Transcriptome Data Mining Of Procambarus Clarkii And Preliminary Functional Study Of Its Gonadal Development Related Genes

The red swamp crawfish(Procambarus clarkii) has become one of the economically important aquatic animals in China due to its delicious taste and abundant nutrition. In recent several years, P. clarkii culture has developed rapidly. A shortage of juvenile has became prevalent, leading to severe restriction in large-scale P. clarkii culture. Isolation and characterization of genes are an initial step with the aim of developing a method for controlling the quality and quantity of P. clarkii and their eggs. knowledge of mechanisms governing gonadal development processes in P. clarkii has been largely limited. In order to develop molecular resources and expedite gene discovery, we have undertaken 454 pyrosequencing of testis and ovary c DNA libraries to produce a comprehensive transcriptome for P. clarkii. The major results are as follows:1. Sequencing and analysis of the transcriptome of P. clarkiiUsing the 454 pyrosequencing technology, we obtained a total of 1,134,993 high quality reads from the P. clarkii testis and ovary libraries, and then assembled into 22,652 isotigs. Average length of the assembled isotigs was 1,921 bp, and 16,018 isotigs were longer than 1,000 bp. Comparative transcript analysis showed that 1,720 isotigs in the ovary were up-regulated and 2138 isotigs were down-regulated. Several gonadal development related genes, such as vitellogenin, cyclin B, cyclin-dependent kinases 2, dmc1 and ubiquitin were identified. Furthermore, 6,724 SSRs and 91,004 SNPs were identified in this EST dataset. Our findings provided an archive for future research on gonadal development at a molecular level in P. clarkii and other crustaceans.2. Microsatellite marker identification from transcriptome derived sequences of P. clarkiiWe isolated and characterized 6,724 putative microsatellite loci in P. clarkii, from the ESTs generated by 454 sequencing. The dinucleotide repeat was the main type, accounting for 57.56% of all microsatellite loci. AC is the most frequency motifs in all the dinucleotide repeat type. 100 primer pairs were designed for polymorphism detected, and 26 primers had polymorphic PCR products in samples. We chose 8 of these expressed sequence tag derived markers for further analysis. The number of alleles per locus ranged from 3 to 11, with an average of 8.25. The observed heterozygosity ranged from 0.333 to 0.708, while the expected heterozygosity varied from 0.325 to 0.806. Most of the microsatellite loci had a high level of polymorphism information content(PIC > 0.5). These novel polymorphic EST-SSR markers should be particularly useful for further investigation of population and conservation genetics.3. Identification and characterization of reference genes for normalizing expression data in P. clarkiiEight commonly used crustacean reference genes(ACTB, GAPDH, EF-1α, UB, TUB, TBP, EIF and 18S) were chosen and investigated as potential candidates for normalization of q RT-PCR data. Expression level of these genes under eight different tissues(Eyestalk, gill, hepatopancreas, heart, intestine, muscle, testis and ovary) and six different ovarian developmental stages(no developmental stage, early developmental stage, previtellogenic stage, vitellogenic stage, mature stage and post-spawning stage) was examined by q RT-PCR, and the stability of their expression was evaluated using three commonly used statistical algorithms, ge Norm, Best Keeper and Norm Finder. A final comprehensive ranking determined that EIF and 18 S were the optimal reference genes for expression analysis in different tissues, while TBP and EIF were optimal for expression analysis in different ovarian developmental stages. EF-1α and UB proved inappropriate for normalization of expression data in P. clarkii. These results would facilitate more accurate and reliable expression studies in the crustacean species.4. Cloning and expression of gonadal development related genes for P. clarkiiThe full length of P. clarkii pcna c DNA sequence was 1387 bp, encoding a protein of 260 amino acids. Multiple sequence alignments showed high identity and conservation with pcna sequence in other species. The expression level of pcna was significantly higher in ovary than other tissues. In different ovarian developmental stages, the expression level of pcna showed higher in the early developmental stage. These results suggested that pcna played an important role in the ovarian development, especially in the process of cell proliferation.The full length of P. clarkii fox L2 c DNA sequence was 1771 bp, encoding a protein of 339 animo acids. Multiple sequence alignment with known fox L2 sequences confirmed the conservation of the P. clarkii fox L2 sequences, specifically the forkhead domain. The expression level of fox L2 in ovary was extremely higher than other tissues, and showed higher expression in the vitellogenic stage, suggesting the participation of P. clarkii fox L2 in the ovary development and vitellogenesis process.The full length of P. clarkii cyclin B c DNA sequence was 2589 bp, encoding a protein of 402 animo acids. The deduced amino acid sequence shared high similarity with other species, and composed of the the cyclin destruction box, signature motif and PKA site. The full length of P. clarkii cdc2 c DNA sequence was 1620 bp encoding a protein of 299 animo acids. The deduced amino acid sequence composed of GXGXXGXV, PSTAIRE and DFG, the dephosphorization/ phosphorylation sites and tryptophan residues. The expression level of of cyclin B and cdc2 in P. clarkii ovary were much higher than other tissues, and showed higher expression in early developmental stage and mature stage, suggested that cyclin B and cdc2 might play important role in ovary development and oogenesis of P. clarkii.