Identification Of Pax Genes And Function Study Of Pax7 Gene In Silkworm

Pax genes are an evolutionarily highly conserved gene family,which is widespread in invertebrates and vertebrates.The encoded Pax protein is an important transcriptional regulator and belongs to the Hox family of transcription factors.Pax genes play important functions in cell proliferation,maintenance of precursor cells,cell fate determination,anti-apoptosis and neuronal differentiation.Pax genes have been identified in other species,and related functional studies have also been reported,but there are few reports in silkworms.In this study,the Pax gene family of Bombyx mori was identified and analyzed by bioinformatics,and the function of a gene specifically expressed in silk gland tissue of Bombyx mori was studied to explore its role in silk gland development and silk protein gene regulation in Bombyx mori.In this study,related experiments were carried out through research methods such as bioinformatics analysis,gene interference,transcriptome data analysis,q RT-PCR and immunohistochemistry,and the following research results were obtained:1.Identification and expression pattern analysis of Pax gene family in silkwormIn this study,8 Pax genes were identified in the silkworm genome database Silk DB3.0 and the Japanese silkworm database KAIKObase,and they were classified into 6 subfamilies.The domain prediction of the members of the silkworm Pax genes showed that some members of the silkworm Pax genes contained both Pax and Hox domains,and some family members only contained the Pax domain.The analysis of phylogenetic tree construction found that the members of the Pax genes of Bombyx mori are highly homologous to the Pax genes of other species,and are highly conserved in evolution.Embryo expression profile analysis found that the Pax genes of the silkworm is expressed in high amounts at all stages of embryo development,which implies that the Pax genes may play an important function in the development of the silkworm embryo.The results of tissue expression profiling showed that Pax genes has specific expression patterns in different tissues of silkworm,laying a certain biological foundation for the future study of the function of this gene family.2.The Bmpax7 gene interference in SilkwormEmbryo expression profile analysis found that the BmPax7 gene was expressed throughout the embryonic period of the silkworm,and the results of early embryo intervention showed that compared with the control,as the concentration of injected double-stranded RNA increased,the embryonic hatching rate after BmPax7 gene intervention showed a downward trend.Interference with the BmPax7 gene may cause embryo death.Tissue expression profile analysis found that the BmPax7 gene was specifically expressed in silk glands of silkworms,so the method of larval intervention was used for research.Double-stranded RNA injection was performed continuously after the larvae developed to 3rd instar 2 days and 4th instar 2 days.The q RT-PCR test results showed that the expression of BmPax7 gene in the interfered individual was significantly down-regulated,proving the success of the intervention.When the larvae developed to the 5th instar,their silk glands were dissected and observed on 1 day.The results showed that the front part of the middle silk gland of the interfering individuals was significantly shorter,and when the larvae grew to the upper cluster stage,the silk glands could not spin and cocoon.The anterior junction of the middle silk gland is broken.The results of DAPI staining showed that the nucleus of the front part of the middle silk gland of the experimental group was obviously disordered.The white light observation revealed that the silk protein channel in the lumen of the silk gland was broken at the junction of the front part of the front part of the silk gland and the front part of the middle part of the silk gland.The results of HE staining showed that there were a lot of cavities in the anterior silk gland and the anterior silk gland cell layer of the middle silk gland of the interference individual,the cell nucleus had obvious signs of shrinkage and degradation,and there was no silk fibroin in the silk gland cavity.3.Transcriptome analysis of Bmpax7 gene interferenceIn order to explore the molecular mechanism of down-regulation of BmPax7 gene affecting silk gland development of the silkworm,the transcriptome sequence analysis of the interference individual silk gland was sent.The results showed that after interference with BmPax7 gene,Fib-L,Fib-H,P25 and Ser-1 genes were significantly up-regulated.The q RT-PCR test results showed that the expression of silk fibroin genes and other related genes were up-regulated,which was consistent with the results of the transcriptome data.The expression of nuclear hormone receptor HR3,glucose dehydrogenase,ecdysone inducible protein 78 C,and other genes were significantly down-regulated.This study further investigated the expression of genes in related pathways,and the results showed that apoptosis and autophagy-related genes were not up-regulated,while genes related to cell hydrolysis pathways,E3-2(ubiquitin proteolytic enzyme),ATG9,and cellular Heat shock response related genes Hsp19.5and PDK1 were up-regulated in the front and middle silk glands.Transcriptome analysis results showed that BmPax7 gene inhibits the expression of silk protein genes in the front and middle silk glands,providing a new target for the study of silk protein gene transcription regulation.4.Immunofluorescence analysis of Bmpax7 gene interferenceTranscriptome data showed that the silk protein gene was highly expressed in the front and middle silk glands,and the immunofluorescence experiment was further designed to discuss the relevant results.Analysis showed that the interfering individual silk fibroin gene Fib-L had obvious signal in the anterior silk gland cell layer,but no signal in the silk gland cavity.The Fib-L gene antibody was incubated in the front of the middle silk gland and found that the Fib-L signal of the interfering individual was detected in the silk gland cell layer,and the lysate in the silk gland cavity was in a diffuse state and there was no obvious hierarchical structure of sericin and silk fibroin.Incubation of Ser-2 antibody interferes with the signal of sericin in the anterior silk gland of the individual,and the signal of lysate in the anterior cell layer of the middle silk gland and the silk gland cavity is obvious.The results of incubating silk fibroin hydrolase gene(Bcp)antibody showed that the Bcp gene of interfering individuals was highly expressed in the anterior silk gland and the anterior cell layer of the middle silk gland.The above results confirm that the abnormal expression and accumulation of silk protein in the front and middle silk glands of the interfering individuals will cause the cell hydrolysis reaction,which in turn causes the silk gland cells to be broken down and die by hydrolytic enzymes.