Identification Of Odd Gene Family And Exploration Of The Mechanism For Apodal(ap)mutation In Silkworm

Pest control has a great significance in agriculture and forestry production.Lepidoptera is the major group of agriculture and forestry pests,thus attracting widespread attention.The silkworm B.mori,as the representative of Lepidoptera,occupies an important position in the studies of insects.Moreover,B.mori has significant advantages in the study of the genetic development of insects because of its rich genetic resources,various morphology and clear background.Among the genetic mutant resources of silkworm,ap mutant presents composited mutation characteristics such as loss of thoracic legs and defect of female reproductive system,etc.Therefore it is a valuable material to investigate the appendage development and the formation of reproductive system of insect.In addition,Researches on the development of thoracic legs and the female reproductive system can provide a new theoretical support for the Lepidoptera pest control.In previous studies,the ap mutant has been mapped on the region between nscaf2930 and nscaf2924 of the third chromosome and the possible candidate gene Bmsob and its downstream genes were also preliminarily studied.Bmsob is one member of the odd family.Genes in this family play important role in the regulation of the development of insect legs and external genitalia.However,identification and functional research on odd gene family in silkworm remains elusive.In this research,to screen and analyze the candidate genes and possible downstream genes for ap mutant,we performed bioinformatics analysis,quantitative Real-time PCR and hybridization in situ by using ap mutant and wildtype Dazao as the metarial.Furthermore,the candidate gene function was also analyzed by RNAi and transgenic knock-out to contribute the understanding of the formation mechanism of ap mutation in silkworm,and provide a new target for biological control of pests.The main results of this study are as following.1.Analysis on the characteristics of odd gene family in silkwormDistribution of the odd family genes in the genome of silkworm.Through bioinformatics analysis,we filled the gap area between nscaf2930 and nscaf2924 on the third chromosome and found four odd family genes in this region which distributed serially.What is more,Bmsob kept a relatively close distance from Bmdrm and Bmodd,107.5 KB and 107.7 KB respectively.However,Bmbowl was far apart from other family genes.Synteny analysis on the odd family genes in six species insects genomes,including silkworm,showed that the distribution of odd family genes on the insect genome was highly conserved with only a reverse arientation of bowl in Drosophila compared with its counterparts in other insects.These indicated that genes in this family may evolve from the same sequence.Analysis on protein structure and evolution of silkworm odd family genes.It is reported that ORF of Bmsob was 1311 bp and encoded 436 amino acids.In this research,we cloned and analyzed ORF of other odd family genes.The results showed that the length of Bmdrm,Bmodd and Bmbowl ORF was 243 bp,1044bp and 2172 bp respectively.And they encoded 80,347 and 723 amino acids respectively.Protein structure prediction showed that all of these proteins were C2H2 type zinc finger proteins.The proteins of BmDrm,BmOdd,BmSob and BmBowl contained two,four,five and five C2H2 type zinc finger motifs respectively.By predicting the protein secondary structure of silkworm odd family genes,we found that their secondary structures mainly included random coil,the alpha helix and beta fold while nearly no other structures.Meanwhile,homology analysis showed that odd family genes were highly conservative in insects.Thus,we speculated that functions of odd family genes may also so conserve in insects.Expression pattern of odd family genes.QRT-PCR results showed that odd family genes had a similar tissues expression patterns,mainly in head,wing disc,malpighian tube and ovary even with a varied expression level.Temporal expression analysis showed that the expression pattern of them had a remarkbale similarity with a high expression in the moth.Besides that,the highest expression of Bmdrm,Bmsob and Bmbowl occurred in early embryonic stage,and the trend of their expression were similar.Nevertheless,Bmodd had an expression peak both in embryonic early and late stage.These results indicated that the Bmodd genes may have an association with the development of silkworm embyo and female reproductive system.2.Exploration of the cause for ap mutantBy identification of the odd family genes in silkworm and analysis on the mapping region,another odd family gene,Bmodd,except for Bmsob,was also found in the ap locus.We dissected silkworm embryo during the whole embryonic period and found that during the period from No.2.5 day to 4.5 day was the occurrence stage of thoracic legs and the stage from No.2.5 to 3.5 day was the key period for thoracic legs development.At the same time,according to the female reproductive system continuous anatomical observation,period from No.1 day to No.3 day of pupa was a key stage of the metamorphosis development of female reproductive system,which also was the significant period of mating capsule formation.We determined the expression level of Bmsob and Bmodd in the key developmental stages for thoracic legs occurrence and mating capsule formation and discovered that the Bmsob was expressed highly in the both periods and expression level of it is significant lower in embryo and the female reproductive system of ap mutant.While no expression difference of Bmodd was detected between wildtype and ap mutant.In situ hybridization results showed that Bmsob gene had a higher level expression in the thoracic and abdominal legs,egg chamber,mating capsule and ovipositor.Hence,we speculated that Bmsob gene may be the candidate gene for the deficiency of thoracic legs and mating capsule in ap mutant.Determination of the expression of the possible downstream genes for Bmsob showed a striking down-regulated expression of BmAntp and BmUbx gene during thoracic legs occurrence stage in ap mutant embryos and the expression trend of BmAntp gene was similar with Bmsob,which suggests that it may participate in silkworm thoracic legs development.Bmabd-A gene had a significant up-regulated expression in the mating capsule formation period of ap mutant female reproductive system and its expression tendency contrasted with Bmsob during pupa No.1 day to No.3 day.So we conclude that Bmabd-A may be one of downstream genes of Bmsob and it may take part in the regulation of mating capsule formation.In order to conform whether the down-regulation of Bmsob is able to cause the ap phenotype,we performed the double-strand RNA interference study aiming at Bmsob gene,and made a striking down-regulated expression of Bmsob.However,we did not observe thoracic legs deficiency.In order to further explore Bmsob gene function,we designed the Bmsob knock-out gRNA sites and constructed transgenic knock-out vectors.The knock-out activity of these gRNA construct were verified by transfecting in the BmN-SWU1 cell lines and the result showed that the site of sob-gRNA-1 has a better knock-out activity.Thus we constructed piggBac(A3-dsRed)-BmU6-sob-gRNA-1 recombinant plasmids and injected it into the new layed eggs of Dazao.We acquired sob-gRNA-1 site positive individuals in G1 generations.Afterwards,we crossed positive individuals with Cas9 transgenic line and positive individuals were screened.In the F2 generation of double positive cross,we obtained the individuals with the deficiency of thoracic legs.Genomic sequencing of these thoracic legs deficient individuals showed that these are the mosaic with Bmsob knock-out.Furthermore,these individuals also showed a significantly down-regulated Bmsob expression.Collectively,we studied odd family genes of Bombyx mori and found that they were all the kind of gene coding C2H2 type zinc finger proteins.The odd family genes were highly conserved in the insect genomes.Furthermore,odd family genes had a remarkably similar expression pattern in silkworm.Exploration of the cause for ap mutant and the functional validation by knock-out indicated that Bmsob gene was an important gene for thoracic legs and mating capsule formation.Additionally,BmAntp as well as Bmabd-A may be the downstream genes of Bmsob which might participate in the regulation of thoracic legs and mating capsule formation separately.However,the functional mechanism of Bmsob gene was still unknown and it needs to be further explored.