Identification And Functional Study Of Gradient Expressed Genes In The Silk Gland,Bombyx Mori

Silk fiber is one of the most mysterious and attractive materials in nature and has been widely used in biomedicine,soft tissue engineering,biosensors,textiles,cosmetics and other fields.More than 23 species of insects and at least 48,353 spiders secrete silk fibers in nature.Usually,silk secreting animals build cocoons to protect themselves,or spin silk nets to catch prey.Bombyx mori and Nephila clavipes are the two most studied silk-secreting species.Natural spider silk has extraordinary mechanical properties such as high tensile strength and toughness.However,spiders display extremely aggressive behavior and not social,making it difficult to obtain natural spider silk by mass breeding of spiders.To this end,researchers have made great efforts to develop recombinant spider silk proteins,including expression of recombinant spider silk proteins in bioreactors such as bacteria,yeast,plants and transgenic animals.However,due to the limitation of protein yield,solubility,stability,artificial spinning process and other factors,the recombinant spider silk obtained in vitro is always difficult to reach the quality of natural spider silk.Since the beginning of breeding silkworms by lei zu to the present day,silkworms have been domesticated for more than 5,000 years.Natural silk is also loved by the people for its comfort and luster.Bombyx mori is an important economic insect and a model organism for studying lepidopteran insects and arthropods.Silkworms like to live in large groups,and can be reared on a large scale,and the silk protein yield is very high.A silkworm with a dry weight of about 2g can produce up to500mg of silk protein,accounting for about 25%of its own dry weight.Silk proteins are stored in soluble and high concentrations?up to 25%?in the silk gland lumen without protein aggregation and denaturation.This extraordinary protein synthesis and storage capacity provides broad prospects for the research and utilization of silk.The synthesis and secretion of silk protein is a dynamically controlled process.During the transport of silk proteins from the posterior silk gland to the anterior silk gland,sericin wraps fibroin layer by layer to form an orderly assembly pattern of silk proteins in the silk gland lumen.This orderly assembly mode also creates silk fibers with strength and toughness inside,and viscosity and hydrophilicity outside.The silk gland lumen of Bombyx mori has a pH gradient.From the posterior silk gland to the anterior silk gland,The pH drops from 8.2 to 6.2,with a change from alkali to acid.The ions in the silk gland lumen also have a gradient change.From the posterior silk gland to the middle silk gland,the content of Na,K,Mg,Cu,and Zn is gradually increased,and the content of Ca is gradually decreased.So how are the orderly composition and gradient changes in the silk gland lumen formed and regulated?In this study,the silk gland of 5^th instar mature larvae was selected as the research object.Through the fine segmentation of single silk gland,transcriptome sequencing was used to analyze and screen the gradient expression genes involved in silk protein synthesis and secretion and silk fiber formation.Then regulate candidate gradient expression genes at the individual level and explore their effects on silk protein synthesis.The main findings are as follows:1.Transcriptome analysis of finely segmented single silk gland of the silkworm,Bombyx moriIn order to deeply analyze the genetic basis of silk protein synthesis,we used RNA-seq technology to carry out sequencing analysis on single silk gland samples with fine segmentation,of which three groups of middle silk gland samples were MSG-A,MSG-M,and MSG-P;Four groups of posterior silk gland samples:PSG-1,PSG-2,PSG-3,PSG-4.Genes with FPKM value?5 were defined as expressed genes.The results showed that the number of genes expressed in the three groups of MSG samples were 3,658,2,860,2,888,and the number of genes expressed in the four groups of PSG samples were 2,987,2,778,3,720,and 2,658,respectively.Further screening and comparison analysis revealed that a total of 3,121 differentially expressed genes?DEGs?were identified in seven groups of silk gland segmented samples.Hierarchical cluster analysis of DEGs using pheatmap revealed that these DEGs exhibited obvious tissue-specific expression characteristics in the silk gland,with the highest differential gene expression levels in MSG-A sample.Co-expression analysis was performed on all DEGs using TCseq,and 430 MSG-A highly expressed genes,252 MSG highly expressed genes,544 PSG highly expressed genes,and 773 genes with gradually upregulated expression were