Functional Study Of BmEOs In The Synthesis Of Silk Protein

Bombyx mori is a model insect of lepidoptera and an important economic insect.Silk gland is the only organ of silk secretion of Bombyx mori.According to its shape and function,it can be divided into Anterior division of silk gland(ASG),Middle division of silk gland(MSG)and Posterior division of silk gland(PSG).ASG has no function to secrete silk.The MSG can synthesize and secrete sericin.The main function of PSG is to synthesize silk fibroin.Studies have shown that the growth and development of silk gland and the synthesis of silk protein are co-regulated by juvenile hormone and molting hormone,and the realization of this regulation is closely related to the hormone titer.The titer of molting hormone is determined by the synthesis and metabolism of molting hormone.The ecdysone C3-epimerization pathway is important to keep the hormone titer in bombyx mori,and the ecdysone oxidase(EO)is the key enzyme in this pathway.There are two known EO genes in bombyx mori,one of which is expressed specifically in midgut and the other in silk gland.The specific EO in midgut of bombyx mori can degrade 20 E,thus regulating the consistency of Bombyx mori development.Through transcriptome analysis in the early stage,we found that EO specifically expressed in silk glands was highly expressed in the MSG at the 4th instar of bombyx mori,which was speculated to be involved in silk protein synthesis.In order to study the function of silk gland EO in silk protein synthesis,the m RNA level and protein level of EO were analyzed in this study.At the same time,the function of RNAi technology was discussed.The main results are as follows:1.Bm EOs gene expression pattern analysisIn order to confirm the expression pattern of Bm EOs gene in silk gland,RT-PCR,RT-q PCR,Western blot,immunohistochemistry and other methods were used for expression analysis and localization study.Tissue expression pattern showed that Bm EOs was highly expressed specifically in the silk glands,but almost not in other non-silk glands.The expression level was highest in the A-MSG.In terms of expression time,the expression level of Bm EOs was higher in the peak feeding stage of larvae,while it was almost not expressed in the 4th molting stage and 7 days in the5 th instar.In addition,we also used Bm EOs antibody for immunofluorescence experiments to accurately locate the expression of Bm EOs gene on the day 3 of the fifth instar larvae of Bombyx mori in silk gland and found that Bm EOs had A strong fluorescence signal in the cell layer of A-MSG,and the fluorescence signal gradually decreased with the backward extension of the silk gland.Meanwhile,obvious fluorescence signal was also detected in the glandular lumen of A-MSG,indicating that Bm EOs was also secreted into the glandular lumen.Fluorescence signal was also detected in the lumen of the anterior silk gland,but not in the cell layer,indicating that Bm EOs was transported to the anterior silk gland along with the secretion of silk protein.2.Bm EOs gene response to hormones and in vitro activity determinationIn order to test whether the expression of Bm EOs gene is regulated by hormones,this study investigated the response of Bm EOs gene to injection of 20 E and application of JHA.The results show that injection of ecdysone can inhibit the expression of Bm EOs gene,while application of JHA can promote the expression of Bm EOs gene.Meanwhile,in order to test whether Bm EOs protein has ecdysone oxidase activity,in this study,prokaryotic and pichia pastoris expression systems were used for expression of Bm EOs gene,and Bm EOs protein was successfully purified from expression supernatant.Using 20 E as substrate,the enzyme activities of Bm EOs were determined,and the reaction products were identified by HPLC.The results showed that no new peak appeared after the 20 E elution peak,indicating that the heterologous expression of Bm EOs protease could not convert substrate 20 E to 3-dehydroecdysone.We further detected 20 E in the silk gland,and found no correlation between the titer of 20 E and the expression level of Bm EOs gene.3.The role of Bm EOs gene in silk protein synthesisIn order to explore the function of Bm EOs in the silk gland,we conducted an RNAi experiment on Bm EOs in larval stage.ds RNA was injected from the 3th instar of Bombyx mori larvae for 3 consecutive times,and EGFP ds RNA was used as control.It was found that the intervention was successful.At the day 3 of the fifth instar larvae,the silk glands were dissected and observed.It was found that the development of the silk glands in the experimental group was abnormal,the silk glands became flat,and A-MSG was shortened and constricted.After DAPI staining,the number of cells in constricted area was significantly reduced.The silk glands were further sectioned for hematoxylin-eosin staining,and it was found that the sericin content in the lumen of the silk glands in the experimental group was significantly degraded,and there was no obvious stratification between sericin and silk fibroin.Further investigation on the expression of silk protein genes showed that,compared with the control group,the expression levels of Sericin1 and Sericin2 were significantly down-regulated,and the expression levels of fibroin-L and P25 genes were significantly up-regulated in the middle silk glands of the experimental group.In posterior silk glands,the expression levels of fibroin-h and fibroin-l were significantly down-regulated,while the expression level of Sericin1 was significantly up-regulated.The above indicated that after Bm EOs gene interference,the localization of MSG expressing sericin and PSG expressing silk fibroin was broken.In addition,we also investigated key genes in the proteolytic pathway,and found that the expression levels of E3-1 and E3-2 genes,as well as Hsp19.5 and PDK1 genes were significantly up-regulated,which may be the reason for the disorder of silk protein secretion in the experimental group.In conclusion,we confirm the expression pattern of Bm EOs gene in larval stage and confirm that the expression of Bm EOs gene is regulated by ecdysone and juvenile hormone.It was found that Bm EOs can affect the synthesis of silk protein by affecting the expression of silk protein genes,and it plays an important role in regulating the localization mode of "sericin expression in MSG and silk fibroin expression in PSG",as well as the expression of key genes in the proteolytic pathway.These results provide guidance and reference for us to understand the regulation mechanism of silk protein synthesis in Bombyx mori.