Study On Genes Related To The Ovary Development Of The Mitten Crab (Eriocheir Japonica Sinensis)

Part 1 Screening of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Chapter 1 Construction of subtractive cDNA library of mitten crab (Eriocheir japonica sinensis) ovaryTwo subtracted cDNA libraries of mitten crab (Eriocheir japonica sinensis) ovaries at two successive developmental stages were constructed by suppression subtractive hybridization (SSH). Two-directional (forward and backward) SSH was performed on cDNAs of the mitten crab ovaries which were either at stage II or at stage III. Two-directional subtracted cDNA fragments were cloned into PinPoint plasmid vectors, and the vectors were transformed into E. Coli JM109. Finally, the forward and backward subtracted cDNA libraries including 863 and 360 clones respectively were obtained. The length of the inserted fragments of the forward and backward subtracted cDNA libraries was 360 and 160 base pairs in average respectively by PCR detection. The results showed that the subtracted libraries constructed were suitable for further study on the genes related to the ovary development of the mitten crab.Chapter 2 Screening of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis) by cDNA macroarray analysisPlasmids were extracted from all the clones in the subtracted cDNA libraries with alkali lysis method, then the inserted fragments in plasmids were amplified by PCR. cDNA macroarray was made as follow: products of PCR were condensed and denatured prior to being robotically printed onto nylon membrane. mRNA from the mitten crab ovaries at stages II and III were made as 33P probes by reversetranscription reactions respectively, then two identical membranes were hybridized with the two kinds probes. Washing, pressing and autoradiography were performed before signals scanning and analysis. In total, 167 clones whose signal difference was >2 fold were obtained. Of the differentially expressed clones, 104 clones were highly expressed in the ovary at stage III, the remaining clones were highly expressed in the ovary at stage II.Chapter 3 Sequencing cDNA fragments of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Partial clones were sequenced which were selected from the differentially expressed clones screened by cDNA macroarray analysis. Seven independent ESTs were obtained from the forward subtracted cDNA library. Homology analysis showed that all the ESTs were novel ESTs, and all of them were accepted by dbEST. The accession numbers are: CA591892, CA591893, CA591894, CA591895, CA591896, CA591897 and CA591898.Part 2 Cloning and analysis of the full-length cDNAs of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Chapter 1 Construction of RACE cDNA library of mitten crab (Eriocheir japonica sinensis) ovaryIn order to get full-length cDNA sequences of the differentially expressed genes, a RACE cDNA library of the mitten crab ovary at stage III was constructed successfully with SMART technique. The result of the agarose gel electrophoresis showed that the length of the full-length cDNAs in the library was pooled mainly between 500 and 2 000 base pairs. By RACE PCR, amplified products were obtained with all the gene-specific primers and the adaptor primers. This verified that the quality of the RACE cDNA library was high. It was appropriate for cloning the full-length cDNAs of the genes related to the ovary development of the mitten crab.Chapter 2 Cloning and analysis of the full-length cDNA of EJO1 gene related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Primers were designed according to the sequence of EST004 (dbEST accen-ssion number: CA591895) which was gotten using SSH and cDNA macroarrayapproach. The full-length cDNA sequence was finally generated by 5'RACE and 3'RACE respectively. At the same time, structure and function of EJOl gene were primarily analyzed by bioinformatics method. RT-PCR was made to study the expression profile of EJO...