条石鲷促性腺激素β(FSH,LH)亚基cDNA克隆及表达特性分析  

Cloning and expression pattern of gonadotropin hormone β( FSH,LH)subunit in Oplegnathus fasciatus

作　　者：陈圣毅[1,2] 柳学周[1] 史宝[1] 徐永江[1] 李晓晓[1,2] 王珊珊[1] 臧坤[1,2] 

机构地区：[1]中国水产科学研究院黄海水产研究所,农业部海洋渔业可持续发展重点实验室,青岛市海水鱼类种子工程与生物技术重点实验室,山东青岛266071 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2014年第10期1673-1686,共14页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2012AA10A413);国家鲆鲽类产业技术体系专项(CARS-50);国家自然科学基金(31201982);山东省优秀中青年科学家科研奖励基金(BS2013SW042)

摘　　要：采用同源性克隆和cDNA末端快速扩增(RACE)方法克隆了条石鲷GtHβcDNA序列,并分析了GtHβmRNA在条石鲷组织中的表达差异和在卵巢发育不同时期的垂体、卵巢、脑中表达水平变化。结果发现,条石鲷FSH cDNA全长554 bp,ORF长363 bp,编码了120个氨基酸,前18个氨基酸为信号肽。条石鲷LH cDNA全长857 bp,ORF长450 bp,编码了149个氨基酸,前24个氨基酸为信号肽。FSH和LH包含了12个保守的半胱氨酸残基(Cys)和一个N-糖基化位点,在LH的第4和第5个Cys之间存在着硬骨鱼类特异性的Cys-Ser-Gly-His(CSGH)区域。同源性比较和进化分析显示,条石鲷FSH和LH与鲈形目鱼类的进化关系较近。采用实时荧光定量PCR法检测发现,FSH mRNA和LH mRNA在垂体中的表达量最高,其次是性腺;FSH mRNA在头肾和肾脏中不表达,LH mRNA在头肾中不表达。周期表达分析显示,垂体中的FSH mRNA在卵巢发育的Ⅳ期达到最大值,LH mRNA在Ⅴ期达到最大值;卵巢中的FSH mRNA在Ⅴ期达到最低值,而LH mRNA在Ⅴ期达到最大值;脑中的FSH和LH mRNA在Ⅴ期达到最低值。激素分析显示,血清中FSH在卵巢发育的Ⅳ期达到最大值,而LH在Ⅴ期达到最大值,血清中FSH和LH水平与垂体中FSH mRNA和LH mRNA表达趋势一致。Full length cDNA sequences of gonadotropin hormone β subunits were cloned from the pituitary of Oplegnathus fasciatus by homology cloning and RACE techniques. The expression of GtHfl mRNA was analysed in different tissues and different ovarian development stages of pituitary, ovary, and brain. The FSH cDNA sequence is 554 bp and its open reading frame is 363 bp, which encoded a precursor protein of 120 amino acids preceded by a signal peptide of 18 amino acids residues. The LH cDNA sequence is 857 bp and its open reading frame is 450 bp,which encoded a precursor protein of 149 amino acids preceded by a signal peptide of 24 amino acids residues. Both of the putative peptide of FSH and LH contained 12 cysteine residues and 1 N-glycosylation sites residues. Moreover, there was a special area of Cys-Ser-Gly-His （CSGH） in the middle of 4 and 5 cysteine of LH. Sequences comparison and phylogenetic analysis showed that the O. fasciatus FSH and LH had the close relationship with Perciformes. The Real-time Quantitative PCR results suggested that the expression levels of FSH and LH mRNA were the highest in pituitary, followed by ovary. The FSH mRNA was not found in kidney and head kidney and the LH mRNA was not found in Head kidney. The GtHI3 expression level in the reproductive cycle showed that the level of pituitary FSH mRNA was maximum in the IV ovarian development stage and the LH mRNA was maximum in the V ovarian development stage. In ovary,the level of FSH mRNA was the lowest in the V ovarian development stage, however, the LH mRNA was maximum in the V ovarian development stage. Both of the brain FSH and LH mRNA were the lowest in the V ovarian development stage. The hormone analysis indicated that the concentration of FSH was maximum in the IV ovarian development stage in serum,but the concentration of LH was maximum in the V ovarian development stage in serum. The content of FSH and LH in serum showed a consistent trend of regular changes with the level of FSH mRNA and LH mRNA in pituitary. The research is be

关 键 词：条石鲷 促性腺激素β亚基 CDNA克隆 表达 卵巢发育周期 

分 类 号：S917.4[农业科学—水产科学]