Study On The Function Of CircRNAs In The Silkworm Hox Gene Cluster

Living things are constantly evolving under natural selection.The higher the organism,the more complex and precise the regulation mechanism of gene expression.The Hox gene belongs to the homeobox family and is highly conserved in both vertebrates and invertebrates.It specifically regulates genes related to the development of organisms.Hox gene expression regulation has the principle of spatiotemporal collinearity.In different tissues and different stages of development and differentiation,different Hox genes have diverse expression regulation modes,and their expression regulation is very complicated.In human and mouse studies,it was found that the non-coding RNA present on the Hox gene cluster plays an important role in the regulation of Hox expression.Non-coding RNA refers to RNA transcripts that cannot encode proteins.In the process of Hox gene expression regulation,non-coding RNA can participate in regulation through cis and trans functions,making Hox gene regulation more precise.With the development of high-throughput sequencing technology,it was discovered that there is another special non-coding RNA in the body of eukaryotic cells,circRNAs(circular RNAs).CircRNA exists widely in eukaryotic organisms,which is not easily degraded by exonuclease RNase R and has important biological functions.Full-length transcriptome data of the whole organization and period of the silkworm showed that there are many circRNAs in the silkworm.The generation mode,function and mechanism of these circRNAs have not been reported.In order to explain whether the Hox gene of silkworm is also regulated by circRNA,we analyzed the non-coding RNA in the Hox gene cluster and identified two circRNAs.In order to explore the biological functions and regulatory mechanisms of these two circRNAs,we conducted quantitative,localization,interference,and overexpression experiments on these two circRNAs.The main research results we have obtained are :(1)Identification and conservative analysis of non-coding RNA in Hox gene cluster of silkwormBased on the whole-organized and full-length transcriptome data of silkworm,we analyzed the types,number,and location of non-coding RNA in the Hox gene cluster of silkworm,and compared them with fruit flies,and found that lncRNA in the Hox gene cluster The largest number,the number of microRNA is small and highly conservative.In addition,two circRNAs were found in the Hox gene cluster of the silkworm,which has not been reported in the Hox gene cluster of humans,mice and fruit flies.(2)Cloning and identification of silkworm BmcircAntp and BmcircUbxThrough sequence alignment analysis,it was found that the two circRNA sequences in the Bombyx mori Hox gene cluster were consistent with the second exon sequence of BmAntp and the second exon sequence of BmUbx;verification by linear RNA digestion and T clone sequencing confirmed that these 2 A circRNA does exist.We named these two circ RNAs BmcircAntp and BmcircUbx.(3)Expression patterns and functional analysis of BmcircAntp and BmcircUbxWe extracted RNA from the whole period and tissues of the silkworm,and detected the spatiotemporal expression patterns of BmcircAntp and BmcircUbx by RT-PCR experiment.RT-PCR results showed that BmcircAntp was highly expressed in Bombyx mori,and had a peak expression at 144 h of embryonic development.It was also highly expressed in the epidermis,fat,muscle and silk gland at the age of 3 days in the fifth laval stage Bonibyx mori,which was related to the expression of BmAntp;BmcircUbx has low expression in silkworms,and has a correlation with BmUbx expression.In order to explore the expression sites of BmcircAntp and BmcircUbx in silkworm embryos,we conducted in situ hybridization experiments using embryos of silkworm 144 h.The results of in situ hybridization showed that BmcircAntp was expressed in the chest and feet and A1,A2,A7 and A8 abdominal segments,and BmcircUbx was expressed in the chest and feet and T2 and T3 thoracic segments.To explore the functions of BmcircAntp and BmcircUbx,we conducted interference and overexpression experiments in silkworm embryo cells.Cell experiments showed that both BmcircAntp and BmcircUbx can be successfully overexpressed.BmcircAntp overexpression had no significant effect on the expression of its parent gene BmAntp;after BmcircAntp interference,BmAntp expression increased,and after BmcircUbx overexpression,BmUbx expression decreased.(4)A preliminary study on the mechanism of action of silkworm BmcircAntp and BmcircUbxThrough subcellular experiments,it was found that BmcircAntp and BmcircUbx are both expressed in the nucleus and cytoplasm;through RNA pull down experiments,no proteins that significantly bind to BmcircAntp and BmcircUbx were found;miRnada,PITA,and RNA22v2 software respectively predicted the binding to BmcircAntp and BmcircUbx In microRNA,a total of 44 microRNAs that may be combined with BmcircAntp and 61 microRNAs that may be combined with BmcircUbx are found,which shows that BmcircAntp and BmcircUbx may act as microRNA sponges.Conclusion: There are lncRNA,microRNA and circRNA in the Bombyx mori Hox gene cluster;the number of lncRNA is large,but it is basically not conserved among species;the number of microRNA is small,but it is highly conserved among species;circRNA has not been reported in Hox gene clusters of other species.It may be unique to silkworms.Through sequence analysis and cloning and sequencing,we confirmed that these two circRNAs do exist in the Hox gene cluster of Bombyx mori,which were generated by the circularization of exon 2 of BmAntp and BmUbx,and were named BmcircAntp and BmcircUbx,respectively.Through RT-PCR and in situ hybridization,we determined the expression period and expression site of BmcircAntp and BmcircUbx.The expression is related to the expression of BmAntp and BmUbx.It is speculated that they may be involved in the regulation of silkworm appendage development;through interference,overexpression and interacting molecules In experiments such as fishing,we speculate that BmcircAntp and BmcircUbx may be used as microRNA sponges to regulate the transcription of target genes by competitively binding microRNA.