Research On The Correlation Of Cocoon Silk Trait And The Part Of Genes Related With Silk Production In Silkworm

Silkworm is one of lepidoptera model insects with high economic value,.As the main benefit product of sericultures,cocoon production is the main determinant of yield and quality benefit in sericulture industry.Therefore,it is very important to make full understanding and research of the application of silk quality in traditional and molecular breeding.A large number of silk proteins are synthesized in the silk gland during the fifth instar larvae.However,there is a big variation in the yield of silk in different silkworm strains.The study of regulatory mechanism of silk production in silkworm,especially some important regulatory factors function,will be useful for the increase of silk yield and construct of silkworm bioreactor.In recent years,with the development of high-throughput sequencing technology,the study of genomics and functional genomics has been promoted.With apparent difference of silk yield,Jingsong?JS?and Lan 10?L10?are research targets in this study.Analyzing of RNA-Seq data of the silk gland of the two strains,and combining with the information of QTL mapping,we studied the regulatory functions of some genes related to the silk yield.1.We constructed an F₂ generation group by using JS and L10 strains,statistically surveyed the cocoon quality traits of F₂ group.The results showed that the characters of cocoon layer and total cocoon in the 1179 F₂ were obviously separated.The distribution of these two cocoon traits showed a normal curve in male and female cocoons respectively.It accorded with the basic characteristics of continuous variation of quantitative trait,and the maximum and minimum of cocoon layer and total cocoon are similar to that of two parents respectively.Meanwhile,correlation analysis of four cocoon traits,including total cocoon quantity,cocoon layer quantity,pupa weight and cocoon layer rate of F₂ generation group were analyzed.The results showed that the correlation coefficients between these cocoon traits were very high,except that the correlation between cocoon layer rate and pupa weight was low.2.Trascriptome of silk gland of the 3^rd day 5^th instar larvae in JS and L10 had been sequencedand 1411 genes were differentially expressed between the two strains,including some of the transcription factors related to protein biosynthesis.Combining the screen of silkworm transcription factors and bioinformatics analysis,transcription factor E2F expression patterns in the five-age silk gland of the silkworm JS and L10 were analyzed.Additionally,the upstream regulatory gene of E2F,Ras1 is invloved in the cell cycle signal pathway,and we analyzed the expression profile of the gene in the silk gland of silkworm,JS and L10.the expression of transcription factor E2F and the expression of Ras1 in Js and L10 were analyzed based on the expression value data obtained from qRT-PCR,.3.Proto-oncogene Abl encoding tyrosine kinase is ubiquitous in nucleus and cytoplasm,and participates in a variety of signal transduction pathways,which play an important role in cell proliferation and cell cycle.In silkworm,Abl gene located on 1^st chromosome,activates Ras1 gene in the cell cycle signaling pathway.The expression of Abl gene in the silk gland of JS is higher than that of L10.Abl gene was knocked out by using the CRISPR-Cas9system in silkworm.The transgenic double fluorescent individuals screened were measured the total cocoon quantity and cocoon layer compared with the wild type silkworm.The results showed that the total cocoon weight of transgenic mutants with double fluorescent was 11.54%lower than that of wild type,and the cocoon layer was 25%lower than that of wild type.These results indicate that the Abl gene has a certain effect on the cocoon production in silkworm.In this paper,we screened the transcription factor E2F,the silkworm proto-oncogene Ras1 and proto-oncogene tyrosine kinase Abl by analysis the differentially expressed genes obtained based on the early transcription group sequencing combining with bioinformatics analysis and related literature knowledge.We have a quantitative analysis of these three candidate genes to determine the expression pattern of five-age silk gland in silkworm.At the same time,we analyze the upstream-downstream relationship between the three genes in the cell cycle signaling pathway,and speculate that the Abl gene is in the upper reaches of the other two genes.We obtained Abl Transgenic silkworm strains with double fluorescence by use CRISPR/Cas9 technology to perform functional validation of candidate Gene Abl.According to phenotypic analysis and genotype analysis,we can determine that the Abl gene has a certain effect on the function of silk gland in silkworm.This study provides an insight into the molecular mechanism of silkworm silk production,and it also plays a guiding role in selecting and cultivating multiple silk varieties.