Cloning And Expression Of Differentially Expressed Genes From Epinephelus Akaara Gonads During Sex Reversal

Red spotted grouper (Epinephelus akaara) is a kind of marine fish. It is very famous and precious with high economic value. The grouper has the phenomena of hermaphroditism, propogyny and sex reversal. Generally, more than six years old the female reverse to the male, so the mature phase of the male does not match with that of the female and there is a low fertility rate. Because of this, commercial production of artificial rearing of the grouper is not achieved in a large scale. Currently, the method of artificial sex inversion is to give the female food containing male hormone or inject male hormone to it, but too much hormone application will bring us many disadvantages. In order to control sex of the grouper better, it is necessary to expound the molecular mechanism of sex reversal.Red spotted grouper {Epinephelus akaara) females between two and four years of age were successfully reversed to functional males by feeding 17α-methyltestosterone (17α-MT) over 42 days. A systemic study was initiated to identify differentially expressed genes in the process of sex reversal of the grouper by using suppression subtractive hybridization (SSH) technique. Some differentially expressed genes between female gonad (FG) and male gonad (MG) in the grouper were obtained. Two SSH plasmid libraries for gonads of pre- and post-reversed grouper were constructed. And then differentially expressed genes were screened byPCR and dot blotting. 800 MG positive clones and 400 FG positive clones were obtained by PCR amplification, and 520 MG and 250 FG PCR positive clones were selected to carry on dot blotting. 51 MG and 20 FG dot blotting positive clones were cloned and sequenced. Searching GenBank by using these nucleotide sequences indicated that 51 cDNA fragments could not be found homologous sequences in the database. Of the 20 known genes, 3 may be closely relevant to sex reversal of the grouper according to reported papers, and they are respectively calmodulin (CAL1), receptor for activated protein kinase C (RACK1) and protein inhibitor of neuronal nitric oxide synthase (PIN). They were designated ECAL1 (GenBank accession: AY281363) , ERACK1 (GenBank accession: AY281364) and EPIN (GenBank accession: AY281365), respectively.A new gene, called EPIN, that may be involved in male formation was cloned from sex-reversed male gonads by using the combinative methods of suppressive subtraction hybridization (SSH), SMART cDNA synthesis and RACE-PCR. The full-length cDNA of EPIN is 499 bp, containing a 270 bp open reading frame that encodes an 89 amino acid protein. It has a 5' untranslated region(UTR) of 74 nucleotides(nt) and a 3' UTR of 155 nt. The start codon is located in region that conforms to the Kozak consensus sequence. The gene has no typical vertebrate polyA tailing signal. EPIN has numerous highly conserved homologs across species. Virtual Northern blotting shows that EPIN is transcribed in sex-reversed male gonads but only slightly transcribed in normal female gonads. RT-PCR and Western analyses indicate that transcription levels of EPIN in gonads increase gradually during the transformation from female to male. RT-PCR analyses of various tissues show that EPIN mRNA can be detected in the brain, heart, liver, spleen, and kidney but not in the muscle. Immunohistochemistry indicates EPIN proteins are distributed in the cytoplasm of spermatocytes, spermatids, spermatozoa and Leydig cells. The results imply EPIN may play an important role in female-to-male sex reversal.The E. akaara CaM (ECaM) cDNA was screened from the subtractive cDNA library of sex-reversed male gonads and its full-length cDNA was obtained by usingRACE-PCR technology. The full-length cDNA of ECaM is 582 bp, containing a 450 bp open reading frame that encodes a 149 amino acid protein. It has a 5' untranslated region (UTR) of 74 nt and a 3' UTR of 58 nt. Virtual Northern blotting shows that ECaM is transcribed in sex-reversed male gonads but only slightly transcribed in normal female gonads. Semi-quantitative RT-PCR analyses from various tissues indicate that m...