中华鲟促甲状腺激素β亚基基因（TSHβ）cDNA序列的克隆及其表达分析
Cloning and expression analysis of TSHβ subunit gene in Chinese sturgeon,Acipenser sinesis

【目的】克隆中华鲟（Acipenser sinesis）促甲状腺激素β亚基(TSHβ)基因，分析其序列特征和进化特征，研究其在中华鲟中的组织表达特征和不同年龄垂体中的差异表达，为中华鲟生长发育调控研究提供基础数据。【方法】采用反转录聚合酶链式反应（RT-PCR）和cDNA末端快速扩增（RACE），克隆中华鲟TSHβ，对其cDNA序列及其推导的氨基酸序列进行序列特征分析和系统发生分析；采用实时荧光定量PCR（Real-time PCR）方法，对TSHβ mRNA在中华鲟肝、心、脾、性腺、肠、垂体、下丘脑、中脑、端脑、小脑和延脑等组织中的表达状况,以及在1，3，5龄中华鲟个体垂体中的表达差异进行研究。【结果】克隆获得TSHβ基因，该cDNA序列全长649 bp,5′和3′端非编码区分别为142和75 bp;开放阅读框432 bp,编码143 个氨基酸。TSHβ亚基成熟多肽含有12个半胱氨酸（Cys）和2个N连糖基化位点。氨基酸序列多重比对表明，中华鲟TSHβ与促滤泡激素β亚基和促黄体激素β亚基的一致性分别为36%和35%，但与糖蛋白激素α亚基的一致性约为10%。与其他脊椎动物的TSHβ氨基酸序列进行比对发现，中华鲟TSHβ亚基与西伯利亚鲟一致性最高（98%）；与鱼类、两栖类、鸟类和哺乳类的序列一致性分别约为44%，55%，56%和60%；脊椎动物TSHβ的Cys和N连糖基化位点位置高度保守。TSHβ亚基仅在中华鲟垂体中有表达；3龄雄性个体垂体中TSHβ的表达量约为1龄的9倍，3龄和5龄雌性个体垂体中的表达量均约为1龄的5倍左右。【结论】成功获得了中华鲟TSHβ亚基基因，该基因具有与其他动物TSHβ相似的结构和表达特征；雄性个体表达量高于雌性个体，其在中华鲟个体的生长发育过程中表达量上升。
【Objective】The thyroid stimulating hormone β (TSHβ) gene in Acipenser sinesis was cloned to analyze its sequence characterization,phylogenetic specificity,tissue expression pattern,and differential expression in pituitaries of Chinese sturgeons at different ages.This study would provide basic data for further research on the growth metabolism regulation of Chinese sturgeon.【Method】The reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used for the isolation of the full length cDNA of TSHβ subunit from pituitary of Chinese sturgeon.Multiple sequence alignment and phylogenetic analysis were conducted on the deduced amino acid of TSHβ.Then,the distributions of TSHβ mRNA in different tissues (liver,heart,spleen,testis,ovary,kidney,intestine,pituitary,hypothalamus,medulla oblongata,cerebellum,midbrain and endbrain) and in pituitaries at different ages were examined by real time PCR.【Result】The full-length cDNA of TSHβ was 649 bp,containing a 142 bp 5′-untranslated region (UTR), a 75 bp 3′ UTR and a 432 bp open reading frame (ORF) with a peptide of 143 amino acids.The deduced amino acid of TSHβ subunit contained a putative signal peptide of 18 amino acids,with the mature protein encompassing 12 cysteine (Cys) residues and 2 putative N-glycosylation sites.Multiple sequence alignment showed that,in Chinese sturgeon,TSHβ subunit showed 36% and 35% sequence identity to the follicle stimulating hormone β subunit (FSHβ) and the luteinizing hormone β subunit (LHβ),followed by only 10% sequence identity to the glycoprotein hormone α subunits.Moreover,Chinese sturgeon TSHβ subunit shared the highest sequence identity (98%) with Siberian sturgeon,and showed only 44%,55%,56% and 60% identity to teleosts,amphibians,birds and mammals,respectively.The 12 Cys and 1