Study On The Effect Of The 3’UTR Of The Vitellogenin Receptor On Gene Expression In Silkworm

The main function of insect vitellogenin receptor(Vg R)is to mediate the uptake of vitellogenin cells into eggs,providing nutrients for the formation of insect eggs and the development of embryos,while there are relatively few studies on the regulation of Vg R expression.In Bombyx mori(B.mori),it is mainly the study of Guanwang Shen et al.in this research center that seven POU regulatory elements upstream of BmVgR promoter can be directly combined with transcription factor POU-M2 to enhance the activity of BmVgR promoter.Chen et al found that bmo-mi R-2739 and novel-mi R-167 could bind to binding elements of BmVgR 3’UTR and inhibit BmVgR expression.Based on previous studies,this study focused on the regulatory function of BmVgR3’UTR in B.mori.Through research,the following main research results are obtained:1.Effect of BmVgR 3’UTR on exogenous gene expression driven by BmVgRP1.8KThe 2.5kb regulatory element upstream of BmVgR promoter was found by bioinformatics analysis,and it was found that the elements affecting promoter activity and gene specificity were almost in the 1.8 kb region of promoter.Therefore,the BmVgRP1.8K was cloned and the cell expression vector was constructed.The results of double luciferase assay showed that BmVgRP1.8K had promoter activity.Then,the cell expression vector of Ds Red driven by BmVgRP1.8K was constructed and transfected into Bm E cell line.The cell fluorescence results at 96 h showed that no obvious red fluorescence signal was observed in the cells driven by BmVgRP1.8K,while weak red fluorescence was observed in the cells driven by BmVgRP1.8K containing the BmVgR 3’UTR.The m RNA and protein expression were further detected,the q PCR results showed that the expression of Ds Red driven by BmVgRP1.8K containing BmVgR 3’UTR was significantly up-regulated,but Western blot(WB)failed to detect protein.To analyze the biological function of BmVgR 3’UTR in regulating gene expression,the transgenic silkworm strains of Ds Red driven by BmVgRP1.8K promoter and Ds Red driven by BmVgRP1.8K containing BmVgR 3’UTR were established.The q PCR was used to detect the tissue expression profile of transgenic B.mori on the 3rd day of upper cluster.The results showed that the endogenous BmVgR was only highly expressed in the ovary,but the exogenous Ds Red was expressed in all tissues,indicating that the gene expression driven by BmVgRP1.8K did not have tissue specificity;Furthermore,the q PCR was used to analyze the expression profiles of transgenic B.mori on the 3rd day of fifth instar,the 3rd day of upper cluster and the 4th day of pupation.The results showed that the expression of Ds Red driven by BmVgRP1.8K containing the BmVgR 3’UTR was significantly up-regulated compared with that of BmVgRP1.8K transgenic strain,but it was not stage-specific,while the endogenous gene BmVgR was up-regulated with the change of stages,indicating that the BmVgR 3’UTR could up-regulate the expression of genes at the transcriptional level.WB results showed that the protein content of Ds Red driven by BmVgRP1.8K containing the BmVgR 3’UTR was significantly down-regulated in the ovaries of transgenic silkworms on the third day of fifth instar and the third day of upper cluster.The above results showed that BmVgR 3’UTR up-regulated the expression of Ds Red m RNA,but the translated protein was inhibited.This study further detected the reported levels of bmo-mi R-2739 and novel-mi R-167 in cells and individuals.The q PCR results showed that the contents of bmo-mi R-2739 and novel-mi R-167 in the cells were significantly lower than those in the ovarian tissues on the 3rd day of fifth instar,which were almost the same as those on the 3rd day of upper cluster and the 4th day of pupation.It suggested that mi RNAs in cells had a weak regulatory effect on the expression and protein synthesis of exogenous genes containing BmVgR 3’UTR,which was similar to the vitellogenesis of silkworm.2.Effect of BmVgR 3’UTR on expression of target genes driven by exogenous promotersAccording to the previous results,the BmVgRP1.8K driver activity was not high and the target protein could not be detected by WB,so in this study,BmVgR promoter with 2.5 kb longer upstream of the promoter was selected to construct the cell expression vector,and after cell transfection,the cell fluorescence observation after 96 h showed that the Ds Red driven by BmVgRP2.5K could observe obviously red fluorescence,and the Ds Red driven by BmVgRP2.5K containing the BmVgR 3’UTR could be observed stronger fluorescence;The expression of target genes was detected at m RNA and protein levels,and the results showed that the Ds Red driven by BmVgRP2.5K containing BmVgR 3’UTR was significantly up-regulated.The above results showed that BmVgRP2.5K had stronger driving activity than BmVgRP1.8K,and further confirmed that BmVgR 3’UTR could enhance the expression of exogenous gene driven by endogenous promoter BmVgR at the cell level.How is the expression of the exogenous promoter-driven gene by BmVgR 3’UTR?In this study,we further constructed the cell vector expression of Ds Red driven by B.mori vitellogenin promoter 1.0kb and Bm Vg P1.0K driven by Ds Red containing BmVgR 3’UTR.After 72 h of transfection,the fluorescence of cells was observed,the results showed that the cells driven by the Bm Vg P1.0K containing BmVgR3’UTR had stronger fluorescence signals;WB results also showed that the content of Ds Red protein driven by promoter containing BmVgR 3’UTR was significantly increased;At the same time,the cell expression vector of Rluc driven by T7 promoter was constructed.The dual luciferase activity results showed that the target gene expression and synthesis protein driven by T7 promoter containing BmVgR 3’UTR were significantly upregulated.The above results showed that BmVgR 3’UTR could enhance the driving activities of endogenous promoters,such as Bm Vg P1.0K and T7.It has been reported that 3’UTR not only regulates m RNA translation into protein,but also regulates m RNA stability.Therefore,this study further analyzed the effect of BmVgR 3’UTR on the stability of target gene m RNA.Cells transfected with BmVgRP2.5K and BmVgRP2.5K-driven Ds Red containing BmVgR 3’UTR were first collected,and then total RNA from the corresponding cells was extracted separately,and the extracted total RNA was placed in an incubator at 37°C,and a certain amount of total RNA was removed at 0,24,48 and 72 h,respectively,and uniformly reverse transcribed into c DNA,finally,RT-PCR as well as grayscale maps were used for analysis.The results showed that the m RNA of Ds Red and Actin3 in BmVgRP2.5K transfected cells degraded suddenly from 0 to 24 h,and the m RNA of Actin3 in BmVgRP2.5K transfected cells with BmVgR 3’UTR also degraded completely at 72 h,while the m RNA of Ds Red and degraded slowly at 72 h.It suggested that the formation of BmVgR 3’UTR multi-loop structure could enhance the stability of target gene m RNA.