CLONING AND EXPRESSION CHARACTERIZATION OF DMRT5 IN P INCTADA MARTENS II

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.