identified.KEGG pathway enrichment analysis of specific and highly expressed genes in MSG-A showed that the tryptophan metabolic pathway,glyoxylate and dicarboxylate metabolic pathway,FoxO signaling pathway,and peroxisome pathway were significantly enriched.Comparative analysis of MSG high-expressing genes and PSG high-expressing genes,GO annotations show that genes in the MSG high-expression group were mainly involved in lipid metabolism,lipid transport,negative regulation of cellular processes,dephosphorylation and antibiotic metabolism.PSG high-expression group were mainly involved in macromolecular catabolism,ethanol metabolism,vesicle-mediated transport,chemical homeostasis,and proteasome assembly.The significant enrichment of the KEGG pathway in the MSG high expression group and the PSG high expression group were co-displayed and found that most of the KEGG pathways were co-enriched by MSG and PSG,including the tricarboxylic acid cycle pathway,glycolysis/gluconeogenesis pathway,carbon metabolism pathway,fatty acid metabolism pathway,amino acid biosynthesis pathway,pyrimidine metabolism pathway,endocytosis pathway,MAPK signaling pathway,mTOR signaling pathway,peroxisome pathway,RNA transport pathway,apoptotic pathway.Besides,the unsaturated fatty acid biosynthetic pathway and lysosomal pathway were only enriched in the MSG high expression group;the tyrosine metabolic pathway,ribosome pathway,proteasome pathway,and glutathione metabolic pathway were only enriched in the PSG high expression group.These results imply that both MSG and PSG are involved in a large number of amino acid metabolism and energy metabolism in the silk gland of fifth-instar mature larval.In addition,we identified 9 lipid metabolism-related genes and 1 lipid transport-related gene that are highly expressed in the MSG,indicating that MSG is also involved in the utilization of lipids.The genes that show a gradually increasing expression pattern from PSG to MSG are refered as gradually upregulated genes.In order to clarify the biological pathways involved in silk protein synthesis and secretion,we used KEGG pathway enrichment analysis on DEGs in the gradient expression group.A total of 17 significantly enriched pathways were identified?q-value<0.05?.We found that these 17 significantly enriched KEGG pathways can be roughly divided into three categories:energy metabolism-related pathways,protein synthesis-related pathways,and protein secretion-related pathways.There are four pathways related to energy metabolism:pentose phosphate pathway,2-oxocarboxylic acid metabolism pathway,glycolysis and gluconeogenesis pathway,carbon metabolism pathway;seven pathways related to protein synthesis:phenylalanine metabolism pathway,Aminoacyl-tRNA biosynthetic pathway,spliceosome pathway,protein processing in endoplasmic reticulum pathway,mRNA surveillance pathway,RNA degradation pathway,and RNA transport pathway;two pathways related to protein secretion:N-glycan biosynthesis pathway,protein export pathway.These results indicate that protein synthesis and secretion are the main activities of the silk glands of the 5^th instar larva.Meanwhile,the results of GO enrichment analysis showed that proton transport ATPase activity was the most significantly enriched in the silk gland.We identified 33 H⁺-transporting ATPase in the silk gland,of which seven V-type H⁺-transporting ATPase have gradually upregulated expression characteristics.Then,we used ion-selective microelectrode?ISM?to measure the pH of silk gland cells.We found that from PSG to ASG,pH gradient also exists in silk gland cells.The pH of PSG cells was in the range of 8.05 to 8.2 and was alkaline;from MSG to the front end of ASG,the pH of silk gland cells dropped from7.8 to 7.6,showing slightly alkaline.2.Carbonic anhydrase regulates the pH gradient of silk glandCarbonic anhydrase?CA?can efficiently catalyze the hydration reaction of CO₂ to regulate pH.We identified two carbonic anhydrase genes in the silk gland:BMgn002647 and BMgn003357.BMgn002647 is annotated as erythrocyte carbonic anhydrase,short for CA2.The similarity between BMgn002647 and the human CA1 is46.07%,and the similarity with the human CA2 is 45.69%.Phylogenetic tree analysis showed that CA2 gene was closely related among Lepidoptera.BMgn003357 is annotated as?carbonic anhydrase 1 and abbreviated as?-CA1.The similarity between BMgn003357 and Drosophila?-CA was 71.76%.Phylogenetic tree analysis showed that the?