| As a lepidopteran model insect,Bombyx mori has extremely high economic value and extensive biological research value.The silk gland is only the silk secreting organ of the silkworm.A large amount of silk protein is synthesized during intermolt,and stopped during molt.Moreover,the synthesis of silk protein has spatial specificity in addition to temporal expression specificity.Among them,silk fibroin is synthesized in the posterior silk gland,and sericin is synthesized in the middle silk gland.They are all coded by corresponding genes,among which silk fibroin genes include fib-h,fib-l and P25 genes.Sericin genes are mainly composed of sericin 1,2,3,4 genes.The spatiotemporal specific expression of silk protein genes is mainly achieved at the transcription level.Therefore,the transcriptional regulation of silk protein genes is an ideal model for studying the spatiotemporal specific expression of genes.Among them,there are many studies on the transcription regulation of fib-h,and multiple transcription factors have been identified to participate in the transcription regulation of this gene.The transcriptional regulatory factors that promote its expression during intermolt include Bmsage,SGF-1,BmDimm,etc.During molt,it is transcriptionally inhibited by factors such as FMBP,Ftz,and MBF2.The transcription factor Broad Complex(BrC)is an early response transcription factor of the 20 E signaling pathway,which is involved in the molting and metamorphosis of insects.BrC is highly expressed in the silk glands at the end of molt period,and basically not expressed in intermolt,which is speculated to participate in the regulation of silk fibroin gene expression.Cong Jiangshan and others in our laboratory overexpressed BmBrC-Z2 in the posterior silk gland,the silk fibroin gene expression was significantly down-regulated,and the amount of cocoon silk was reduced.Moreover,the EMSA experimental results show that BmBrC-Z2 can directly bind to the corresponding binding sites of fib-h and P25 gene promoters,indicating that it can directly inhibit the transcription of silk fibroin genes.The study also found that BmKr-h1 and BmDimm,the key factors involved in the positive regulation of silk fibroin gene expression in the JH signaling pathway,were significantly inhibited.Whether BmBrC-Z2 regulates these two factors directly or indirectly is currently unknown.In this regard,this study used promoter binding site prediction,EMSA technology,and dual luciferase reporter system to study the transcriptional regulation of BmBrC-Z2 on BmKr-h1 and BmDimm and determine their regulatory sites.At the same time,transcriptome sequencing and differential gene analysis were performed on the posterior silk glands of the transgenic lines overexpressing BmBrC-Z2.In order to reveal the molecular mechanism of BmBrC-Z2 overexpression inhibiting silk fibroin gene expression more fully.The main findings are as follows:1.The transcriptional regulation of BmBrC-Z2 on BmKr-h1In order to explore whether BmBrC-Z2 has a direct regulatory relationship with the BmKr-h1 gene.In this study,the promoter region of BmKr-h1 was used to predict the potential binding elements of BmBrC-Z2 through the promoter region binding site prediction website.The prediction results show that there are multiple BmBrC-Z2 potential binding elements in the upstream promoter region of BmKr-h1.In this experiment,the-312,-588,-756,and-1164 sites were selected for EMSA experimental verification.The results showed that among the binding sites-312,-588,-756,and-1164,only the probe signal at-588 can be dose-dependent with the BmBrC-Z2 prokaryotic expression protein and posterior silk nucleoprotein.Moreover,it can be weakened by the cold probe gradient competition,and it can also compete with the BmBrC-Z2 antibody for binding to form an ultra-hysteresis band.The above results show that BmBrC-Z2 can specifically bind to the-588 site of the BmKr-h1 promoter.In order to further explore the inhibitory and regulatory effects of BmBrC-Z2 on BmKr-h1.In this study,BmBrC-Z2 was overexpressed in the silkworm embryonic cells(BmE cells)successed,and the BmKr-h1 promoter activity was detected by the dual luciferase gene reporter system analysis.The results showed that both the addition of 20 E or the overexpression of