Fine Mapping And Functional Analysis Of Candidate Genes Of The Pale Red Egg Mutant(re~p-1) Of Bombyx Mori

The silkworm（Bombyx mori）is an important economic insect.It is s an ideal model organism for lepidopteran insect research with a short growth cycle and high reproduction rate.Normal silkworm eggs are yellowish white immediately after oviposition and eventually turn into dark brown.Our research team previously found a new type of pale red egg mutant（pale red egg,re^p-1）,which eggs exhibite a yellowish-white at begging and become brown around 36 hours.In this study,the re^p-1 mutant was taken as material to searh genes related to re^p-1mutant and analyze the structure and function of the candidate genes,by map-based cloning,to obtain 3′UTR of the candidate genes within the mapping interval by 3′RACE technology,to characterize the function of the candidate genes through RNA interference（RNAi）and CRISPR/Cas gene editing techniques combining with transcriptome sequencing（RNA-seq）of eggs 36h after oviposition of re^p-1 mutant and its wild-type silkworm C1（H）.Our research provided the possibility of elucidating the molecular formation mechanism of re^p-1 mutant.The main research contents are as follows:1.Fine positioning of re^p-1mutant genes.The P₁（p50）and P₂（re^p-1）was used as parents to construct F₁,F₂,BC₁M and BC₁F populations,in which BC₁F populations are used for linkage analysis.By using SSR polymorphic molecular markers,the major gene was located in the fifth linkage group of the silkworm,B.mori with a fine location between the molecular marker S2674-N53 and S2674-N4.The genetic distance between the two markers is 0.23c M and the physical distance is 88kb.Based on the Silk DB genome database query,there are three candidate genes in the locating interval,including BGIBMGA003693,BGIBMGA003500 and BGIBMGA003501.2.Structure analysis of candidate genes of the re^p-1 mutant.The structure of candidate genes was analyzed by CDS sequencing.Comparing with the wild type,the CDS of the above three candidate genes showed no changes in the mutant;Differences in the size of the electrophoretic fragment of the seventh pair of primers were observed through electrophoresis detecting of the genomic sequence between S2674-N53 and S2674-N4.The cloning and sequencing revealed that there was a 52bp deletion occurred between the candidate genes BGIBMGA003501 and BGIBMGA003693,both of which with coding regions locating on the complementary strands.Analyze indicated that the deleted fragments were located at the 3’terminal of the two genes so that the candidate gene BGIBMGA003500 was initially excluded.The 3′UTR of BGIBMGA003693 was obtained by 3′RACE amplification.However,the 3′UTR of BGIBMGA003501 was finally been cloned after repeating several times.It is speculated that the actual structure of BGIBMGA003501 may be different from the predicted one.The major genes responsible for the mutant traits of the re^p-1mutant need further study and confirmation.3.Expression analysis of candidate genes of the re^p-1 mutant.Combining the results of map-based cloning,two candidate genes BGIBMGA003501 and BGIBMGA003693 were obtained.Then their expression levels were analyzed by q RT-PCR technology and C1（H）served as the control.The results showed that in the egg stage of re^p-1 mutant the expression level of BGIBMGA003693 increased first and then decreased later within 24 to 96 h after oviposition.The expression level of C1（H）reached the peak at 48 hours,while in the mutant re^p-1 BGIBMGA003693expression reached the highest expression level at 36 h.The gene expression profile of BGIBMGA003501 showed the same tendency as BGIBMGA003693.The level of wild type and mutant reached the peak 48h after oviposition.The expression level of early mutant was significantly higher than that of wild mutant.The expression profile of BGIBMGA003693 in tissue indicated that there are differently expression genes in most tissues between wild type and mutant.While there are no differences in gene expression level between male and female.In the tissues with different expression levels,the differences of expression level between male and female individuals are opposite.Overall,the expression levels are higher in the Markov tube and midgut.The gene expression profile of BGIBMGA003501 in tissue showed that there are same differences in many tissues of male and female.The overall expression level of BGIBMGA003501 is similar to BGIBMGA003693 that there are higher levels in the Martensitic tube and midgut.4.RNAi analysis of candidate genes of the re^p-1 mutant and gene editing by CRISPR/Cas9.RNAi analysis was performed to the two candidate genes by injection of synthetic si RNA into the wild-type eggs within 2 h after oviposition.The results showed that the control group injected with EGFP si RNA revealedno change after injection,and the phenotype of the group injected with BGIBMGA003693 gene si RNA also showed no visible change.The egg color of the group injected with BGIBMGA003501 gene si RNA was changed into a pale red compared with the control group egg phenotype.The expression levels of BGIBMGA003501 in the pale red eggs resulted from RNAi were significantly lower than those of the control group.The gene knockout analysis of the candidate genes BGIBMGA003501 and BGIBMGA003693 was performed by CRISPR/Cas9 editing technology.Two positive individuals were obtained by knocking out the BGIBMGA003501 gene,both were heterozygous after genotype identification.And they will be hybridized in G2generation to obtain homozygous mutants for verifying the function of BGIBMGA003501.While the BGIBMGA003693 gene knockout failed.5.RNA-seq of of eggs from the re^p-1 mutant and wild-type C1（H）of 36h after oviposition.After analysis of The RNA-seq results,totally 800 differentially expressed genes（DEGs）were identified,of which 475 genes were down-regulated and 325 genes were up-regulated in the re^p-1 mutant compared to wild-type.The results revealed a majority of DEGs distributed in the growth and development,and pigment synthesis and metabolism pathways.In a word,our research has laid a foundation for elucidating the formation mechanism of pale red egg mutant re^p-1.