Functional Study Of Bmel-1/-2 Gene Encoding RNA-binding Protein In Bombyx Mori

RNA binding protein(RBP)plays an important role in transcription,post-transcription and translation level regulation of multi-cell biological genes.The ELAV/Hu family is a type of RNA binding protein family containing RRM domains.This protein family has conserved neural development regulation function in multiple species while evolving different functions in different species.In the post-genome era,the functional study of the silkworm BmELAV-like protein family has also been rarely reported.This study focuses on the Bmelav-Like-1 and Bmelav-like-2 genes in silkworms,and preliminarily explore the biological functions in silkworms.The main research contents and results are as follows:1.Our research group used CRISPR/Cas9 genomic editing technology to obtain Bmel-1~-and Bmel-2~-mutants of Bombyx mori.In this study,the phenotypic analysis of the two mutants was firstly conducted.The results showed that the body weight of the two mutants in the larval stage and the pupal stage were down-regulated by 5.9%-31.7%and 13.0%-28.0%respectively,and compared with the control group,and the weight loss of the Bmel-2~-mutant was greater than that of the Bmel-1~-mutant.The cocoon layer ratio of Bmel-1~-and Bmel-2~-mutants was increased by 1.4%-1.3%,0.8%-0.4%,respectively.The expression of four silk protein genes,including Fib-H,Fib-L,P25 and Ser-1,were up-regulated by 1.2-6.8 times.In addition,the oviposition quantity of Bmel-2~-mutant female silkworm decreased by 19.8%.These results indicated that Bmel-1 and Bmel-2 genes were involved in the growth and development of silkworm and the regulation of silk protein expression.2.To further analyze the regulatory functions of Bmel-1 and Bmel-2 genes in the growth and development of silkworm,transcriptome sequencing of the second instar larval silkworm of Bmel-1~-and Bmel-2~-mutants was performed,and metabolome analysis of the fat body of the mutants on the third day of the fifth instar was performed.According to the surface type of the mutants,combined with transcriptome and metabonomics analysis,it was speculated that the regulation of fatty acid metabolism pathway in the two mutants was the key reason leading to the inhibition of their development.The q PCR results showed that the expression level of fatty acid synthase FAS gene,a key enzyme in fatty acid metabolism,was significantly down-regulated in both mutants,especially in the Bmel-2~-mutant.At the same time,the key catalytic genes in the fatty acidβ-oxidation pathway were also inhibited to different degrees.Metabolomics data showed that the contents of diacylglycerol and phosphatidic acid,the upstream raw materials of fat synthesis,were significantly decreased in the adipose bodies of the two mutants,while the glycoside metabolites and amino acid metabolites were increased.The above data results revealed that the loss of fat synthesis and accumulation in the two mutant silkworms affected the overall energy metabolism,and ultimately led to the inhibition of the growth and development of the mutant silkworm.3.In the endoplasmic reticulum of the two mutant silkworm silk gland cells,the expression of protein folding auxiliary protein genes OST,UGGT,and PDIs were up-regulated to varying degrees,and the endoplasmic reticulum stress response factor ATF6 gene was also significantly up-regulated.It is speculated that the changes of these genes are due to the increase of protein synthesis in the endoplasmic reticulum.However,in BmN cells with overexpression of Bmel-1 and Bmel-2,The expression of these genes was significantly down-regulated.These results suggest that Bmel-1 and Bmel-2 genes play important regulatory roles in protein processing.4.Finally,Bmel-1 and Bmel-2 overexpressed silkworm strains were successfully constructed by using the transgenic technology of silkworm,which provided the basis for further study on the regulation of Bmelav-like gene in the development,protein synthesis and new biological functions of silkworm.