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草鱼（Ctenopharyngodon idellus）促性腺激素及其受体基因的克隆及表达分析
Molecular Cloning, Characterization and Expression of Gonadotropin and Their Receptors from Grass Carp (Ctenopharyngodon Idellus)
【作者】 周毅；
【作者基本信息】 湖南师范大学 ， 动物学， 2009， 硕士
【摘要】 促性腺激素(Gonadotropin Hormone,GTH)和促性腺激素受体(Gonadotropin receptor,GTHR)对鱼类性腺发育及排卵排精起重要调控作用。本研究克隆了草鱼(Ctenopharyngodon idellus)GTH两种激素三个亚基的cDNA全长序列和GTHR两种受体的部分序列。采用RT-PCR分析了不同组织中两种GTHβ亚基的表达模式,并通过实时定量PCR技术对不同卵巢发育时期的草鱼GTH表达水平进行了分析。旨在探讨草鱼GTH和GTHR的表达与其生殖发育的关系。获得的主要研究结果如下:1.获得了GTHα,FSHβ及LHβ三个基因的全长序列,为844bp,739bp和576bp,它们分别编码118,130和146个氨基酸。2.获得了草鱼的FSHR及LHR的部分序列。其中FSHR为509bp编码169个氨基酸。LHR获得了两个片段,LHR1为869bp编码289个氨基酸;LHR2为1204bp编码253个氨基酸。3.与其它物种的GTH氨基酸序列比对表明:鱼类中GTH的三个亚基相似性较高,且GTHα较FSHβ和LHβ更为保守。构建的NJ进化树与形态分类学的系统进化相一致。4.RT-PCR证实,草鱼GTH仅在脑垂体中特异性表达。5.实时定量PCR对草鱼卵巢不同时期的FSH和LH分析表明:FSH和LH在草鱼性腺发育早期表达量很低,而在性腺成熟期则同步高表达,且LH的表达量高于FSH。
【Abstract】 GTH(Gonadotropin Hormone) and GTHR(Gonadotropin receptor) play a central role in reproduction in fish gonad development and ovulation.The three GTH subunits and two partial GTHR cDNA were cloned and characterized from grass carp.RT-PCR was employed to check tissue distribution of FSH and LH.Real-time PCR showed the expression pattern of GTH in different period of ovary.This study showed some results of GTH and GTHR gene cloning and characterization.1.Full length cDNA of GTHα,FSHβand LHβcDNA was 844bp, 739bp and 576bp,with open reading frame encoding proteins of 118,130 and 146 amino acids,respectively.2.Partial cDNA of FSHR and LHR were cloned.FHSR was 509bp encoding 169 amino acids.LHR obtained two partial fragments.LHR1 was 869bp encoding 289 amino acids,and LHR2 was 1204bp encoding 253 amino acids.3.Phylogenetic analysis of the proteins of GTH showed a high homology with other fishes.Homology analysis also indicated that GTHαhas the highest conservation.The Phylogenetic tree was identical with morphologic.4.RT-PCR analysis indicated that GTH were only expressed in the pituitary. 5.Real-time quantitative PCR protocols were employed to measure FSHβand LHβmRNAs during ovarian development.The FSHβand LHβmRNA level was very low in the pituitaries of early-pubertal fish and significantly increased during ovulation period.These results suggested that in grass carp the gonadotropins synthesized synchronously in order for asynchronous oogenesis to take place.