTR,a 415 bp 3′UTR and a 1 449 bp ORF coding a polypeptide of 482 amino acids.Quantitative Real-time PCR results showed that sox9a was primarily expressed in gonad,eye,brain,liver,and the expression level in testis was significantly higher than that in ovaries.sox9b was widely expressed in multiple tissues in large yellow croaker;the expression level was the highest in testes,but can be barely detected in ovaries.At early developmental stages of fry,sox9a/b was expressed at a lower level.Sox9 a/b peaked at 84 dph(day post hatch)and 123 dph,then their expression declined and gradually rose again at 10 mph.In addition,17β-estradiol can significantly down-regulate the expression of sox9a and sox9b in testes.17α-methyl testosterone can significantly elevate the expression of sox9a and sox9b in gonads.The study demonstrated that sox9a/b may play important roles in sex determination and differentiation in the large yellow croaker.However,the functions of the two genes may be different.