| Silkworm(Bombyx mori.) is an important economic insect. Bombyx mori nuclear polyhedrosis virus disease is a silkworm disease caused by Bombyx mori nuclear polyhedrosis virus(BmNPV), has been badly harming sericultural production. Breeding of high anti-BmNPV varieties becomes an important research area in silkworm. Transgenic RNAi is one of the new techniques in recent years, which combines transgene advantage with RNAi advantage, is expected to become an effective way to cultivate varieties of anti-virus silkworm. The constitutive promoter could produce more siRNA, as a result, RNAi efficiency could be improved.However, exogenous gene fragments sustained high level expression, not only resulted in vivo waste of resources, but also caused side effects of organisms.Therefore, the pathogen-inducible promoter, which could drive the expression of exogenous genes only in pathogenic infection conditions, will gradually become a hot topic.The main purpose of this study is to screen an efficient BmNPV inducible promoter for creating the anti-BmNPV transgenic material of silkworm.The BmNPV inducible promoter was obtained and reformed. The transgenic promoter interference vector was constructed based on the BmNPV inducible promoter. The main findings are as follows:1The selection and clone of BmNPV gene promotersBased on the reports in the literatures and baculovirus cascade regulation characters, the seven viral gene Bm122, Bm21, P143,39k, P33, VP1054, P6.9Bm122, Bm21, P143,39k, P33, VP1054, P6.9,whose promoters were initially identified as objects of screening of virus inducible promoter. According to genomic sequence of BmNPV (T3strain), the800-1000bp region of ATG upstream in seven genes were cloned and analyzed. The firefly luciferase recombinant plasmid PGL3-Bm122, PGL3-Bm21, PGL3-P143, PGL3-39k, PGL3-P33, PGL3-VP1054, PGL3-P6.9were successfully constructed. Each recombinant plasmid along with the internal reference plasmid P-IE1-Rlucp were co-transfected into the silkworm BmN-SWUl cells. The results showed that compared with the uninfected group, the luciferase relative ratio of seven promoters had increased in different degrees at72h after BmNPV infection, indicating that BmNPV infection could increase the transcriptional activity of these promoters. The39k promoter transcriptional activity increased71.9times after infection, and had the highest virus-induced transcriptional activity among these promoters. Thus,39k promoter was chosen as the object for further study.2Sequence Features And Functional Analysis of39k PromoterBy sequence analysis, we found that39k promoter contains enhancer-like elements [searching mode A (A/T) CGT (G/T), or CGTGC]、capping sites (CAGT-motif, which is the characteristic sequence necessary for the immediate-early and delayed-early genes) and TATA-box, indicating that39k promoter has typical features of eukaryotic promoter.The5end UTR segment missing vectors of39k promoter was constructed by PCR and was transfected into BmN-SWU1cells, relative enzyme activity was measured at the time points of24h,48h,72h after BmNPV infection. The highest zone of39k promoter transcriptional activity is the full-length promoter sequence. The important transcriptional activity section was located upstream of the TSS (Transcription Start Site)-610～-419bp and basal transcription active region is located upstream of the TSS-419to TSS at the downstream31bp. The total length of39k promoter plasmid PGL3-773luc was transfected into BmN-SWU1cells, the relative luciferase activity increased from24hours to72hours gradually after virus infection and reached highest at72h. The relative luciferase activity difference was extremely significant (P＜0.01) between24h and72h, the relative luciferase activity difference was significant (P＜0.05) between48h and72h.This was consistent with the fact that39k gene is delayed-early phase gene and39k promoter transcriptional activity had a cumulative effect in period of24h to72h after BmNPV infection.3Reformation of39k promoterThe homologous regions (hrs)of the baculovirus were commonly used enhancer elements. Baculovirus polyhedrin upstream gene polh-up (pu) has also been confirmed to be able to enhance promoter transcription. Hr3, hr5,the polh-up (pu) and hr3-pu from BmNPV were cloned and inserted upstream of39k promoter with Bombyx mori Actin4promotor as positive control, the enhancement effect on39k promoter was analyzed. The results showed that the transcriptional activity of39k, hr3-39k, hr5-39k, pu-39k and hr3-pu-39k promoter was very low without BmNPV infection; After BmNPV infection, luciferase relative ratio raised to varying degrees; Hr3enhancement effect was stronger than pu and hr5, the relative activity increased4.38times compared with39k promoter; the enhancement effect of hr3and pu joint action was more obvious; the enzyme activity had14.3times compared with the original promoter; Compared with the strong constitutive promoter A4, the relative activity increased2.147times. These results indicated that the combined effects of polh and hr3would significantly enhance BmNPV39k promoter transcriptional activity. At the same time, the DsRed expression driven by39k promoter in BmN-SWU1cells was consistent with luciferase activity analysis. Thereby, hr3-polh has the strongest enhancement effect, and the hr3-polh-39k promoter will be used for subsequent RNAi research.4The39k promoter driving shRNA interference effect on the cellular levelIn order to detect whether hr3-pu-39k promoter could drive shRNA transcription to achieve specific gene silencing in BmN-SWU1cells, three gene-specific target sequences of BmNPV lef-1were synthesized, the PIZ/V5-hr3-pu-39k-shRNA interference vector and PGL3-A4-luc-lef-1expression vector were constructed.BmN-SWU1cells were infected with PIZ/V5-hr3-pu-39k-shRNA, PGL3-A4-luc-lef-1and the internal control plasmid P-IE1-Rlucp, the cells were collected and the luciferase activity was measured at72h after transfection. The date showed that compared with the control group, the relative luciferase ratio in experimental group (PIZ/V5-hr3-pu-39k-shRNA) significantly decreased at72h after BmNPV infection,the difference of the relative activity was extremely significant (P＜0.01) between before and after the infection of the experimental group. The interference sequence could inhibit lef-1gene expression at mRNA level and the effect of suppressing was more than85%. These indicated that with BmNPV effection, BmNPV39k promoter could be induced in cell level, driving shRNA vector to express enough interferometric fragents, specifictly inhibit BmNPV gene transcription on the cellular level to certain extent. |
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