Expression Regulation Mechanism Analysis Of Vitellogenin Receptor In Silkworm,Bombyx Mori

Insect,which is the largest group of animals,has been living and multiplying on the earth for more than 400 million years.The super reproductive ability is one of the important "stunts" for insects,which they gained during the evolution of over billions years;so that they can adaptat to various ecological environments and maintain the species.Obviously,the super reproductive ability of insect is closely related to the unique structure of ovariole,as well as the oogenesis of female insects.Therefore,the study on the reproductive process of female insects is crucial for expanding the breeding of economic insects and searching the molecular targets for the pest control.Identifying key genes affect the oogenesis in insects and analyzing its regulatory mechanism are important to improve artificial regulation of insect reproduction and provide effective molecular targets for pest control and breed improvement.Vitellogenin receptor(VgR),which plays an important role in oocyte maturation and embryonic development,is considered as the key molecule that helps oocytes uptake the nutrients such as Vitellogenin(Vg)into oocytes.Previous studies show that the structure and function of VgR is highly conserved among many Lepidopteran species.Not only VgR transfers Vg into the ovary by endocytosis,but also transfers haemolymph proteins into the ovary.In addition,rencent studies have shown that VgR is also be able to participate in the vertical transmission of the virus in insects.As the Bombyx mori(B.mori)is considered as the most important economic insects as well as the mode organism of Lepidoptera,the studies of VgR and Vg in B.mori have been carried out for several decades.Lin et al have report the first full-length CDS sequence of BmVgR in 2012 and demonstrat BmVgR can bind to Bm Vg protein to enter ovary by endocytosis;furthermore,the mutation of BmVgR induces the vit(scanty vitellin)mutant that lead to recessive homozygous embryo lethal.There are only few studies on the expression regulation of insect VgR and only a little transcriptional regulation of VgR of Aedes aegypti and Solenopsis invicta,and almost no information on the regulation mechanism of post-transcriptional or translation level is aviable so far.So the comprehensive studies on the regulation mechanism of VgR will provide reliable method to regulate the reproductive process of insects accurately.In this study,we investigated the mechanism of expression regulation of BmVgR in silkworm.First,the types and abundance of small RNAs in ovariole were determined by small RNA sequencing.Subsequently,miRNAs that are involved in post-transcriptional or translational regulation of BmVgR were identified by means of dual luciferase reporting system,miRNAs transfection and transgenes.Further,to investigate whether the identified miRNAs were associated with the transcriptional regulation of BmVgR,studies on transcriptional regulation of BmVgR were carried out.Finally,the key transcription factors affecting the expression of BmVgR at the transcription level were identified by promoter cloning analysis,and their relationship with miRNAs were further analyzed,so as to improve the regulation network of BmVgR expression.The main research results are as follows: 1.Identification of small RNA in the ovariole of B.mori.Many follicles at different development stages that arranged to “beadeds” structure are the main component of the insect ovariole,so the ovariole is an ideal model for the study on regulation of gene expression.The ovariole of silkworm were used for the research model in our study,the follicular tissues in vitellogenesis and choriogenesis were collected for constructing the small RNA libraries and sequencing analysis.Totally,13143251 and 16743213 clean reads were obtained from two libraries;meanwhile,999665 and 999551 small RNAs that with a length of 18-30 nt were identified.The statistical analysis of the length of all small RNAs in two libraries showed: small RNAs that are 21-22 nt were dominated in vitellogenesis,but 27-28 nt small RNAs were abundant in choriogenesis.As reported previous,the length of classical mature miRNAs is 21-22 nt and the small RNAs that have 27-28 nt are recognized as the pi RNAs(PIWI-interacting RNAs).Our results indicated that there were a large number of pi RNAs and miRNAs in the developing ovariole;otherwise,the total number of miRNA was increased from vitellogenesis to choriogenesis,however,the total number of pi RNAs was decreased.Western blot results showed the expression of BmVgR proteins reduced from vitellogenesis to choriogenesis.Moreover,miRNAs that may be involved in the regulation of BmVgR expression were analyzed.There were 383 identified miRNAs and 257 new miRNAs detected from the two small RNA