-CA1 gene was closely related to Lepidoptera.The tissue expression profile of V7^thD showed that BMgn002647 is highly expressed in epidermis and blood cells,and less expressed in the silk gland;BMgn003357 is also highly expressed in epidermis and blood cells,but its expression level is relatively high in the silk gland.We obtained mutant individuals whose BMgn002647 and BMgn003357 genes were overexpressed in ASG,MSG,and PSG,respectively.Transcription level and protein level detection showed that the target genes achieved a certain degree of overexpression.The statistical results of cocoon layer rate showed that there were no significant changes in cocoon layer rate in ASG and MSG overexpression of two carbonic anhydrases.Overexpression of two carbonic anhydrases in PSG significantly increased the cocoon layer rate.PSG overexpression of BMgn002647 gene increased the cocoon layer rate by 1.28-1.7 percentage points;PSG overexpression of BMgn003357 gene increased the cocoon layer rate by 0.69-2.3 percentage points.Using ISM to measure the pH of PSG cells found that overexpression of BMgn002647 gene caused the pH of PSG decreased from 7.8-8.0 to 7.3-7.5,that is,from alkaline to neutral.Quantitative detection of silk protein expression level found that overexpression of BMgn002647 gene caused an increas in Fib-H expression.This suggests that regulating the pH of PSG can promote the secretion of silk proteins.3.Effects of candidate gradient expression gene-angiotensin-converting enzyme BmACE on silk protein synthesisAngiotensin-converting enzyme?ACE?has the effect of raising blood pressure and regulating ion balance in the human body.We identified an ACE gene with a gradually upregulated expression pattern in the silk gland,with the gene number BMgn006539.The amino acid similarity between the BmACE and the human ACE is 24.02%,and the similarity with the Drosophila ACE is 22.52%.The amino acid sequence similarity of their key domain parts is high.Tissue and period expression profile showed that BmACE gene was specifically overexpressed in the silk gland and testis,with the highest expression in molt stage and spinning stage for 48h.Using CRISPRa up-regulation expression technology,we screened a gRNA that can significantly increase BmACE gene expression at the cellular level,and injected and obtained mutant lines that up-regulated BmACE gene expression in the PSG.At the same time,using traditional overexpression methods,we obtained mutant lines that overexpressed BmACE gene in the ASG,MSG,and PSG,respectively.Quantitative detection showed that BmACE gene was effectively improved,whether based on CRISPRa or overexpression technology.Cocoon layer rate statistics showed that there was no significant change in cocoon layer rate in when BmACE gene was overexpressed in ASG and MSG.However,when the expression of BmACE gene in PSG was increased,the cocoon layer rate was significantly increased,in which the cocoon layer rate of PSG over-expressed BmACE gene increased by 0.52-1.16 percentage points,and the cocoon layer rate of PSG up-regulated BmACE gene increased by 1.16-2.3percentage points.This suggests that increasing the expression of BmACE gene in PSG can promote silk protein synthesis.4.Effects of candidate gradient expression gene-Reticulon BmRTN3L on Silk Protein SynthesisReticulon?RTN?is mainly localized in the endoplasmic reticulum,which can promote the curvature of the endoplasmic reticulum membrane.We identified a BmRTN3L gene with a gradually upregulated expression pattern in the silk gland,with the gene number BMgn005860.Tissue expression profiling showed that BmRTN3L gene was highly expressed in blood cells and relatively low in the silk gland.Using CRISPR/cas9 knockout technology,we screened a gRNA with high knockout efficiency at the cellular level,injected it and hybridized with the nanos-cas9 line to obtain a stable inherited BmRTN3L gene knockout line.Sequencing results showed that the knockout efficiency of the F1 generation was as high as 42.86%-74.28%,and the short segment knockout was the main type of knockout.Quantitative results showed that the BmRTN3L gene was significantly knocked out in MSG,and the mRNA level decreased by 25.24%.Quantitative results of silk protein showed that the expression of Ser2 gene decreased,and the expression of Ser3 gene increased.Phenotype observation revealed that the F1 generation cocoon shells became thinner and the cocoon layer rate was significantly reduced by 2.03-5.74 percentage points.This indicates that knockout of the BmRTN3L gene results in blocked silk protein synthesis.In conclusion,we screened a large class of gradient expression genes involved in silk protein synthesis and secretion using transcriptome sequencing technology,and explored the factors affecting silk protein synthesis and secretion by regulating silk gland pH and candidate gradient expression genes.This study will provide new ideas for elucidating the mechanism of silk protein synthesis and secretion and improving silk protein yield.