马氏珠母贝Dmrt5基因的克隆及时序表达模式分析
CLONING AND EXPRESSION CHARACTERIZATION OF DMRT5 IN P INCTADA MARTENS II
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摘要: Dmrt(Double sex and Mab-3 related transcription factor)基因是一类含有高度保守的DM(Double sex and Mab-3)结构域的基因家族,该基因家族的很多成员都参与了性别决定和分化过程的调控。目前已研究了很多物种的Dmrt基因,但对于具有重要进化地位和经济价值的贝类,却鲜见相关报道。Dmrt5作为Dmrt基因家族的重要成员,是否也参与了动物的性别决定与分化的调控,至今为止没有定论。采用RACE-PCR技术,从马氏珠母贝(Pinctadamatensii,Dunker)精巢的SMARTcDNA中克隆了Dmrt5基因的全长cDNA序列。同源性比对显示,pmDmrt5编码的氨基酸序列与海胆、线虫、青鳉、斑马鱼、爪蟾和小鼠的Dmrt5基因的一致性分别为17.2%、26.7%、24.8%、18.7%、20.8%和15.8%。虽然各个物种间同源性并不高,但他们的DM结构域是高度保守的。RT-PCR结果表明,pmDm-rt5基因在雄性性腺中的表达量显著高于鳃、外套膜、闭壳肌、消化盲囊和雌性性腺;在早期雄性性腺、成熟期雄性性腺中的表达量显著高于性腺发育其他时期。这意味着pmDmrt5基因可能参与了马氏珠母贝性别发育的调控。Abstract: The Dmrt genes are a new gene familywhich make a high focus in recent years. The Dmrt genes family compri-es several transcrip tion factors that share a highly conserved DM (Double sex andMab-3) domain and has been studied 1994--010 China Academic Journal Electronic Publishing House. All rights reserved.many species,including a variety of vertebrates and several of invertebrates such as fruit fly,nematode,freshwater rawn and sea squirt. But there are only few reports on Dmrt gene family of bivalve sofar,which have important value involution and production. Pinctada martensii. is the most important aquaculture marine bivalve for producing pearl in Chinand holds very high value in production. A majority of Pinctada martensii. are male in juveniles of 1 year and convert grad-ally intofemale during the second or the third year. The hermaphroditic individuals can be found in this period. This im- lies that sex reversal has happened in the development of P. martensii. But as an important aquaculture marine bivalve,here is not any report about itsDmrt gene family. Dmrt5,which also has highly conserved DM domain,is conceived as anmportant member ofDmrt gene family. But it is controversialwhetherDmrt5 involves in sex determination and differentia-ion. In this study,pmDmrt5 has been screened from testis cDNA library of dunker (Pinctada martensii./i>) by RACE-PCRnd DNA sequenceswere analyzed byBLAST and CLUSTALW programs. The full-length of pmDmrt5 cDNA is 873bp andas a ORF of 6-1bp ecoding a pep tide of -06 amino acids. It has a 1-6 bp of 5’-untranslated region (UTR) and a 1-6 bp’-untranslated region (UTR). Amino acid alignments and their identities of pmDmrt5 were compared between Pinctadaartensii and other species. The deduced amino acid sequence shows 17. -%,-6. 7%,-4. 8%,18. 7%,-0. 8% and5. 8% identity to Dmrt5 of sea urchin,elegans,medaka,zebrafish,clawed frog and mouse respectively. The homologyf N-terminal among different species is higher obviously than C-terminal. Although the sequence identity is not high,theighly conserved DM domain was predicted to exist in the pmDmrt. Phylogenetic tree ofDmrt in vertebrates and inverte-rates is determined to clarify pmDmrt status in the evolutionary history. Phylogenetic tree is constructed with MEGA 3oftware. The result indicates phylogenetic tree is basically agreed with the known taxonomic relationships among thesepecies. The pmDmrt has higher identitywith Dmrt of sea urchin and Dmrt of invertebratesmay have more far evolutionistory than vertebrates. The temporal and spatial expression patterns of pmDmrt among different tissues are analyzed byemi-quantitative RT-PCR. The result reveales that pmDmrt mRNA is transcribed mainly in male gonad,less in gill,hereas,only very slightly pmDmrt transcrip t is detected in mantle,digestive diverticulum,adductormuscle and femaleonad. The analysis on expression patterns of pmDmrt among gonads of different phases indicates that the expression ofmDmrt in early male gonad and mature male gonad is higher obviously than gonad of juveniles,gonad during transitionrom male tofemale,early female gonad and mature female gonad. The results indicated that pmDmrt5 might involve inex development regulation of Pinctada martensii.
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Keywords:
- Dunker /
- Dmrt5 /
- Sex development /
- Temporal exp ression
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