BmBrC-Z2 by means of transfection plasmid resulted in a significant down-regulation of the promoter activity of the BmKr-h1 gene.After truncating the-588 position upstream of the BmKr-h1 gene promoter,the inhibitory effect of overexpression of BmBrC-Z2 on the BmKr-h1 promoter disappeared.The above results indicate that BmBrC-Z2 inhibits the transcription of BmKr-h1 gene by binding to the-588 site upstream of the BmKr-h1 promoter.2.The transcriptional regulation of BmBrC-Z2 on BmDimmIn order to explore whether BmBrC-Z2 has a direct regulatory relationship with the BmDimm gene.The same as the above-mentioned study method of BmBrC-Z2 on BmKr-h1 transcription regulation.First,the BmDimm promoter region is predicted for the potential binding elements of BmBrC-Z2.The results show that there are multiple BmBrC-Z2 binding elements in the promoter region of BmDimm.This experiment selected-576,-679,-937 sites for biotin labeling to carry out EMSA binding experiments.The results showed that only the-937 BmBrC-Z2 binding element upstream of the BmDimm promoter can specifically bind to BmBrC-Z2.The results of dual luciferase activity analysis show that overexpression of BmBrC-Z2 can significantly inhibit the promoter activity of BmDimm,and the inhibitory effect disappears after truncating the site.The above research results also indicate that BmBrC-Z2 can inhibit the transcription of BmDimm gene through the-937 position upstream of the BmDimm promoter.3.Analysis of transcriptome data of posterior silk gland-specific overexpression of BmBrC-Z2Previous studies have shown that BmBrC-Z2 can directly inhibit the two important transcriptional regulators BmKr-h1 and BmDimm,which in the juvenile hormone pathway of the positive regulatory pathway of silk fibroin genes.In addition to these two factors,what genes and pathways does BmBrC-Z2 use to inhibit the expression of silk fibroin genes? In order to fully obtain the genes related to the overexpression of BmBrC-Z2 in the posterior silk gland that inhibit silk protein synthesis.In this study,transcriptome sequencing and differential gene analysis were performed on the posterior silk glands of transgenic lines overexpressing BmBrC-Z2.The results of transcriptome data analysis showed that BmBrC-Z2 was highly expressed in the posterior silk glands of the overexpression transgenic lines,but was not expressed in the control,which further verified that the foreign inserted BmBrC-Z2 was indeed successfully expressed in the posterior silk glands.Among the top 10 genes with the highest expression levels in the two strains,the genes with the highest expression levels in the silk glands after overexpression of BmBrC-Z2 are mainly silk fibroin genes and ribosomal proteins involved in silk fibroin translation,but three Silk fibroin are significantly downregulated.In order to further explore the role of BmBrC-Z2 overexpression in the two important pathways of JH and 20 E.We screened important genes and transcription factors in these two pathways by log2 Fold Change(D9L vs BrC)value.The analysis results show that,in addition to the significant inhibition of BmKr-h1 and BmDimm in the previous study.Genes such as JH degrading enzyme,juvenile hormone epoxide hydrolase-like protein 2,juvenile hormone esterase-like protein 3,and juvenile hormone epoxide hydrolase-like protein3,were significantly up-regulated.The synthesis of JH synthase,farnesic acid o-methyltransferase-like protein 1,farnesic acid o-methyltransferase-like protein and other genes were down-regulated.In the 20 E signaling pathway,most of the downstream genes and key regulatory factors of the 20 E signaling pathway are significantly up-regulated,including BrC,FTZ-F1,E75 A,E74,E74 B,etc.It is worth noting that MBF2,which is involved in inhibiting the transcription of fib-h in the 20 E signaling pathway,has been significantly up-regulated.Transcriptome data show that the posterior silk gland overexpression of BmBrC-Z2 not only inhibits the synthesis of JH and the expression of BmKr-h1 and BmDimm genes in the JH signaling pathway,but also promotes the decomposition of JH and promotes the 20 E signaling pathway genes and the inhibitor MBF2 To achieve indirect inhibition of silk fibroin gene in a way. |
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