Studies On The Differentially Expressed Genes In Accessory Sex Gland And Function Of Eriocheir Sinensis

Eriocheir sinensis commonly known as the Chinese mitten crab, is an economically important aquaculture species in China. Although E. sinensis reproductive biology has received increasing attention in recent years, accessory sex gland reports remain limited to structural descriptions. With the beginning of growth occurring in August, the size of the accessory sex gland at maturity in late November, which is significantly delayed compared to that of the testes. The shape and the size of accessory sex gland both have some changes at the course of development, and the secretion matter also increases greatly. The function of the accessory sex gland has not been reported in the reproductive system of Crustaceans, and the research is still no progress in the molecular field.Suppression subtractive hybridization (SSH) technology is a simple and efficient differential gene cloning technology that can overcome the high false positive rates in Differential Display (DD) and the complications of Representational Difference Analysis (RDA), yet can also enrich some genes that are expressed in low abundance. In the present study, we constructed the forward and reverse subtractive cDNA libraries, in order to permit the identification and targeted analysis of genes that are differentially expressed during accessory sex gland development. SSH results were validated by the real-time PCR analysis of five candidate genes, confirming the SSH success. Results reported here provide a foundation for future molecular analysis, and the theoretical analysis of sex accessory gland function in the male reproductive system of E. sinensis. The main results and conclusion in this thesis are as follows.1, The cDNA library construction of accessory sex gland in Eriocheir sinensis and EST sequence analysisIn the present study, we performed suppression subtractive hybridization (SSH) experiments in the crab, Eriocheir sinensis, and constructed the forward subtractive cDNA library with cDNAs from the early stage (tester) and the peak stage (driver) of the E. sinensis accessory sex gland. The reverse subtractive cDNA library was constructed with cDNAs from an accessory sex gland during the peak stage (tester) and the early stage (driver). A total of 175 ESTs were obtained from 180 randomly picked clones in the two libraries, with an average insert size of 450 bp, and a cloning efficiency 92%. Comparative sequence analysis of ESTs with sequences reported in public genomic databases identified88 unigenes, 35 of which were homologous to reported proteins, with the remainder unidentified. Unigene annotation via the Gene Ontology classification, identified unigenes included those with an inferred function in cellular processes (involved in cell proliferation and differentiation), which were greater in the early stage of gland development, and genes with inferred functions in protein synthesis, energy metabolism, and signal transduction, which were more prevalent during the peak stage of gland development. Thus, genes elevated during the early stage may play roles in the process of rapid cell growth, while those elevated during the peak stage may play important roles in protein synthesis and secretion, as well as sperm capacitation and fertilization.2, Identification of subtractive librariesExpression of the five genes, one (NEK6) from the forward and four (AK, PPIB, Ubiquitin, transmembrane protein) from the reverse library, was quantified in early and peak accessory sex gland stages by real-time PCR using species-specific primers to validate the SSH results. The results showed that the relative level of gene expression differed significantly, by a magnitude of 5.2 to 10.3 fold, in the two stages. NEK6 was up-regulated in the forward library compared to the reverse library expression levels, while the other four genes were up-regulated in the reverse library, confirming the SSH success.3, Expression of some differentially expressed genes in accessory sex gland at different stagesUsing Real-time quantitative PCR to study the five differentially expressed genes changes in abundance during the different accessory sex gland development stages, such as the early stage, the preperiod, the middle stage, the late stage and the end stage, to further more in-depth understand of their functional characteristics and expression of the relationship. The results showed that: NEK6 expression increased rapidly from early development to the preperiod, the expression is up to the maximum at the preperiod development, lower abundance at the medium-term, followed by a rapid decline, the level of expression was the minimum during the end development, the maximum value is the minimum value of 14.4 times. The relative abundance changes of four genes AK, PPIB, ubiquitination and transmembrane protein were in accordance with the accessory sex gland development, the levels of gene expression were increased with the increased secretion gradually. The expression level was the highest in the late stage, followed decreased with reproductive activities and the secretion of gonadal lower activity. Relative abundance of the highest changes in expression are as follows: 7.06 times, 6.10 times, 14.01 times and 6.25 times.4, The role of accessory sex gland content on spermatophore ruptureHere we report that accessory sex gland protein concentration was the principal factor affecting spermatophore digestion, and have identified optimal incubation temperatures, durations, and protein concentrations. We identified incubation with 15.4 mg/ml accessory sex gland protein at 32℃for 7 min as conditions for rapid and complete spermatophore digestion. The accessory sex gland protein method of spermatophore digestion yielded free sperm with low mortality (1.94%) compared to the traditional methods of trypsin digestion and mechanical homogenization, followed by enzyme digestion (7.36%), with greatest mortality observed with the homogenization method (22.3%), sperm mortality differed significantly among isolation methods (P<0.05).