| Vitellogenin (VG) is a member of the large superfamily of lipid transfer protein(LLTP),the LLTP is a macromolecular glycoprotein, the molecular weight is about200-700ku. Vitellin (Vn) comes from the vitellogenin and provides amino acids,carbohydrates, fats, vitamins, sulfur,phosphorus and other substances to the embryo.Structure and function of economics crustacean VG is widely ignored by scholars,andthe cDNAsequence is the basis for molecular studies.Vitellogenin receptor (VGR) is a member of low-lipoprotein receptor superfamily,during the ovary development, vitellogenin receptor combines the vitellogenin asreceptor mediated effect, so vitellogenin receptor is important for the process of theembryo maturation. At present, the research of economy crustaceans in vitellogenin andvitellogenin receptor focus on eucaridas, prawns and crabs, Procambarus clarkii as animportant economic crustacean,the synthes and distribution of yolk protein playacrucial role for the development of embryonic and larval. Reports about Procambarusclarkii mainly stay in aquaculture methods and morphology,the studies of vitellogenincDNA sequence, functional analysis and expression has not been reported. In thisstudy,we choose Procambarus clarkii as the research object,the study was focused onthe cloning of vitellogenin cDNA sequence,the structure and function of Procambarusclarkii,cloning the vitellogenin receptor and the expression analysis of vitellogeninreceptor synthesis on the oogonia phase by semi-quantitative PCR and real-timefluorescence quantitative PCR.In this experiment,it was found that the full-length vitellogenin cDNA of Procambarus clarkii is7674bp by RT-PCR and RACE techniques.It has25bp of5’UTRand129bp of the3’UTR;when translated the open reading frame into amino acidsequences in the NCBI website to BLASTP.The identities of the amino acid sequence ofvitellogenin were about33%-62%compared with other known crustaceanvitellogenin.Structural analysis of this sequence and the phylogenetic tree show thatProcambarus clarkii has a closer relationship with Cherax quadricarinatus andHomarus americanus.The further analysis showed this sequence encodes a2506aminoacid residues.May contain two domains: von Willebrand factor D-type domain (VWD)and vitellogenin N-terminal domain (Vitellogenin N) participated in lipid transport.Theprediction of vitellogenin transmembrane structure of the Procambarus clarkii showdthat Procambarus clarkii contains six kinds of membrane to the outer transmembranestructures and four kinds of outer membrane to the membrane structures. Such astructure may be more conducive to transport both within and outside its membrane,andrelated to its biological functions and reproduction.In the experiment of cloning partialvitellogenin receptor,we get a506bp sequence,after BLASTP in NCBI,we found thesequence has a higher similarity with the vitellogenin receptor mRNA sequence ofMacrobrachium rosenbergii, Marsupenaeus japonicas, Penaeus monodon, Penaeussemisulcatus. Establish phylogenetic tree based on the sequence of vitellogenin receptorof other decapods and insects, the result showed that: compared with insects,Procambarus clarkii has a closer relationship with decapod,but independent cluster,which is different from other decapod.In this paper,vitellogenin receptor was found in ovary and hepatopancreas bysemi-quantitative PCR and real-time fluorescence quantitative PCR,From thedevelopment process of the ovary,the expression level of VGR in ovary was higherthan hepatopancreas, indicating that ovary is the main site of VGR synthesis during thegrowth and development of Procambarus clarkii.Vitellogenin gets into the oocyte bythe receptor-mediated endocytosis with vitellogenin receptor from ovary,it was digestedinto small molecular into yolk-associated protein and provided nutrition. In ovary,the relative expression of vitellogenin receptor is highest in Oval cell proliferation period, itstarts gradually decreased in the Pre vitellogenesis period and the lowst in the Maturestage,picked up during the Recovery period.It has higher level in Oval cell proliferationperiod and Pre vitellogenesis period, the difference was not significant (P>0.05).Primary vitellogenesis period is much lower compared with Pre vitellogenesis period,the difference was significant (P <0.05).The relative expression reached its lowest levelin Mature stage,significantly different from Secondary vitellogenesis stage andRecovery stage (P<0.05). With the development of the ovary, consider about thechanges in the quality of the ovary, The total amount of expression was lowest in theOval cell proliferation period,keep increasing from Pre vitellogenesis period,and peakedin Mature stage, reducing in Recovery period, but still slightly higher than the Oval cellproliferation period. Pre vitellogenesis period is about3times compared with Oval cellproliferation period, the difference was significant (P<0.05). The total amount of VGRexpression peaked in Mature stage, compared with Secondary vitellogenesis stage andRecovery stage, the differences were significant (P <0.05). VGR has a lower relativeexpression in the hepatopancreas,the trend is not significant.In each developmentalstages of the ovary, total expression levels of VGR from ovary were higher thanhepatopancreas.The contrast between the main expression of the synthesis part of VGR and VGfound:the overall trend is the same about the vitellogenin receptor in ovary andvitellogenin in hepatopancreas.It expresses lowest in Oval cell proliferation period,andbe the top in Mature stage,declined in Recovery stage.VG is much higher than VGRfrom the total value of the expression,both expression of VG and VGR are the highest inMature stage,but the expression of VG is nearly150times higher than the VGR.Themain function of VGR is as a combination to transfer VG into oocytesof by the way ofreceptor-mediated endocytosis, the experimental results verify that the expression ofVGR demands to rise up when VG rising up, in order to play a role in mediatingreaction mechanism. |
PDF Full Text Request