The Function And Molecular Interaction Mechanism Of Body Color Control Gene Bmcortex In Silkworm,Bombyx Mori

The rich body color patterns of insects are both ornamental and have been a focus of biological research.Melanization is the main coloring mode of insects and plays an important role in the adaptive evolution of insects.It is closely related to insect courtship,camouflage avoidance and body temperature regulation.At present,the pathways of insect melanin biosynthesis and metabolism have been systematically studied,but many related networks and their molecular mechanisms remain to be further studied.Previous studies have found that the cortex gene controls the body color patterns of various Lepidoptera insects such as Biston betularia、Heliconius and more.Our previous studies on the sml melanosis moth of silkworm have also proved that the cortex genes of silkworm control the melanosis phenotype of silkworm,however,the function and molecular mechanism of the cortex gene is unclear.In this study,sml melanogaster and wild-type strains were selected as the research objects.The microstructure was analyzed by scanning electron microscopy and transmission electron microscopy.Bmcortex gene function and molecular mechanism were analyzed by means of transgene overexpression technology,CRISPR / Cas9 genome editing technology,and Co IP Preliminary research,the main findings are as follows:1.Microscopic structure of scales of wild type and sml melanophora and expression characteristics of Bmcortex in silkworm,Bombyx mori Scanning electron microscope and transmission electron microscope were used to analyze the microstructure of silkworm moth scales.It was found that: it was aligned with wild type Dazao,and there was indeed a large amount of melanin on the surface of smlmelanin moth scales.However,the attachment of melanin significantly affected the size and arrangement of the scales,and there is no obvious structural difference between the male and female individual scales.In order to comprehensively investigate the spatiotemporal expression pattern of Bmcortex gene,the wild-type Dazao was taken,and the tissue and period expression profiles of Bmcortex gene were analyzed.It was found that the expression level of Bmcortex gene was relatively low in the whole period,and was 3h,12 h and 24 h in the embryo.In the early embryo,there is an expression peak,in addition to 9 days and the moth stage there is an expression peak;the tissue expression profile shows that the expression of Bmcortex gene in the testis and ovary is significantly higher than other tissues,suggesting that Bmcortex may be It also has important functions in reproduction.2.Bmcortex transgene overexpression and gene knockoutWe constructed a Bmcortex overexpression transgene vector fused with the Flag tag protein,and successfully obtained transgenic positive individuals who overexpressed Bmcortex in sml melanoglyphs.We investigated the phenotype of the transgenic individuals and found that: The Bmcortex gene does cause lightening of the sml melanophora moth.In addition,a Bmcortex gene g RNA transgenic vector for CRISPR / Cas9 gene knockout experiments was constructed,and the positive g RNA transgenic individuals of Bmcortex gene were successfully obtained in wild type Dazao.Currently,g RNA transgenic positive individuals have been crossed with Cas9 transgenic positive individuals for screening of Bmcortex gene knockout individuals(in progress).3.Preliminary study on the molecular mechanism of Bmcortex gene regulating body colorPrevious analysis found that the Bmcortex gene encodes a typical WD40 domain protein,which is usually used as a scaffold for protein interaction in functional protein complexes.We speculate that the Bmcortex protein may combine with other proteins to form protein complexes,which may indirectly or directly regulate melanin synthesis.Therefore,we used Bmcortex transgenic overexpression-positive individuals fused with Flag-tagged proteins as materials.At day 7 before the sml black moth was stained,we took out the wing and used Co IP technology to fish for the Bmcortex interaction protein.The Q Exactive protein profile identifies proteins that specifically bind to Bmcortex protein.The results show that proteins that may specifically bind to Bmcortex proteins include: phenol oxidase subunit 2,protein kinases,catalase,heat shock proteins,and a large number of epidermal and ribosomal proteins.The analysis found that phenol oxidase proteins are involved in the pigment biosynthesis process of many insects.It is speculated that Bmcortex protein may indirectly control the depth of body color of the silkworm by combining with key functional proteins on the melanin synthesis pathway such as phenol oxidase in the pupal stage.We carried out a preliminary study on the function and molecular mechanism of the Bmcortex gene in silkworm.The related experimental results and the obtained transgenic materials have laid an important foundation for the in-depth study of the molecular mechanism of Bmcortex gene regulation of body color.