Studies On The Function Of Cocoon Quality Traits Related MiRNAs And Abl Genes Of Bombyx Mori

Bombyx mori is an important economic insect,which has developed silk glands and strong ability to secrete silk protein.Silk has important economic value for the benefit plays a leading role in the economic benefit of sericulture industry.Therefore,it is of great significance to fully understand and study the biological mechanism of cocoon silk synthesis for breeding excellent silkworm varieties and improving cocoon production efficiency.There is a gap with different silkworm strains in the ability of silk gland to synthesize silk protein is different,which leads to significant differences in silk production.Nd and Nd-s,as the mutants of silkworms secreting fibroin,are naked pupae and sericin cocoons respectively.Micro RNA(mi RNA)plays an important role in many biological processes,such as cell proliferation,differentiation and apoptosis.Taking the Dazao strain as a control,the mi RNA expression profiles in u02(nd)and u05(Nd-s)silk glands were studied to reveal the potential function of mi RNA in the expression of silk protein and the development of silk glands.The mi RNA libraries of the three strains were sequenced.260,236,233known mi RNAs were identified from Dazao,u02,u05,respectively.53 mi RNAs were differentially expressed between Dazao and u02,51 mi RNAs were differentially expressed between Dazao and u05,while 16 mi RNAs were differentially expressed between u02 and u05.Go/KEGG analysis showed that most of the predicted target genes of these mi RNAs expressing differentially were related to cell proliferation,organ development and cell regional structure.The mi RNA expression profile of naked pupa will provide a new way to understand the development of silk gland and the regulation of silk protein expression.On the basis of QTL mapping of cocoon silk characters in the early stage of the laboratory,it was found that Abl was located near the QTL region of the first chromosome.Abl gene,a highly conserved proto-oncogene encoding tyrosine kinase,participates in a variety of cellular signal transduction pathways and plays an important role in cell proliferation and cell cycle.Using CRISPR/cas9technology,we obtained the mutant Abl~-,whose Abl was knocked out,and investigated the cocoon quantity and cocoon layer quantity between the mutant and the wild-type.It was found that there were obvious differences between the both.In Abl~-females,the whole cocoon quantity and cocoon layer quantity decreased by 14.29%and 15.38%,while in males,the both quantities decreased by 12.77%and23.08%respectively.After the silkworm was treated with GNF5,compared with the silkworm fed with mulberry leaves,the cocoon layer decreased by more than 10%.The above results indicated that Abl had a great influence on the cocoon quality.The expression of fibroin genes in Abl~-and wild-type varieties was compared by q RT-PCR,the expression of Fib-L and Ser-1 in the Abl~-is decreased obviously.In order to further study the regulatory mechanism of Abl in cocoon quality traits,transcriptome sequencing of silk glands of wild-type individuals and Abl individuals had been done,revealed 492 up-regulated genes and 683 down-regulated genes in Abl mutants.Go analysis showed that the differential genes were mainly enriched in the items of catalytic process and metabolic process.Compared with the control,CSP4 and JHEH2 were significantly up-regulated.The former affected the concentration of ecdysone in silkworm,while the higher concentration of ecdysone would lead to the premature occurrence of autophagy in silk gland cells,which led to the decrease of silk production to a certain extent;the latter promoted the degradation of juvenile hormone,inhibited the synthesis and secretion of silk protein,and led to the decrease of silk production.The expression of Dgt6 involved in the development regulation process also increased significantly,which may make up for the influence of Abl deficiency on the development process of silkworm to some extent,so that there is no significant difference in the individual growth and development cycle between the mutant silkworm and the wild type silkworm.In addition,the loss of Abl results in a significant decrease in the expression of Ras1 gene,which further proves that Abl plays a regulatory role in the signal transduction pathway of cell cycle.