libraries;and 242 miRNAs that have different expression pattern were found in the two libraries.These results provide important data to support the research on expression regulation of miRNAs in oogenesis and build a solid foundation for following study on miRNAs regulation of BmVgR expression.2.Identification and verification of miRNAs targeting to BmVgRAfter obtaining the sequences of BmVgR 3' untranslated region(3'UTR),the possible binding sites for 640 identified miRNAs in the ovariole were predicted by the online website Target Scan.We found there were 112 miRNAs had binding sites in the region of BmVgR 3'UTR;among them,27 miRNAs showed differentially expression pattern in vitellogenesis and choriogenesis.Subsequently,qRT-PCR was performed to present the expression pattern of those 27 miRNAs,the results suggested the transcripts of 10 miRNAs accumulated from vitellogenesis to choriogenesis;and their expression patterns showed negatively correlated with the expression of BmVgR.The function of these 10 miRNAs were further verified by Dual-Luciferase Reporter Assay System(DLR),and we found bmo-miR-2739 and novel-mir-167 could significantly inhibit the activity of luciferase,indicating that bmo-miR-2739 and novel-miR-167 may participate in the expression regulation of BmVgR by binding to BmVgR 3 'UTR.Subsequently,synthesized miRNA mimics and inhibitors were used for interference the expression of bmo-miR-2739 and novel-miR-167 in the silkworm ovarian cell line Bm N4-SID1 resulted in down-regulated and up-regulated expression of BmVgR correspondingly that showed by qRT-PCR and western blot at the transcriptional level and protein level.Meanwhile,the depletion was found more severe in protein synthesis indicating that these two miRNAs may plays more important roles in the translation inhibition process of BmVgR.Moreover,it was found that after inhibiting the endogenous bmo-miR-2739 and novel-miR-167,the ability of cells to uptake Bm Vg was enhanced due to the up-regulated expression of BmVgR.After the injection of miRNA antagomir to the at pupal day 2 and 4,the expression of bmo-miR-2739 and novel-miR-167 in the ovary were down-regulated by 48.2% and 28.6% respectively;however the amount of BmVgR and Bm Vn proteins were induced in the egg compared to the control,while m RNA of BmVgR was reduced.These results indicated that bmo-miR-2739 and novel-miR-167 may have more important roles in the translation inhibition of BmVgR,but the detailed m RNA degradation or translation inhibition of these two miRNAs still needs further study.3.Spatiotemporal expression analyses of BmVgR,bmo-miR-2739 and novel-miR-167The qRT-PCR and western blot were performed to obtain the expression the expression pattern of BmVgR in various tissues at different stages.The results showed the transcripts of BmVgR were increased from the 2nd day of wandering(W2D)and had a maximum at the 3rd day of wandering(W3D)in ovary;BmVgR protein showed a higher expression at pupal day 3(P3D)and pupal day 4(P4D).However,the expression pattern of bmo-miR-2739 and novel-miR-167 showed they had a higher expression level at larval stages compared to pupal stage.These results suggested the expression of bmo-miR-2739 and novel-miR-167 were negatively correlated with the synthesis of BmVgR proteins.The transcripts of BmVgR could be detected in different tissues at W2 D,but dominated in ovary,and the protein of BmVgR was only detected in ovary by western blot.Interestingly,bmo-miR-2739 was only highly expressed in the ovary,while novel-miR-167 was highly expressed in non-ovarian tissues.The expression of novel-miR-167 reached a peak earlier than that of bmo-miR-2739 in ovariole at different development stages,suggesting that novel-miR-167 played an inhibitory role on BmVgR protein first,and with the increase of the expression of bmo-miR-2739,both of them coordinated to complete the fine regulation of BmVgR protein.4.Construction and analysis of transgenic overexpression strains of bmo-miR-2739 and novel-miR-167In order to analyze the functions of bmo-miR-2739 and novel-miR-167 in vivo,we constructed and obtained transgenic overexpression strains of bmo-miR-2739 and novel-miR-167 using the piggy Bac system(U6-2739 and U6-n167).The qRT-PCR assay showed that the miRNAs were up-regulated by 2.1 times and 1.8 times to wild type(WT),respectively.Interestingly,the expression of novel-mir-167 was up-regulated in U6-2739 strain,and the expression of bmo-mir-2739 was also up-regulated in U6-n167 strain,which suggested that there might be a mutually reinforcing mechanism between the two miRNAs.Further observation showed that the development of ovariole of transgenic strain individuals was delayed compared with control.In the overexpression strain,we found Bm Vg accumulated around the follicular tissue,there was less vitelline(Bm Vn)in oocytes