The Quantitative Examination Of Vitellogenin MRNA In The Marsupenaeus Japonicus And Procambarus Clarkii

The accumulation of yolk is prerequisite for the mature of Crustaceans oocytes. In vitellogenic stage, Vitellogenin（Vg）is accumulated rapidly,which can provide nutrition for the development of embryo and early larvain Crustaceans. At present, the researches that reported on the variousexpressions of Vg mRNA at the whole ovarian development in shrimp wererelatively less. Among the researches on Vg in Marsupenaeus japonicus,the sites of Vg biosynthesis were confirmed, but the variations ofVg-mRNA at the whole ovarian development were not clear yet. Thevariation for Vg synthesis was investigated in M. japonicus. But forProcambarus clarkia, there are little research about the expression of Vg，both the sites of Vg biosynthesis and the expressional condition is unclear.Firstly, according to the observation of anatomy and histology in ovary ofM. japonicus and P.clarkia, we devide the whole ovarian development intosix stages, namely oogonium proliferation stage, previtellogenic stage,primary vitellogenesis, secondary vitellogenesis, mature stage and recoverystage, then we compare the difference between basing on the presence ofovary and the observation of histological section.Secondly, we analyze the expression of Vg-mRNA in Ovary andhepatopancreas at different development stages with Semi-quantitative RTPCR and Real Time PCR in the experiment. Semi-quantitative RT PCR gelelectrophoresis results are analyzied with Bio-Rad Quantity Onesoftware,finding that both in Ovary and hepatopancreas of M.japonicus are present of PCR product. In ovary the relative expression of Vg-mRNAincreases with the development of ovary. In hepatopancreas, the relativeexpressions of Vg mRNA showed the similar tendency. Both in ovary andhepatopancreas, the relative expression of Vg mRNA showed peakinsecondary vitellogenesis, and mature stage, while having a decline in therecovery stage. Then with SYBR Green I RT-PCR analyze, the relativeexpressions of Vg mRNA at each developmental stage was1.01.716.345.624.8and9.9in ovary respectively, while1.8，4.6，16.0，3.8，7.9，and1.5in hepatopancreas respectively. Combining the wight of Ovary andhepatopancreas at each developmental stage, relative expression quantityand contributive proportion was calculated. The result of RT-PCR analyzehad a similar tendency comparing to Semi-quantitative RT PCR. Finally,Northern Hybridization analysis detected the hybridization band of muchmore than6kb from both ovary and hepatopancreas. Strong signals wereobserved in especially in primary vitellogenesis, secondary vitellogenesisand mature stage. The signals increase with the development of ovary wecan find the conclusion is that the synthesis site is ovary andhepatopancreas at ovarian development in M.japonicus, and according tothe relative expression quantity and contributive proportion of Vg-mRNAin ovary and hepatopancreas, it was demonstrated that ovary dominated inthe Vg synthesis process. For P. clarkia, Semi-quantitative RT PCR gel electrophoresis resultsare analyzed with Bio-Rad Quantity One software and finding that both inOvary and hepatopancreas of P.clarkia are present of PCR product. Inovary the relative expression of Vg-mRNA on the increase with thedevelopment of ovary, the expression increased in the four stage, and in themature stage,the value was465.5,however the value was152.3with asharply decline in the recovery stage. In hepatopancreas, the relativeexpressions of Vg-mRNA showed the similar tendency. Both in ovary andhepatopancreas, the relative expression of Vg-mRNA showed peak inmature stage and primary vitellogenesis, while having a decline in therecovery stage. Then with SYBR Green I RT-PCR analyze, the relativeexpressions of Vg-mRNA increased in the four stage, and in the maturestage,the value was37.6, in hepatopancreas the elative expressions ofVg-mRNA also increased in the four stage, and in the mature stage,thevalue was2315.4.Combining the wight of ovary and hepatopancreas ateach developmental stage, expression quantity and contributive proportionwas calculated.The result of RT-PCR analyze had a similar tendencycomparing to Semi-quantitative RT PCR. Finally, Northern Hybridizationanalysis detected the hybridization band at the size of8kb fromhepatopancreas. Strong signals were observed in mature stage. The signalsincrease with the development of ovary we can find the conclusion is that the synthesis site is ovary and hepatopancreas at ovarian development inP.clarkia and according to the relative expression quantity and contributiveproportion of Vg-mRNA in ovary and hepatopancreas, it was demonstratedthat hepatopancreas dominated in the Vg synthesis process.