Cloning And Expression Patterns Of Gonad Related Genes From Large Yellow Croaker

To clone the gonad-specific or gonad-related expressed genes of large yellow croaker Larimichthys crocea, a normalized gonad cDNA library was constructed using the combination of SMART and TRIMMER techniques. A total of 3961 clones randomly picked from the library were single-pass sequenced and 2916 unigenes were clustered from the 3535 high quality ESTs (expressed sequence tags). The content of cDNA library was 4.3×106pfu/mL, the recombinant percentage of the normalized cDNA library was 95.8%, the percentage of unique sequence of the library was 82.49% and the average length of unigenes was 355 bp. Searching Genbank by using these nucleotide sequences indicated that 1785 ESTs showed significant homology with known genes in the NCBI database. Among the 1785 known genes, about 66 significant gonad-related genes were found, accounting for 3.70% of the total unigenes. Six gonad-related microsatellite-containing ESTs were identified from the 129 ESTs containing a total of 149 microsatellites. Expression patterns of 11 gonad-related homologues in testes and ovaries were selected for quantitative relative-time PCR (qRT-PCR). The results showed that gene encoded testis-specific LRR protein was expressed in testes but not ovaries (P<0.05), and MSL3L1, ARINPK, SARP, HSP70 and cyclin A1 were expressed more in testes than ovaries (P<0.05). Conversely, Cathepsin C, NASP and cyclin B1 were expressed more in ovaries than testis (P<0.05), and expression levels of MAPK1 and UCH-L5 in ovaries and testes were not significantly different (P>0.05).Based on the above results, full length cDNAs of cypl9a, cyp19b, cyclin B1, CDC2, UBE2D and UBC9 from the large yellow croaker were obtained by using SMART-RACE PCR or RT-PCR. The full length cDNAs are 1805 bp,2268 bp,1882 bp,1151 bp,798 bp and 846 bp respectively, and the lengths of deduced protein sequence of these genes are 518,500,397,303, 147 and 148 amino acids respectively. The results of qRT-PCR showed that the expression level of cyp 19a in ovary was significantly higher than in testis (P<0.01), and the expression level of cypl9a in both ovary and testis was also much significantly higher than in other organs (P<0.01). However, cypl9b has lower expression levels in gonads, and has higher expression levels in midbrain, forebrain, hypothalamus, spleen and liver, and the highest expression level in blood. Cyclin B1 and CDC2 were expressed the most in gonads, and the expression level in ovaries is significantly higher than in testes (P<0.05). UBE2D was most expressed in spleen and gonads, and the expression level of UBE2D in testis is significantly higher than in ovary, and the expression levels in gonads were also significantly higher than in other organs (excluding spleen) (P<0.01). The expression levels of UBC9 in gonads are significantly higher than in other organs (P<0.01).