三角帆蚌Dmrta2-2基因的鉴定与表达分析

Identification and Expression Analysis of Dmrta2-2 Gene in Hyriopsis cumingii

为了研究DMRT基因家族中Dmrta2-2(Doble-sex and mab-3 relatated transcription factor a2-2)基因在淡水蚌的性腺发育和性别分化等过程在发挥的作用,本研究从本实验室已有的三角帆蚌(Hyriopsis cumingii)cDNA库中筛选出Dmrta2-2(HcDmrta2-2)短片段,利用RACE技术扩增和鉴定出了Hc Dmrta2-2基因。获得核苷酸序列全长为1682 bp(登录号:MK372348),ORF(Open reading frame)区为1170 bp,编码389个氨基酸。NCBI在线预测该基因保守位点,发现存在一个超保守的DM和DMA区域,氨基酸序列比对及系统发育树表明三角帆蚌与池碟蚌(Hyriopsis schlegelii)亲缘关系高达90%,在进化树中聚为一小支。通过实时荧光定量(qRT-PCR)和原位杂交检测Hc Dmrta2-2基因在各组织和发育时期的表达水平,发现该基因在成熟蚌的鳃、肝、肾、外套膜、闭壳肌、斧足及性腺中均表达,除了肝,其它各组织中雌雄表达差异均显著(p<0.05),雌性性腺表达明显高于雄性;早期表达显示在第3天和3月龄表达量显著高于其它时期,推测Hc Dmrta2-2参与了三角帆蚌早期胚胎和幼龄发育过程;1~3龄雌雄性腺定量显示在1龄表达水平最高且雌雄差异极显著,而后随着时间雌雄各表达量逐级递减。原位杂交结果显示雌性性腺中有明显信号产生,在雄性中较弱。研究推测三角帆蚌Hc Dmrta2-2基因可能参与了早期幼龄发育过程,且对卵巢的分化起到重要作用,同时可能对于精巢的维持发挥着一定作用。本研究结果为进一步开展三角帆蚌Hc Dmrta2-2基因的功能特性研究提供了基础。

In order to study the role of the Dmrta2-2(Doble-sex and mab-3 relatated transcription factor a2-2)gene in DMRT family in the process of gonadal development and sex differentiation in freshwater carp.In this study,a short fragment of Dmrta2-2(HcDmrta2-2)was screened from the cDNA library of Hyriopsis cumingii,and the HcDmrta2-2 gene was amplified and identified by RACE.The full-length cDNA was 1682 bp(Accession number:MK372348),including 1170 bp open reading frame(ORF),encoding 389 amino acids.The protein molecular weight was predicted to be 42568.48 and the theoretical isoelectric point was PI=6.75.There didn’t exist any signal peptide or transmembrane structures.NCBI predicted the conserved site of this gene online,indicating that HcDmrta2-2 gene had a highly conserved DM and DMA protein domain.Amino acid sequence alignments and phylogenetic trees indicated that the relationship between H.cumingii and H.schlegelii was as high as 90%,and it was clustered in a small tree in the phylogenetic tree.Real-time quantitative PCR(qRT-PCR)and in situ hybridization were used to study the expression of HcDmrta2-2 gene in different tissues and different developmental stages of H.cumingii.The results showed HcDmrta2-2 expressed in gonads,gill,liver,kidney,mantle,adductor muscle and foot,simultaneously all tissues except for liver had significant differences of male and female(p<0.05),and female gonad expression level was distinctly higher than male;the early expression showed that the expression level was significantly higher at the 3 rd and 3 th than other periods,speculating that HcDmrta2-2 was involved in the early embryo and young development of H.cumingii;The expression of 1 to 3-year-old male and female gonads exhibited the highest level on 1 years gonad of H.cumingii,and subsequently decreased evidently.The results of in situ hybridization showed that signals were detected significant in female gonads and weak in male.It was speculated that HcDmrta2-2 gene may be involved in the early and young development,and play a role in the differentiation of ovary,simultaneously may play an important role in the maintenance of testis.The results of this study provide a basis for further studies on the functional characteristics of HcDmrta2-2 gene in Hyriopsis cumingii.