Gene Clone And Functional Analysis Of Vitellogenien And Vitellogenin Receptor In Macrobrachium Nipponense

Oriental river prawn,Macrobrachium nipponense,subordinated to Arthropoda,crustaceans,Decapoda,Palaemonidae,Macrobrachium,is an important shrimp species in fresh water aquaculture.In recent years,with the scale expansion of shrimp farming industry and increase of output a series of problems to be solved during production of"precocious" is the most prominent problem.The autumn shrimp propagate together in one aquaculture ponds,male and female gonad development ahead of time,lead to a premature of baby shrimp gonads mature,smaller shrimp individuals,largely breeding density,lead to a series of problems such as multigenerational together et al,restricts the sustainable and healthy development of the shrimp industry.Gonad maturity is closely related to the reproductive regulation genes,a lot of genes participate in reproductive regulation,of which Vitellogenin(Vitellogenin,Vg)and Vitellogenin receptors(Vitellogenin receptor,VgR)play an important role in the oocyte maturiation during oogenesis.At present,a short fragment of Vg was cloned from others but no gene function research was reported.Few studies of VgR were developed in crustacean.In current study,we carry out the genetic molecular research of vitellogenin and vitellogenin receptor in the M.nipponens.Full length cDNA sequences of VgR an Vg were obtained using cDN A end Rapid Amplification(Rapid Amplification of cDNA Ends,RACE)on the basis of their partial sequences acquiried in the ovary and testis cDNA library.The expression pattern of the different tissue organizations,different development period and different ovary development stage were analysised by real-time PCR Explore the gene function of Mn-VgR and Mn-Vg and regulation relationship between each other under eyestalk ablation and RNAi.The cDNA encoding the Vg gene from the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag(EST)analysis and the rapid amplification of cDNA ends(RACE)approach.The transcript encoded 2536 amino acids with an estimated molecular mass of 286.810 kDa.Quantitative real-time PCR indicated high expression of Mn-Vg in the female ovary,hemocytes,and hepatopancreas.As ovaries developed,the expression level of Mn-Vg increased in both the hepatopancreas and ovary.In the hepatopancreas,the expression level rose more slowly at the early stage of vitellogenesis and reached the peak more rapidly compared to the expression pattern in ovary.The observed changes in Mn-Vg expression level at different development stages suggest the role of nutrient source in embryonic and larval development.Eyestalk ablation caused the Mn-Vg expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages.Ablation accelerated ovary maturation by removing hormone inhibition of Mn-Vg in the hepatopancreas and ovary.In adult females,Mn-Vg dsRNA injection resulted in decreased expression of Mn-Vg in both the hepatopancreas and ovary,and two injection treatment dramatically delayed ovary maturation.Vg RNA interference down-regulated the vitellogenin receptor(VgR)expression level in the ovary,which illustrates the close relationship between Vg and VgR in the process of vitellogenesis.A cDNA encoding the vitellogenin receptor(VgR)of the oriental river prawn Macrobrachium nipponense was cloned using expressed sequence tag(EST)analysis and the rapid amplification of cDNA ends(RACE)approach.The coding region consisted of 5920 bp flanked by a 45 bp 5-untranslated region(UTR)and a 166 bp 3-UTR,which encoded a 1902-residues mature protein with a predicted molecular mass of 209 kDa.The deduced amino acid sequence of the Mn-VgR cDNA have typical conservative domains,such as an extracellular,lipoprotein-binding domain(LBD),epidermal growth factor(EGF)-like,and O-glycosylation domains,a transmembrane domain,and a short C-terminal,cytosolic tail.Quantitative real-time PCR(qPCR)indicated Mn-VgR highly expressed in the female ovary.Expression analysis by qPCR demonstrated its larval developmental stage-specific,and ovary developmental stage-specific expression pattern,respectively.As ovaries developed,the expression level of Mn-VgR gradually increased from reproductive cycle(stage I)then increased and reach the peak in the(stage Ⅲ)then dropped afterwards and stepped in to a new development cycle after reproduction molting.Eyestalk ablation caused the Mn-VgR expression level to increase significantly compared to eyestalk-intact groups during the ovary development stages(P<0.05).In adult females,VgR RNAi dramatically delayed the maturation of the ovary according to the gonad somatic index(GSI).After 4 ug/gVgR dsRNA injection,the expression of VgR in ovary was significantly decreased dropped 92%in the fifth day.In addition,Mn-VgR RNAi leaded to the vitellin depletion in the oocytes and accumulation of vitellin in the hepatopancreas.In this study,we first cloned the complete cDNA sequence of Mn-Vg and Mn-VgR in the M.nipponense,the expression in different tissue,different development stage and differnt development stage of ovary maturiation was identified.The RNAi technology AND eyestalk ablation was applied to study the gene function of Vg and VgR and regulation relationship between each other.The results indicate that Mn-Vg and Mn-VgR have an important function in the manturiation of ovary.The result have big significant in the study of oogenesis.According to the result of gene regulate relationship study between Vg and VgR proved basic data for the research of molecular gene regulate mechanism in crustacean.