Analysis And Identification Of Ng Candidate Genes Of Silkworm,Bombyx Mori Using BSA-seq And RNA-seq

Silkworm,Bombyx mori is a model insect of the second largest order lepidoptera with important economic and scientific benefits.Its collterial gland can secrete and store highly adhesive substances that can fix and protect silkworm eggs.The character of no glue egg is mainly due to mutations in the collterial glands,which secrete less or even unable to secrete viscous substances.In this study,H9,which produced no glue eggs and P50,which produced glued eggs,were used as parents,and offspring with extreme traits similar to the parents were selected to establish a mixed pool for BSA whole-genome resequencing.The BSA resequencing was correlated with H9 and P50 colleterial gland transcriptome sequencing.And biological analysis of differentially expressed genes was investigated.The results are as follows:1.The BSA resequencing of the silkworm strains H9 and P50 and their progeny was performed,a total of 4 candidate regions related to Ng trait through BSA positioning,with a total length of 17.68 Mb,and a total of genes annotated in the related regions 437 based on the intersection of the associated regions obtained by SNP and In Del.2.GO annotation analysis of the differentially expressed genes: According to the significance KS value,the differentially expressed genes corresponding to the top 10 functions with the most significant enrichment were selected.The differential genes mainly concentrated in synaptic vesicles,transmembrane transport,synaptic proteins,etc,and identified a large number of chitin binding and cuticle structural;KEGG pathway annotation analysis: According to the significance Q value,the 10 most significant enrichment pathways were selected.Differential genes mainly related to lipid and sugar metabolism,and related to cell phagocytosis;COG annotation analysis: indicates that these differential genes may be related to replication,recombination,repair and signal transduction.3.In this study,20 differential genes were selected from the candidate genes that functionally annotated of BSA resequencing,and 17 differential genes were selected from the genes obtained of resequencing and transcriptome association analysis.And The expression of these genes in the H9 and P50 collterial glands of the silkworm was detected,and 10 genes were selected to detect their expression in different developmental stages.The results showed that the expression levels of most genes in the H9 and P50 collterial gland were different,and in different developmental stages of silkworm,the expression levels of some genes showed an overall upward trend in the fifth instar,pupa and moth stages of silkworm development.4.Two related candidate genes of YKT6 and STX16 were initially identified,cloned,expressed,and prepared polyclonal antibodies.The expression pattern of target genes in the3 rd day 5th instar tissues and different developmental stages showed that they were expressed in all tissues and ages,but the expression levels were different.The relative expression level of the target gene in H9 and P50,as well as the different development stage of collterial gland was consistent with the expression of protein levels,but the expression levels were also different.By BSA resequencing,BSA and transcriptome association analysis and screening the correlative genes related to Ng trait,the basis for further study on the development and secretion of collterial gland were provided.