Identification And Expression Regulation Of ShxA?ShxC1 Genes Promoters In Silkworm,Bombyx Mori

The regulation of gene expression is the molecular basis for the normal development and life activities of organisms,and it is also one of the central issues in molecular biology research nowadays.Hox(homeotic genes)is a kind of development-regulating gene and is ancient and widespread in organisms.Defects in the function of these genes usually lead to embryonic lethality or homeosis.Hox genes are highly conserved in long biological evolution and rarely undergo gene duplication and amplification.However,In insects,scholars found that Hox genes have undergone gene duplication and amplification.Especially in Lepidoptera,a cluster of new Hox genes with conserved Homeobox domains was discovered between pb and zen,that is Shx(Special homeobox genes).Bombyx mori is an important economic insect and has been domesticated for millennia.The completion of the whole genome seque ncing of silkworm and the publication of the genome re-sequencing data of 29 silkworm strains as well as the gradual improvement of transcriptome and proteome data provide solid foundation for the post-genome study of the silkworm.Recent years,biotechnology developed rapidly,and using transgenic technology in silkworm to study gene function and the regulating mechanisms of gene expression is also increasingly common.At the same time,because of the important developmental regulation function of Hox genes,the newly amplified Shx genes in Lepidoptera insects have important research value.In the previous study of the Shx gene in silkworm,our team completed the identification and expression pattern analysis of the Shx gene and found that there are five Shx genes,namely BmshxA,BmshxB1,BmshxB2,BmshxC1 and BmshxC2,and the expression patterns of the five genes were highly consistent.These genes were specifically expressed in the ovary at the larval stage,and were only exclusively expressed in the eggs of female moths during the adult stage.In situ hybridization showed that Bmshx genes were specifically expressed in larval ovaries and follicular cells in adult eggs,which was supposed to be related to serosal development.Shx genes are newly identified Hox-related genes,their specific functions and molecular regulation mechanisms for the development of ovary or egg are unclear.In this study,ShxA and ShxC1 genes were selected as research objects for further study.First,the promoters of the two genes were identified by bioinformatics,gene cloning and transgenic methods.Then using protein and DNA binding experiments to catch the trans-factors specifically binding to the upstream sequence of BmshxA gene,screen trans-factors involved in regulating the expression of BmshxA gene,and preliminarily explore the regulating mechanism of trans-factors on BmshxA.The research of Shx genes in silkworm can provide important reference for studying the function of Hox genes,the regulatory mechanism of insect ovary and ovum development.The main findings of this article are as follows:1.Identification promoters of ShxA and ShxC1 genes in silkwormThe transcription factor binding sites were predicted and analyzed by bioinformatics within the potential promoter sequence of BmshxA,BmshxC1 gene.Then 934 bp and 1619 bp sequences of the initial codon upstream of BmshxA gene and 1656 bp sequences of the initial codon upstream of BmshxC1 gene were intercepted as candidate promoter fragments.PiggyBac expression vectors with the three candidate promoters driving the red fluorescent protein gene was constructed respectively.Three transgenic lines labeled BmshxA-934,BmshxA-1619,BmshxC1-1656 were obtained by microinjection at the early embryonic stage and fluorescence screening of the next generation of injected individuals.Furthermore,the insertion sites of transgenic lines were analyzed,and it was not found that the gene fragments were inserted into the exons of any gene,which will result in the destruction of gene st ructure and function.No abnormality was found in the course of feeding,exercise,mating and oviposition of transgenic individuals.In order to analyze the activity of candidate promoters intercepted,we investigated the expression level of DsRed gene in ovarian tissues of transgenic individuals and found that they were detected at mRNA level and protein level,indicating that the truncated BmshxA-934,BmshxA-1619 and BmshxC1-1656 fragments were active.Furthermore,we compared the promoter efficiency of the BmshxA-934 and BmshxA-1619 promoters and found that the efficiency of BmshxA-1619 promoter was significantly higher than that of BmshxA-934,which suggests that 935-1619 bp upstream of the start codon of the BmshxA gene may contain elements or trans-acting factor binding sites that enhance gene expression.2.Screening and functional analysis of trans-acting factors upstream of BmshxA geneGiven the significant difference in the activity of DsRed gene expression initiated by the BmshxA-934 and BmshxA-1619 fragments in the transgenic lines,combining the predictive analysis of transcription factor binding sites on the truncated promoter sequences,we selected a total of 282 bp-fregments from 1344 bp to 1626 bp upstream of the start codon of the BmshxA gene as probe.DNA pull down was used to fish the trans-factor combined with the probe.According to the result of DNA pull down and mass spectrometry identification.Eight proteins that specifically bind to the probe were identified in the experimental group.Functional annotation was performed on the eight proteins,and the correlation between the expression patterns of the eight genes and that of the BmshxA gene at the same time was investigated.Finally,two genes significantly related to the expression of BmshxA were screened,encoding the replication protein A large subunit and importin subunit alpha gene,respectively.In order to further explore the regulation mechanism of the expression of BmshxA gene by replication protein A large subunit and importin subunit alpha gene,we conducted interference experiments on these two genes by targeting double-stranded RNA at the stage of pupae formation.The expression level of target gene interferenced and BmshxA gene were detected.Interfering with the replication protein A large subunit gene,the expression of the BmshxA gene in the interfering individuals was down-regulated;when interfered the importin subunit alpha gene,the expression of the gene was down-regulated,whereas the BmshxA gene was significantly up-regulated,suggesting that the importin subunit alpha gene has an inhibitory effect on BmshxA gene.In this study,we successfully constructed three transgenic lines BmshxA-934,BmshxA-1619 and BmshxC1-1656,all of which have the activity of initiating gene expression.Eight proteins were identified binding to the upstream sequence of BmshxA gene by DNA pull down and mass spectrometry.Two proteins,replication protein A large subunit and importin subunit alpha,were focused on.Interference with these two coding genes,it was found that importin subunit alpha may be a negative regulator of BmshxA gene and inhibit the expression of BmshxA gene.It may be involved in regulating the development of BmshxA on the ovaries or eggs.