and the eggs were smaller.Western blot analysis of hemolymph from transgenic lines showed that the expression of Bm Vg increased compared to the control;whereas,Bm Vn and BmVgR in eggs decreased compared: there results indicated that the overexpression of miRNA led to a decrease in the synthesis of BmVgR in eggs,and resulted in less Bm Vg transported into the egg,so Bm Vg accumulation in hemolymph and outside of the follicles.The dissection of the transgenic silkworm moth after oviposition showed that there were eggs that still immature,this is the reason that the egg-laying rate was lower than WT.Two miRNA transgenic strains were hybridized to screen the individuals that expressed both bmo-miR-2739 and novet-miR-167 at the same time.The results showed that the pupae of the female individuals that overexpressed the two miRNAs was larger than that of the normal group,but the development of the eggs was hindered,and there was even almost no egg formation in some pupae.In the eggs which had seriously delayed,the expression of bmo-miR-2739 and novel-miR-167 were up-regulated by 4.8 times and 51.0 times,respectively.Compared with the single miRNA overexpression strain,the expression was significantly increased,which proved that there was indeed mutually reinforcing mechanism between the two miRNAs.In addition,Western blot results showed that BmVgR and Bm Vn proteins in eggs were significantly induced than control.These findings suggest that these two miRNAs significantly inhibit the synthesis of BmVgR that resulting in delayed follicular development and even failure to form in the ovariole.5.Transcription factors POU-M2 and BrC involve in the transcriptional regulation of BmVgR by responding to ecdysoneThe current study illustrated the expression of BmVgR is mainly regulated by miRNAs through translation inhibition or m RNA degradation,however,whether miRNAs affect the expression of BmVgR at the transcriptional level is still unclear.Hormone treatment of ovarian in vitro showed that ecydone(20E)could induce the transcription of BmVgR,while treatment with juvenile hormone(JH)had no significant effect on BmVgR expression.Subsequently,we successfully cloned the 2.5 kb upstream regulatory region of the BmVgR gene as a potential BmVgR promoter sequence.The transcription starting site of this promoter was located at 57 bases upstream of ATG,and TATA box was located at-80~-88.It was found that the number of POU-domian elements(VVL)was the highest among the 59 predicted cis-response elements(CREs).In addition,a comparison of CREs in the VgR promoters of six insects revealed that multiple POU domain ventral veins lacking(VVL)elements were located in the six promoter regions of VgR,especially in Lepidoptera.Subsequently,DLR,EMSA,Ch IP-PCR were used to prove that the transcription factor of POU domain was bound to BmVgR promoter and the high expression of BmVgR was activated at the wandering stage.POU transcription factors are highly conserved in insects and even vertebrates.These findings led to hypothesize that the POU domain family might play a crucial role in regulating VgR expression in insects.In addition,many ecdysone early response gene-binding elements BrC and E74 were also predicted on the promoter of BmVgR.Co-transfection experiment results suggested that BrC-Z1/Z2/Z4 alone had no significant effect on BmVgR promoter activity.However,when BrC-Z1/Z2/Z4 was co-existed with POU-M2,the original enhancement caused by POU-M2 became more significant.DLR and EMSA assays showed that BrC-Z1 could bind the promoter of POU-M2 gene and enhance its expression.Therefore,it was speculated that expression of BrC-Z1/Z2/Z4 was induced by 20 E,as well as further activation of POU-M2 epression.POU-M2 can bound with transcription factors such as BrC-Z2,which resulted in high expression level of BmVgR at the wandering stage.In Sum,we found bmo-miR-2739 and novel-miR-167 inloved in regulating the expression of BmVgR in specifc tissues or specific stages by binding to the 3'UTR of BmVgR directly,thus BmVgR can be systhesied in the ovary at the vitellgenesis.20 E activated the expression of BmVgR in wandering stage by acting on POU-M2 and BrC,then the transcripts of BmVgR were transported to oocyte.The BmVgR was synthesized which the process bmo-miR-2739 and novel-miR-167 inloved,ensure the Bm Vg can be absorpted to the oocyte succesffuly.In this study,we identified bmo-miR-2739 and novel-miR-167 were the key regulatory factors for the BmVgR that is considered as important nutrition transport receptors in the reproduction process of silkworm.In addition,we also found POU-M2 and BrC were key genes in the transcription regulation of BmVgR.Our results will help archiving to artifial regulate the reproduction process accurately and also are beneficial to future application in pest control and breed improvement.