| Long non-coding RNA(LncRNA)refers to non coding sequences longer than 200 nt,which play important molecular and biological functions in the innate immune process of organisms.Cell apoptosis is considered an important defense strategy for insects against viruses,bacteria,and other pathogens.During virus infection,immune related lncRNAs can regulate multiple immune pathways and trigger cell apoptosis through various cell signals.Silkworm is an important economic insect that secretes silk in the order Lepidoptera.As the pathogen of hemophilic sepsis in silkworm,the nuclear polyhedrosis virus(BmNPV)belongs to the rod-shaped virus,which brings huge economic losses to the silk industry every year.Analyzing the infection of BmNPV to the host silkworm is of great significance.Research has shown that lncRNA plays an important regulatory role in the process of cell apoptosis induced by virus infection,and cell apoptosis plays an important role in the process of BmNPV infection in silkworms.However,the role and specific mechanism of apoptosis related lncRNA in the interaction between BmNPV and the host are still unclear.Based on the apoptosis model and transcriptome data of the silkworm previously constructed,this study,taking apoptosis as the entry point,studied the mechanism of lncRNA related to apoptosis in the process of BmNPV infection of the silkworm,analyzed and improved the mechanism of interaction between the silkworm and BmNPV,and provided new targets and ideas for the effective prevention and control of the blood type thick disease of the silkworm.The main research findings and conclusions are as follows:1.Screening and identification of apoptosis-related lncRNAs targeting BmiapThe previously established apoptosis model(BmE-iap-KO),inhibition of apoptosis model(BmE-BmNPV),apoptosis/inhibition of apoptosis model(BmNPV infected BmE-iap-KO)and transcriptome data of the control group were used.Through differential analysis of four groups of transcriptome data,514 differentially expressed genes and 1109 differentially expressed lncRNAs were screened,including the anti apoptosis gene Bmiap.Using the differential gene Bmiap as the entry point to screen for potential lncRNAs,a total of 84 lncRNAs were screened.By analyzing the differential expression between these 84 lncRNA groups,6 candidate lncRNA with significant differences were obtained.Cluster analysis and potential functional prediction were conducted on these 6 candidate lncRNAs,and TCONS＿0003793(Lnc Unc-89-AS1)is transcribed from the intron region of the apoptosis related gene(BGIBMG013531),and subsequent research will focus on Lnc Unc-89-AS1.2.Full-length cloning,expression pattern analysis and functional study of silkworm Lnc Unc-89-AS1In order to study the function of Lnc Unc-89-AS1 in silkworm,a full-length cloning of Lnc Unc-89-AS1 was performed using RACE technology,resulting in a1402 bp full-length sequence located in the intron region of the gene.The secondary structure of Lnc Unc-89-AS1 contains unique structural features of lncRNA,such as helical regions,loops,protrusions,and single stranded regions.The spatiotemporal expression pattern analysis found that Lnc Unc-89-AS1 was highly expressed in all embryonic stages and prepupal stage,and the lowest expression was in hemolymph.Fluorescence in situ hybridization showed that Lnc Unc-89-AS1 was expressed in both nucleus and cytoplasm.In order to investigate the role of Lnc Unc-89-AS1 in cell apoptosis,TUNEL staining,Caspase enzyme activity detection,flow cytometry,q RT PCR and other methods were used to detect the effect of Lnc Unc-89-AS1 on cell apoptosis.The results showed that overexpression of Lnc Unc-89-AS1 could inhibit cell apoptosis,while interference with Lnc Unc-89-AS1 could promote cell apoptosis.Overexpression of Lnc Unc-89-AS1 could inhibit Bmiap-KO induced cell apoptosis.Overexpression of Lnc Unc-89-AS1 significantly decreased the expression of mitochondrial apoptosis pathway related genes BmDronc,BmICE,and Bmbuffy,while inhibition of Lnc Unc-89-AS1 significantly increased the expression of mitochondrial apoptosis pathway related genes.3.Screening of target genes of silkworm Lnc Unc-89-AS1In order to study the role of Lnc Unc-89-AS1 in the process of BmNPV infection in silkworm cells,overexpression and interference of Lnc Unc-89-AS1 in BmE-SWU1 after infection with BmNPV were performed.The copy number of the virus,the expression of viral replication related genes,and the protein level changes of BmNPV replication related gene Vp39 were detected by q RT-PCR and West Blot,and the changes in Caspase enzyme activity before and after BmNPV infection were detected,The results showed that overexpression of Lnc Unc-89-AS1 could promote the proliferation and replication of BmNPV,and could inhibit the proliferation and replication of BmNPV after interference.Caspase activity significantly decreased after BmNPV infection,indicating that Lnc Unc-89-AS1 may promote the proliferation and replication of the virus by inhibiting cell apoptosis.Subsequently,a transgenic silkworm line Lnc Unc-89-AS1 was constructed,and economic traits of the transgenic line were analyzed.It was found that the weight of larvae and pupae of the transgenic line was greater than that of the control line.The effect of overexpression of Lnc Unc-89-AS1 on the proliferation and replication of BmNPV was tested at the individual level,and overexpression of Lnc Unc-89-AS1 had an inhibitory effect on the proliferation and replication of BmNPV at the individual level.4.Effect of silkworm Lnc Unc-89-AS1 on the proliferation and replication of BmNPV In order to further investigate the mode of action of Lnc Unc-89-AS1,enrichment analysis was conducted on the potential cis and trans acting target genes of Lnc Unc-89-AS1.A total of 6 cis acting target genes,356 trans acting target genes,and a total of 7 trans acting target genes related to apoptosis were selected.The expression trends of these 13 lncRNAs related to apoptosis were detected using q RT PCR.According to q RT PCR results and prediction of gene function,BmUbe2 f,which is related to apoptosis and consistent with transcriptome data,was selected as the research object.The subcellular localization of BmUbe2 f was analyzed by immunofluorescence assay.It was found that BmUbe2 f was localized in the nucleus and might participate in the post transcriptional regulation of Lnc Unc-89-AS1.In order to investigate the role of BmUbe2 f in cell apoptosis,overexpression and knockout vectors of BmUbe2 f were constructed.The effects of Lnc Unc-89-AS1 on cell apoptosis were detected using Caspase enzyme activity detection,flow cytometry,and q RT PCR techniques.The results showed that overexpression of BmUbe2 f could promote cell apoptosis,while knockout of BmUbe2 f could inhibit cell apoptosis,and overexpression of BmUbe2 f could promote the mitochondrial apoptosis pathway related gene BmDroncUpregulated expression of BmICE and Bmbuffy.In order to analyze the effect of BmUbe2 f on the proliferation and replication of BmNPV,the copy number of the virus was detected after overexpression and interference with BmUbe2 f.It was found that overexpression of BmUbe2 f inhibited the proliferation and replication of BmNPV,while interference with BmUbe2 f promoted the proliferation and replication of BmNPV. |
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