Screening And Analysis Of Genes Related With The Characters Of Cocoon And Silk Production In Silkworm

Bombyx mori Cocoon and silk yields are the most important characteristics of sericulture.A silkworm with high silk yield over 0.5g,while many silkworm strains is less than 0.1g.Although techno-positioning can find the genetic loci of large fragment which Location Dependent(LOD)values are relatively high,transcriptome analysis may suitably detect minor genes by RNA-Seq technology.The adventage of high-throughput sequencing(HTS)technologies promote the study of genome.In this study,we used RNA-Seq and quantitative polymerase chain reaction(qPCR)were performed on two silkworm strains with different silk yieldsin to determine divergence of the silk gland,which controls silk biosynthesis in silkworms.Our results will provide insights into the molecular mechanisms of silk production and improving cocoon silk yield.RNA-seq will help to discover the key functional gene that associated with silk and cocoon production.1.Silk gland is the most important tissues for silk production.JingSong(JS)and Lan10(L10)are two strains having significantly different cocoon and silk yields,The silk yield of JS is about five-fold higher than that of L10,but the rate of sericin of L10 is more than JS.Comparative transcriptome analysis reveals different silk yields of two silkworm strains.JS exhibited 1411 differentially expressed genes(DEGs;738 upregulated genes and 673 downregulated genes).Nine enriched gene ontology(GO)terms were identified by GO enrichment analysis based on these DEGs.KEGG enrichment analysis results showed that the DEGs were enriched in three pathways,which were mainly associated with the processing and biosynthesis of proteins.The representative genes in the enrichment pathways and ten significant DEGs were further verified by qPCR,consistent with the results of RNA-seq.RNA-seq will help to discover the key functional gene that associated with silk and cocoon production.2.Transcription factor is a kind of nucleli protein factor.Combine with cis element and have interaction with RNA polymerase to regulate transcription activity,and then affect the protein synthesis indirectly.With the results of the transcriptome sequencing analysis,we speculate that regulatory factors influencing these pathways may variant at the transcription level.Bioinformatics method was applied for screening the transcription factor trx and SGF1.We choose the trx and SGF1 and built the expression profiles,finally.3.trx gene complicated existed in the cell cycle and silence of telomerase,centromere and even associated with gene methylation.We speculated that the trx may be an important transcription factor have functions in inhibiting or silencing expression of silk protein gene.The expression level of trx in the L10 was higher than JS in the PSG,which may the reason that silk fibroin of L10 is less than the JS.Besides,we choose SGF1 as an important transcription factor,which control the silk protein genes,e.g.FibH,P25,Ser-1.So,we apply the CRISPR-Cas9 system to konckout the trx and SGF1 gene.We construct the plasmid pBac [IE1-DsRed2-U6-sgRNA],through microinjection method to injected them into silkworm eggs.Until the G1 generation,get the positive individuals,breeding to G2 generation,then crossbreed with Cas9 strains,offspring was detected by fluorescence microscope to get double fluorescent transgenic silkworm strains which target genes were knocked out.Compared the cocoon shell weight between transgenic and wild silkworm strains,we found there has no obvious difference.4.When we compared the different expression transcription factors,we found some other silk protein related genes.Studies have found that these genes(e.g.FibH,P25,Ser-1)may be located in the downstream pathway of SGF1.In order to gain an insight into the expression of silk protein gene in spatial and temporal,qRT-PCR was used to detect the expression level of six silk protein genes(FibH,Fib-L,P25,Ser1,Ser2,Ser3)in the fifth instar of L10 amd JS.In the same strain,the expression level pattern of FibH,Fib-L and P25 were similar,they all exhibited high expession of JS than L10,and sericins' expressions are also exhibited high expession in JS than L10.Then we compared the sericin rate of Ser1/FibH.This result was in accord with our experienment that the rate of sericins was higher in L10 than JS after weight each boiled and dried cocoon.Although the cocoon weight of JS is higher than L10,while the rate of sericins of L10 was 1.2~1.3 times higher than JS.The results of RNA-Seq not only provided the main KEGG pathways that related to silk production,but aslo offered big data of DEGs in silk gland.We have a general understand of silk and prodution genes of KEGG pathways,which palys a fundamental role in molecular mechnism of silk yield genes.Which has an important influence of breeding high-yielding varieties silkworm,and has a guiding significance of silk gland bioreactor.