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Large yellow croaker (Larimichthys crocea) gonadal material, the use of the SMART (switching mechanism at 5'end of RNA transcript) and DSN (duplex-specific nuclease) treatment constructed gonadal linearized cDNA library, and randomly picked from the library 3961 clones were sequenced, a total of 3535 high-quality expressed sequence tags (Expressed Sequence Tag EST). 2916 Unigene sequences were obtained by cluster analysis, including 415 contigs and 2501 singletons were. Preliminary analysis results show that: the linear library storage capacity of 4.3 × 106pfu/mL library recombination rate of 95.8%, non-redundant EST was 82.49%, Unigene average length of 355 bp. Bioinformatics analysis, 1785 is known gene of the total ESTs 61.21%; 66 genes associated with gonadal development, accounting for 3.70% of the known genes. Microsatellite analysis found a total of 129 EST sequences containing 149 microsatellite repeat sequences, including six gonadal development of ESTs. Technical Analysis 11 with gonadal development-related genes in gonadal expression using real-time quantitative PCR (qRT-PCR). Results show that testis-specific LRR gene expression (testis-specific LRR) only in the large yellow croaker testis (P lt; 0.05); male-specific lethal 3-like 1 gene (MSL3L1), androgen the interaction receptor proteins kinase gene ( ARINPK), spermatogenesis apoptosis-related protein gene (SARP), heat shock protein 70 (HSP70) and cyclin A1 gene (cyclin A1) expression in the testis was higher than the ovaries (P lt; 0.05); Instead, organizations Protein C gene (Cathepsin C), the nuclear autoantigen protamine gene (NASP) and cyclin B1 (cycli B1) expression in ovarian higher than the testis (P lt; 0.05); mitogen-activated protein kinase 1 ( MAPK1) and ubiquitin C-terminal hydrolase L5 (UCH-L5) expressed in the testis and ovary were no significant differences (P gt; 0.05) On the basis of these results, the use of the SMART-RACE technology or RT-PCR, cloned genes gonadal development CYP19a the cyp19b cyclin B1, cdc2, UBE2D and UBC9 full-length cDNA sequence, and its length to 1805 bp, 2268 bp, 1882 bp, 1151 bp, 798 bp and 846 bp, can encode 518,500,397,303,147 and 148 amino acid precursor protein. QRT-PCR analysis of these genes in various organs in large yellow croaker expression case, the results display: expression CYP19a amount in the ovary was significantly higher than the testis (P lt; 0.01), and CYP19a in the testis and ovary expression are extremely significant with higher than that of other organs (P lt; 0.01); cyp19b telencephalon, midbrain, hypothalamus, spleen, and liver higher expression levels, the highest expression level in the blood of male large yellow croaker. Very low expression levels in the gonads. cyclin B1 and cdc2 gene expression in the gonads was significantly higher than in other organs (P lt; 0.01), and expression in the ovary than testis (P lt; 0.05) UBE2D expressed in the spleen and gonad high and low expression levels of other organs, expression was significantly higher than in other organs (except spleen) (P lt; 0.01) in the gonads. The UBC9 gonads higher expression levels, and was significantly higher than in other organs (P lt; 0.01).
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