| Vitellin is the most important nutrient and energy source for the development of oocytes as well as developing embryos. The quality and quantity of vitellin in the mature ovary play a vital role on the crustacean reproduction and offspring quality. The swimming crab, Portunus trituberculatus, is a commercially important fisheries resource and mariculture species in East Asian countries. Because of overfishing, destruction of coastal spawning and nursery grounds and marine pollution, the landing of this crab has shown a declined trend in East China Sea since 1990s. The decline in natural stock and increase in market demands has driven substantial aquaculture interests for the crab species. However, the seed production of the crab is dependent on wild-caught broodstock, which could become a constraint to the sustainable development of this crab aquaculture. Our previous studies have shown pond-reared female broodstock have poor ovarian development and worse reproductive performance compared to wild females. Therefore, it is very urgent to understand the mechanism of vitellin synthesis and accumulation for female P. trituberculatus. The present study was conducted to purify vitellin from mature ovary of female P. trituberculatus, and develop a stable and standard ELISA for its determination of female P. trituberculatus. Then, the concentration of Vg/Vn in the ovary, hepatopancreans and Hemolymph were detemined during the ovarian development of female P. trituberculatus. Meanwhile, the vitellogenin gene expression levels in the ovary and hepatopancreans were also studied by real-time PCR. Those results should not only enhance our understanding for vitellogeninsis mechanism, but also provide the background information for the artificial breeding and evaluation of broodstock quality of female P. trituberculatus.1. The vitellin was separated into three major polypeptides with molecular weight of 102, 75, and 66 kDa, respectively by the denatured SDS-PAGE. The western-blotting confirmed all three major polypeptides had specific reactivity with vitellin anti-body. The higher linear correlationship could be found on the sample direct coating ELISA than the antibody sandwich coating ELISA. Then, the optimal dilution rate of purified vitellin antibody and coating period were shown to be close to 1:90000 and 8 hour under 4℃, respectively. Under 37℃, the appropriate reaction duration of the first antibody and the second antibody were 2 hour and 1 hour, respectively, while the best color development solution was around 20 minutes. Furthermore, based on these parameters, the standard linear equation, y=0.0009x+0.3991 (R2=0.9861, x and y represents Vn concentration and OD450 value, respectively), was established for the determination of female P. trituberculatus vitellin concentration with the valid range of 200-900 ng/ml. The susceptibility of this ELISA is around 14.9ng/ml for Vn contents. Finally, the ELISA was used to determine ovarian Vn contents of female P. trituberculatus for the identification of validity of this assay. The results demonstrated the mean coefficient variation of intra-assay and inter-assay were 3.59% and 3.10%, respectively. In conclusion, the developed ELISA of this study is precise, stable and repeatable.2.The results showed that: (1) The ovarian index (GSI) was significantly increased during ovarian development, hepatopancreas index (HSI) first decreased and then increased and then decreased, hepatopancreas index and the ovarian index was not significantly correlated; (2)During ovary period, total soluble protein in the ovary was significantly increasing,and in the hepatopancreas was first increased and then decreased,, total protein in hemolymph was lower in the early period, higher in the mid and late peiod; (3) During ovarian development, Vn in the ovarian increased significantly and the highest value inⅤ, up to 256.93mg / g wet weight, in more than 80% of the the total soluble protein; In hepatopancreas, Vg can not be detected inⅠandⅡ, a significant increase since the beginning, Hemolymph Vn content inⅡ-Ⅳperiod was increased significantly, then decreased significantly inⅤ,only 3.48μg/ml. These results suggest, in the P.trituberculatus , in the late ovarian development, yolk protein synthesized mainly from hepatopancreas, and then trans-ported to the ovary by haemolymph ; After ovarian maturity, hepatopancreas VgmRNA abundance and Vn content in the hemolymph decreased significantly, indicating that exogenous yolk protein synthesis less in the mature P.trituberculatus.3. using real-time quantitative polymerase chain reaction (PCR) method, We analysised vitellogenin gene of the female swimming crab Portunus tritubereulatus hepatopancreas and ovarian tissue with different developmental stages. we found the vitellogenin gene expressed both in the hepatopancreas and ovarian tissue, And with the growth in the ovarian, hepatopancreas and ovarian vitellogenin mRNA express differently, vitellogenin mRNA expression of ovarian significantly belower than this of hepatopancreas, Vitellogenin mRNA expression of ovarian in five period was the highest. but in the hepatopancreas, Vitellogenin mRNA expression reached the highest in the four period and suddenly signifycantly decreased in five period. |
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