Molecular Mechanism Of BmAbl1 On Cocoon Characters Of Bombyx Mori

Bombyx mori is an economic insect that feeds on mulberry leaves and produces silk.Silk yield is one of the important indicators of efforts to improve during the last 5000 years of domestication.In our previous studiy,we used SSR and SNP markers to accurately map the quantitative trait loci(QTLs)of silkworm chromosomes related to cocoon silk,and screened the potential genes with great influence on cocoon silk traits.In previous studies of potential genes affecting silk traits,a nonreceptor tyrosine kinase gene,BmAbl1,located on chromosome 1 was identified.By knocking out BmAbl1,the size of individual silkworm was decreased.The total cocoon weight and cocoon layer mass of female and male were reduced by 14.29% and 12.77%,15.38% and 23.08%,respectively.The silk gland transcriptome results of BmAbl1 knockout strain showed that the glutathione metabolic pathway,amino acid synthesis,and metabolic pathway were significantly affected in BmAbl1 knockout strain.However,the molecular mechanism by which BmAbl1 affects silk fibroin production and amino acid synthesis through the glutathione metabolism pathway is still unclear and needs further study.We investigated whether BmAbl1 deficiency has an effect on glutathione metabolism.Further analysis of KEGG enrichment of the previous transcriptional data revealed that BmAbl1 knocked down the gene encoding glutathione transferase(BMSK0003439,BMSK0003598,BMSK0003984,BMSK00012237,EC2.5.1.18;BMSK0006405,EC1.5.4.1)and gene encoding corydalis acetoacetate hydrolase in Tyrosine metabolism(BMSK0001189,EC4.1.1.25)were significantly down-regulated,so these genes could be used as candidate genes for subsequent studies.Fluorescence quantitative PCR of these candidate genes revealed significant changes in glutathione metabolism and tyrosine metabolism pathway in BmAbl1 knockout strains.The results were basically consistent with the transcriptome sequencing data,indicating that glutathione metabolism and tyrosine metabolism pathway were indeed different from the Control strain.To further verify the effect of glutathione transferase decline on glutathione metabolism,we compared the glutathione content in the posterior silk glands of BmAbl1 knockout strain and Control strain on the fifth and third days.The results showed that the glutathione content in the posterior silk glands of BmAbl1 knockout strain was abnormal,and the content was 1.49 times that of the Control strain.The results showed that glutathione could not be effectively decomposed into glutamic acid,cysteine,and glycine in BmAbl1 knockout strains.To examine how the deletion of BmAbl1 affects the synthesis and production of silk protein,we detected the expression of silk protein gene and the content of silk protein in the posterior silk glands of BmAbl1 knockout strains and Control strain during the fifth age from the transcriptional and protein levels.The transcription levels were detected and there was no significant difference in the expression of silk fibroin gene between BmAbl1 knockout strain and the Control strain.The results of Western-blot analysis showed that the content of light chain,heavy chain,and P25 in BmAbl1 knockout strain was significantly lower than that in the Control strain,suggesting that the effect of BmAbl1 deletion on silk protein production occurred at the stage of protein synthesis.Glutathione can be decomposed into glutamic acid,cysteine,and glycine,and glycine is the main amino acid component of silk fibroin,accounting for 38.04 % of all amino acids in silk fibroin,so the change of free glycine content will affect the synthesis and yield of silk fibroin.Combined with the results of the first part of the work,we speculate that the abnormal metabolism of glutathione in BmAbl1 knockout strains leads to the decrease of glycine content in free amino acids.Therefore,the amino acid analyzer was used to detect the content of free amino acid in the posterior silk glands of BmAbl1 knockout strains and found that the glycine abundance decreased by 22%,which confirmed our speculation above.The effect of BmAbl1 on silkworm cocoon silk yield was further verified,and the foundation was laid for breeding transgenic silkworm strains with high cocoon silk yield.We constructed transgenic silkworm strains overexpressing BmAbl1 based on piggy Bac transposition element.The results showed that,compared with the Control,the body weight of the fifth adult larvae of BmAbl1 overexpressed strain increased significantly,and the total cocoon weight,pupa weight,cocoon shell weight,and cocoon layer ratio increased significantly,especially the cocoon shell weight of male BmAbl1 overexpressed strain increased by 33%.The expression of Fibroin Heavy chain and Fibroin Light chain genes in the posterior silk gland of BmAbl1 overexpression strain was 3.3-fold and 1.3-fold higher than that of Control strain,respectively.These results indicated that overexpression of BmAbl1 could effectively promote silk protein production of bombyx mori.In summary,we explored the role of BmAbl1 in regulating silk protein synthesis and revealed the molecular mechanism of BmAbl1 affecting silk protein production.The results provided the theoretical basis and variety of resources for breeding silkworm strains with high cocoon silk yield.