企鹅珍珠贝Dmrt2基因的克隆及表达分析

摘要

本研究利用RACE-PCR技术获得企鹅珍珠贝(Pteria penguin)一个Dmrt2基因cDNA的全长序列,通过荧光定量PCR分析Dmrt2基因在各组织中的表达特征,以及在早期卵巢、成熟期卵巢、早期精巢、成熟期精巢和排放期精巢中的表达变化.结果表明,Dmrt2基因全长1 257bp,其中开放阅读框(open reading frame,ORF)为951 bp,编码3 1 6个氨基酸,5'非编码区(untranslated region,UTR)为52 bp,3'UTR为254 bp,第20位到第73位氨基酸为DM结构域.预测其分子质量为36.61ku,等电点为9.80.氨基酸序列比对显示该企鹅珍珠贝Dmrt2基因与黑蝶真珠蛤(Pinctada margaritifera)和马氏珠母贝(Pinctada martensii)的Dmrt2基因同源性(identity)最高,分别为46.0％和45.7％.其中在DM结构域高度同源.荧光定量PCR分析组织表达特征显示,Dmrt2在企鹅珍珠贝的外套膜、鳃、消化盲囊、足、精巢和卵巢均有表达,其中在精巢中表达量最高(P＜0.05),足为其次,在闭壳肌中没有检测到Dmrt2表达.对性腺发育不同时期的表达量分析发现,Dmrt2在发育早期和成熟期卵巢中表达量都很低,在发育早期精巢中表达量较高,在成熟期精巢检测到最大表达量(P＜0.05),到精巢排放期表达显著下降,推测Dmrt2可能与企鹅珍珠贝精巢的发育有关,可能参与了企鹅珍珠贝雄性性别分化和性腺发育这一生理过程.%Dmrt2 (doublesex and mab-3-related transcription factor 2) is an important member of the Dmrt gene family with a highly conserved zinc finger-like DM domain and plays an important role in somite formation,organ formation,and skeletal patterning.This study was conducted to investigate the role of Dmrt2 in sex determination and gonadal development in Pteria penguin.The full-length cDNA of Dmrt2 gene was characterized from P.penguin by RACE-PCR,and real-time PCR was performed to assess the expression of Dmrt2 in different tissues and gonads of different growth periods.The results showed that the full-length cDNA of Dmrt2 was 1257 bp,including a 5'UTR of 52 bp,a 3'UTR of 254 bp,and an open reading frame of 951 bp,which encoded a deduced protein of 317 amino acids.The DM domain was from 20 to 73 amino acids.The predicted molecular weight was 36.61 ku,and the isoelectric point was 9.80.The amino acid sequence alignment showed that Dmrt2 of P.penguin shared 46.0％ and 45.7％ sequence identity with Dmrt2 of Pinctada margaritifera and Pinctada martensii,respectively.The real-time PCR results showed that Dmrt2 was constitutively expressed in the mantle,gill,digestive diverticulum,foot,testis,and ovary,except in the adductor muscle.The expression level was highest in the testis (P ＜ 0.05),followed by the foot.The expression analysis in gonads of different growth periods revealed that Dmrt2 had lower expression in early ovary and mature ovary,higher expression in early testis and emission testis,and the highest expression in mature testis (P ＜ 0.05).This study showed that ppDmrt2 might play a role in sex determination and gonadal development in P.penguin.