黄颡鱼LRH-1基因的分离和表达

Isolation and expression pattern of LRH-1 in yellow catfish (Pelteobagrus fulvidraco)

肝受体类似物(Liver receptor homolog-1,LRH-1)是核受体家族Ftz-F1中一个亚家族成员,由于其在哺乳类卵巢中的高表达而被认为在卵巢类固醇合成的调控中起着一定的作用。本研究使用RT-PCR、RACE法分离到了黄颡鱼(Pelteobagrus fulvidraco)肝受体类似物基因(LRH-1)以及一个无活性的LRH-1′cDNA。LRH-1cDNA全长1945bp,包括5′非翻译区207bp,3′非翻译区238bp,开放阅读框1500bp,编码500个氨基酸,推算的蛋白分子量为57.19kD。LRH-1′全长1663bp,5′非翻译区283bp,3′非翻译区330bp,开放阅读框1050bp,编码350个氨基酸,推算的蛋白分子量为39.7kD。与LRH-1相比,LRH-1′缺少存在于基因5′端的锌指结构和3′端起转录激活作用的AF-2基序。不同动物LRH-1氨基酸序列比对结果表明,黄颡鱼LRH-1和其他动物的LRH-1相似性为79%-85%,和其他动物的类固醇生成因子(SF-1)相似性为59%-66%。系统发育树显示,Ftz-F1分为两大支,一支包括鱼类SF-1;另一支又有2个分支,分别包含其他动物的SF-1和所有动物的LRH-1,在LRH-1分支中鱼类LRH-1单独为一小支。以β-actin为内标的荧光实时定量RT-PCR结果显示,LRH-1在雌、雄黄颡鱼脑、肝、肾、性腺均有表达,以肝脏的表达量为最高,雌鱼各组织的表达量均比雄鱼高,其中肝脏和性腺差异明显(P<0.05);在卵巢的不同发育阶段,该基因的表达量也不尽相同,Ⅲ期卵巢表达量最高,明显高于Ⅴ期(P<0.05),Ⅴ期又明显高于Ⅱ、Ⅳ和Ⅵ期(P<0.05);注射催产素促黄体释放激素类似物(LRH-A)12h后,LRH-1在脑的表达量没明显变化,在肝脏和肾脏的表达量明显增加,而在卵巢的表达量明显下降。

Liver receptor homolog-1（ LRH-1） is an orphan member of the Ftz-F1 subfamily of nuclear receptors. LRH-1 is highly expressed in ovary and so it is thought to be involved in the regulation of steroidogenesis in ovary. Here we isolated LRH-1 cDNA and inactive LRH-1′ cDNA in yellow catfish（ Pelteobagrus fulvidraco） using RT-PCR and RACE method. The yellow catfish LRH-1 cDNA（ 1 945 bp） had a 207 bp 5′-untranslated region（ UTR）,a 238 bp 3′ UTR and a 1 500 bp open reading frame,which encoded a 500-amino-acid residues protein with deduced molecular weight of 57.19 kD. However, LRH-I＇ was 1 663 bp with 283 bp 5＇ UTR,330 bp 3＇ UTR and a 1 050 bp open reading frame, which encoded a 350-amino-acid residues protein with deduced molecular weight of 39.7 kD. Compared with LRH-1, LRH-I＇ were absent of zinc-fingers and AF-2 motif. The alignment results of LRH-I＇s amino acid sequences indicated the similarity was from 79% to 85% between yellow catfish LRH-1 and other vertebrate animals＇ counterparts, and was from 59% to 66% between yellow catfish LRH-1 and other animals＇ steroidogenic factor-1 （SF-1）, another member of Ftz-F1 subfamily. Using Clustal W, we constructed the phylogenetic tree of the Ftz-F1 which showed two main branches. All teleost SF-1 clustered into one branches, and the other branches bifurcated into two clear sub ones, one of which contained other animals＇ SF-1 and the other contained two smaller groups of all teleost＇ s LRH-1 and other animals＇ LRH-1, rseparately. Real-time RT-PCR with fl-actin as internal control was used to explore the expression levels of LRH-1 in brain （including forebrain, hypothalamus, pituitary）, liver, kidney and gonad. The results indicated LRH-1 expressed in brain, liver, kidney, and gonad with relatively high level in male and female fish liver. The expression levels in female liver and ovary were significantly higher than in male fish liver and testis, respectively. In ovary, the expression level of LRH-1 varied with the developmental phases. There were two highest expression levels in overy of phase III and V. After spawning or degeneration of ovary, the LRH-1 expression level decreased sharply. And after 12 h of LRH-A intramuscular injection, the expression level of LRH-1 significantly decreased in ovary. However, it significantly increased in liver and kidney.