Study On The Function And Mechanism Of Ecdysone Receptor EcR/USP On Silk Fibroin Synthesis In Bombyx Mori

The silkworm Bombyx mori,an important special economical animal known for silk production,is the biological basis of sericulture industry.Silk gland is a large organ for silkworm to synthesize silk protein exclusively.According to morphological structure and functional characteristics,it is usually divided into three regions:anterior silk gland(ASG),middle silk gland(MSG)and posterior silk gland(PSG).MSG mainly secretes scricin protein and PSG mainly secretes silk fibroin protein.Both of them are the main components of silk protein(fiber)and also the molecular basis of silk quality or performance.The composition of silk protein seems simple,but the contorlcd synthesis mechanism of silk protein in silk gland is quite complex and still unproven.Eedysone(active form:20-hydroxyccdysone,20E)is one of the three major regulatory hormones in insects,and it lead regulation of insect growth and development,metamorphosis,reproduction and other important physiological processes.20E mainly outputs signal through EcR/USP,amplifies signal through a series of response factors and finally acts on target organs.In Bombyx mori,the 20E signaling pathway not only regulates molting and metasmorphosis development,but also plays an indispensable role in silk gland development and silk protein synthesis.However,the specific mechanism is still unclear.This study aimed to explore the molecular mechanism of 20E signal regulating silk protein synthesis by targeting 20E receptor EcR/USP.The main research strategy is to produce transgenic silkworm strains overexpressing EcR and USP at first,and then analyze the main function and mechanism of EcR/USP in regulating silk protein synthesis from individual level and molecular level,respectively.The main results obtained are as follows.1.Production of EcR and USP overexpressed mutantsGAL4/UAS dual expression system is an effective tool to study gene function.Our group previously established a PSG-specific GAL4 transgenic line fibH-GAL4(HG4)with fibroin heavy chain(fibH)promoter as regulatory element.In this study,UAS expression vectors for overexpression of EcR-A(one of the main subtypes of EcR)and USP were further constructed,and corresponding UAS transgenic lines UAS-EcR-A and UAS-USP were obtained.Two UAS transgenic lines were crossed with HG4 transgenic lines respectively,and the corresponding GAL4/UAS positive individuals were screened from their hybrid offsprings,which were named HG4/UAS-EcR-A(EcR-AOE,overexpress EcR-A)and HG4/UAS-USP(USPOE,overexpress USP).Genomic PCR and molecular detection results showed that the overexpression systems were successfully constructed and could be used for further research.2.Mutant phenotype analysis of EcR-AOE and USPOEUsing wild type(WT)Bombyx mori as control,the growth and development characteristics of silk glands,pupae and cocoons phenotypes of EcR-AOE and USPOE were investigated respectively.The results showed that:(1)On day 6 of fifth instar(L5D6),the phenotype of EcR-AOE PSG did not change significantly but the PSG of USPOE became shorter and thinner,with uneven nodular protrusion.(2)The spinning amount of USPOE decreased significantly,the cocoons layer became thinner.There was slightly change in cocoons layer quantity of EcR-AOE individuals.(3)The length of PSG,the weight of whole silk gland and PSG of USPOE were significantly decreased,and the weight of cocoon layer was also decreased,while the weight of pupa increased.(4)Further detection confirmed that the transcriptional expression level and protein synthesis level of silk fibroin gene were significantly down-regulated in the PSG and cocoon of EcR-AOE and USPOE.In conclusion,EcR/USP plays an important role in silk gland,and its aberralt expression will lead to abnormal silk protein synthesis and silk gland development.3.Preliminary study on the mechanism of EcR/USP regulating silk fibroin synthesis The above phenotypic results confirmed that EcR/USP receptor plays an important role in the development of PSG and the expression of silk fibroin gene.How EcR/USP exercise regulatory function in PSG of Bombyx mori?To this end,we carried out transcriptome sequencing analysis of L5D6 PSG using USPOE with more severe mutation phenotype as material.The results showed that there were 4552 differentially expressed genes(2728 significantly up-regulated and 1824 significantly down-regulated)in the PSG of USPOE.Interestingly,at least four differentially expressed genes were found to be closely related to silk protein synthesis and silk gland development,including 20E signal response factors E75,HR3,Br-C and HR39,silk fibroin genes fihH,fibL and P25,silk gland transcription factors Sage,Awh,SGF-1 and Dimm,etc.JH signal related genes SRC,Met1,etc.These results indicate that the regulated gene network of EcR/USP in PSG is very complex.Furthermore,we analyzed the interaction between EcR/USP and three silk fibroin genes by using the dual-luciferase reporting system and EMSA.The results showed that EcR/USP could directly bind the DNA elements in fibH,fibL and P25 promoters and inhibit their activity.These results indicated that EcR/USP regulated numerous downstream target genes in PSG of Bombyx mori,which not only directly regulated silk fibroin genes,but also had close relationship with silk gland transcription factors and JH signaling pathway,which was worthy of further investigation.In conclusion,this study confirmed that EcR/USP is a key factor regulating silk protein synthesis at the phenotypic level by making and analyzing transgenic silkworms overexpressing EcR and USP,and revealed the general picture of downstream target genes regulated by EcR/USP through transcriptome analysis.The molecular interaction revealed that EcR/USP could directly bind to the promoter of silk fibroin genes and regulate their expression.These results provide important clues and theoretical reference for further elucidating the molecular mechanism of 20E regulating silk gland development and silk protein synthesis.