Study On Positional Cloning And Molecular Mechanism Of The Fourth Brown Egg(b-4) In The Silkworm,Bombyx Mori

The diversity of insect egg color in nature is closely related to its environmental adaptability.It is an important clue,provided by exploring the biochemical and genetic basis of insect egg color formation,to reveal the evolution of insect appearance color.The appearance color of organisms can be divided into pigment colors and structural colors.Pigment material is the substance basis of insect pigment color,and the change in its type,content,and spatial positioning may produce a variety of appearance colors.Therefore,understanding the pigment substances involved in egg color formation,their synthesis and metabolism processes in the body,and how these substances are transported and deposited to specific locations is an important basic research to reveal the formation of insect pigment colors and their diversity.As a famous economic insect and an important model organism of Lepidoptera,Bombyx mori not only has a deep genetic research foundation and high-quality reference genome,but also has a wealth of egg color mutants stored in its mutant library,which is provided convenience to study the mechanism of insect egg color formation.The fourth brown egg（b-4）is a mutant of silkworm which has reddish-brown serosa and dark red compound eyes in adult and this mutant of egg color formed naturally.The site of b-4 is located in the 20th chromosome by classical genetic studies.In this study,the molecular mechanism of insect egg color formation was initially explored and its controlling genes were identified through the positional cloning method by using the b-4 mutant of silkworm as research material.The research results obtained are as follows:1.Mapping the b-4 locus to the 310kb genomic region of silkworm chromosome 20According to the genetic law of complete linkage of silkworm females,the molecular positioning was performed by preparation segregation population of the BC₁M generation.The 13 polymorphic molecular markers located on chromosome 20were used to perform genotyping experiments on the DNA of 985 red compound eye individuals from the BC₁M generation,finally,the b-4 was locked to the 310kb genome range of chromosome 20,which contained 14 coding genes.2.The candidate genes of b-4 are locked into Bm-tmem68 and Bm-mah through expression analysis,sequence analysis and functional annotationThe expression patterns of 14 genes in the mapping interval during the egg color formation of Dazao and b-4 mutants were investigated,and it was found that the expression levels of KWMTBOMO12114,KWMTBOMO12115,KWMTBOMO12116,KWMTBOMO12118 and KWMTBOMO12119 in the two strains are significantly different during the key period of egg color formation within 24h-48h after spawning;At the same time,the coding sequence of 14 genes was cloned and analyzed,and it was found that the two genes of KWMTBOMO12116 and KWMTBOMO12119 had different splicing forms of transcripts in Dazao and b-4,and abnormal transcripts would lead to defects in the formation of the encoded protein structure.Based on expression analysis and sequence analysis,the candidate genes of b-4 were locked into KWMTBOMO12116 and KWMTBOMO12119.Annotate the functions of two genes,KWMTBOMO12116 is homologous to Tmem68 gene in the rat and encodes a transmembrane protein;KWMTBOMO12119 is homologous to mah gene in the Drosophila and encodes an amino acid transporter.In Drosophila,the loss of this gene function leads to the formation of compound eye phenotypes defect.3.The upstream of the Bm-mah gene has specific sequence differences in the b-4 mutantThe four single-base substitutions and four-base deletions at 500 bp upstream of the transcription start site of the gene in b-4 was found by investigating regulatory sequence of the Bm-mah genein Dazao and b-4 mutants,then,the sequence of this locus was investigated among 22 silkworm strains with different egg colors,and the results showed that the base substitutions and deletions in the upstream regulatory sequence of the Bm-mah gene are unique in b-4 mutant.4.The red-brown egg appeared in Dazao by RNAi with Bm-mah geneIn order to identify the control genes of the b-4 mutant,we performed RNAi experiments.The result showed that only knocking down the Bm-mah,the egg color turned red,other genes don’t,Therefore,we determined that the Bm-mah gene is control gene for the b-4.5.Bm-mah gene and Bm-re gene may joinly regulate the color formation of silkworm eggThe expression of Bm-KMO,Bm-white,Bm-scarlet,Bm-cardinal and Bm-re in the eye pigment synthesis pathway was investigated in the dazao after the interference gene of mah,the results showed that the expression of red egg control gene Bm-re was significantly reduced.We further investigated the expression of Bm-mah and Bm-re in Dazao,b-4 and re,found that the expression patterns of Bm-mah and Bm-re genes were similar,the expression levels of two genes were significantly lower in b-4 and re mutants during the critical period of egg color formation.In summary,this study has gradually locked the candidate gene of b-4 through molecular location,temporal expression patter and expression difference analysis,and sequence analysis of genes,and conducted functional research on the candidate gene through RNAi,finally,proved that Bm-mah is the control gene of b-4,which encodes an amino acid transporter with 11 transmembrane domains,is a new gene in insect ommochromes metabolism pathway.This study provides important clues to further explore the key steps downstream of insect eye pigment synthesis pathway.