BmPIK3CD Gene Function And Its Effect On Silk Gland Development In Bombyx Mori

Cocoon silk yield is the most important economic trait of silkworm,and obtaining high-yielding silkworm strains has always been an important goal for breeders.After thousands of years of folk selection and breeding and recent systematic breeding,cross breeding,etc.,the cocoon silk yield trait of the silkworm has been extremely significant improvement,such as the highest cocoon shell rate can reach 30%.However,traditional breeding to improve cocoon silk yield improvements have entered a plateau.The silk gland is the only silk-producing organ of the silkworm,and silk gland development plays a decisive role in cocoon yield.Gene editing has become a powerful tool for genetic improvement.It is necessary to use gene editing technology to genetically improve the silk gland,and then to breed new strains of silkworms with higher yield and better quality.In this study,we focused on the correlation between cancer-related genes and silk gland development and expected to screen key genes related to the regulation of silk gland development to provide targets for the creation of high-yielding strains.The main findings are as follows:1.Identification of human cancer-associated genes in the domestic silkwormThe literature analysis focused on 22 oncogenes that have been well studied in humans.By analyzing the expression levels of 22 oncogenes in the posterior silk glands of three high and low silk yield domestic silkworm strains each,differential genes were screened and four significantly differentially expressed genes were targeted:BGIBMGA007238(MAP2K4),BGIBMGA002921(PIK3CD),BGIBMGA011784(RECQL4),and BGIBMGA010110(SMAD4).To determine their association with silk gland development,the genomic variant loci of these four genes in eight low yield and eight high yield strains were analyzed,and the genomic region of BGIBMGA002921(PIK3CD)was found to have dense cocoon silk yield-related variant loci between high and low yield strains,and these variant loci resulted in amino acid sequence,functional domain The association analysis showed that five Indels in the genomic region were significantly associated with cocoon shell quantity.It is hypothesized that BGIBMGA002921(PIK3CD)may be associated with silk gland development and will be named as BmPIK3 CD.2.Effect of BmPIK3 CD gene on silk gland developmentCRISPR/Cas9 knockdown of the BmPIK3 CD gene showed that BmPIK3 CD knockdown was embryonic lethal in pure heterozygotes,but promoted the development of silk glands in the heterozygous state,causing larger silk quantity and significant increases in cocoon shell quantity,whole cocoon quantity,and nymphal weight.Comparative analysis with the control strain showed that the whole cocoon quantity increased by 6.4%,cocoon shell quantity increased by 27.8%,nymphal weight increased by 5.7%,and cocoon shell rate increased by 20.8% in females,while the whole cocoon quantity increased by 7.4%,cocoon shell quantity increased by 8.3%,nymphal weight increased by 9%,and cocoon shell rate increased by 1.7% in males.It was observed that the cocoon size and nymphal body size of G2 generation heterozygous individuals were also significantly larger compared to the control group.The above functional study proved that BGIBMGA002921(PIK3CD)has an important effect on silk gland development.3.A preliminary investigation on the molecular mechanism of BmPIK3 CD affecting the growth and proliferation of silk gland cellsTo investigate the reason why BmPIK3 CD promotes silk gland development in the heterozygous state,we counted the number of silk gland cells in the embryos of BmPIK3 CD knockout heterozygous individuals and non-knockout individuals,and investigated the expression of silk protein gene.The results showed that the number of silk gland cells during the embryonic period was significantly increased in BmPIK3 CD knockout heterozygous individuals compared to the control,with an average silk gland cell number of 477 in the control and 559 in the heterozygous individuals,an elevation of 17.2%.Investigation of gene expression levels showed that the expression level of BmPIK3 CD was significantly increased in both embryonic and larval stages of heterozygous individuals compared to the control group,indicating that the gene transcription was activated in the heterozygous state,which in turn led to an abnormal increase in its transcription level.The expression levels of the filamentous heavy chain gene(Fib-H),filamentous light chain gene(Fib-L),filamentous P25 protein gene,and filamentous glands gene(Ser1)were investigated during the embryonic and larval stages and were all significantly increased compared to the control.Since the development of embryonic filamentous glands mainly involves mitosis of filamentous gland cells,the expression of cell cycle protein family genes and key regulatory genes of cell growth in embryonic and larval stages were investigated.The results showed that the expression of AKT and m TOR were significantly higher in knockout heterozygous individuals than in the control,and the expression of Cy CD was also significantly higher in knockout heterozygous individuals,and Cy CB and Cy CB3 were up-regulated to different degrees.The expression of AKT,m TOR,and Ras1 were significantly higher in knockout heterozygotes than in the control,and the expression of Cy CD and Cy CE were also significantly higher in knockout heterozygotes than in the control,and the expression of Rps6 was significantly higher in knockout heterozygotes than in the control.Therefore,BmPIK3 CD may regulate the development of filamentous glands through the cell cycle pathway and up-regulate the expression of key regulatory genes for downstream cell growth,which in turn promotes the development of filamentous glands.In summary,this study targeted the candidate gene BmPIK3 CD related to silk gland development by differential expression analysis and association analysis between variant loci and cocoon silk yield,and performed functional studies on the candidate gene by CRISPR/Cas9 technology to demonstrate that the BmPIK3 CD gene could promote silk gland development in the knockout heterozygous state.The gene was aberrantly activated in the heterozygous state,resulting in upregulation of expression of key genes for cell growth and cycle regulation,which in turn led to an increase in the number of silk gland cells at the embryonic stage and,by further upregulating the expression of genes related to silk protein synthesis,eventually led to an increase in silk gland size and silk yield.This study provides a basis for further analysis of the molecular mechanism of silk gland development and also provides a new target for the creation of high-yielding strains of silkworm.