Positional Colning And Function Research Of L-e~m(lethal Egg Of Mutant) Gene In Silkworm Variety "ming"

The silkworm is a complete metamorphosis insect and diapaused in egg stage. Eggstage is the first and an important stage of the silkworm (Bombyx mori) life cycle, andthe quality of the silkworm eggs has a direct impact on the development of larvae,pupae and moth. The normal eggs are usually short and elliptic, flattened laterally, thesurface of the lateral side is sometimes hollowed. The object of this study (“Ming”lethal egg mutants, l-em) is a lethal egg of mutant which was found in “ming” duingthe production and preservation of “su·ju×ming·hu”. The eggs laid by l-emmutant lostwater and became concaved around one hour, ultimately exhibiting a triangular shapeon the egg surfaces. Genetic analysis showed that the l-emmutation is dominated by arecessive gene and followed the pseudo-maternal inherited mode, and the hollowplace of surface have obvious rhagades and the cross section have cracks were foundin dead eggs using the scanning electron microscope.In the present study, we performed positional cloning using map-based cloningbased on the phenotypic observation and genetic analysis. Expression analysis andfunctional verification of candidate genes were performed by using qRT-PCR,2-DE,RNAi. The results of the study are as follows.1. Positional cloning of the l-emgeneThe male parent (P1) and the female parent (P2) were selected from thehomozygous recessive individuals and inbred line of p50, respectively. Both of themwere used as parent strains for the mapping panel and a single-pair cross between p50and l-emmutant produced the F1offspring. The female individuals of F1offspringwere used for selfing and back-crossing with P1to produce F2and BC1F progeny. Thepolymorphic SSR markers of28linkage groups were selected using P1, P2and F1, andconfirmed the l-emgene located on the10thlinkage group using BC1F progeny. Thegenetic linkage map of the l-emgene was constructed using2,287F2femaleindividuals which laid dead eggs and13polymorphic SSR markers which linked withthe l-emgene, and a region of~360kb tightly linked to the l-emgene between themarkers S65and S82was identified.24genes within this locus were predicted byusing gene-prediction models. 2. Expression models, gene structures and functional verification of thecandidate genesTwo differentially expressed genes (BmVMP23and BmEP80) were identified ascandidate genes through investigating the expression level of the24initial candidategenes in the eggs laid by virgin WT and l-emmutant moths. The results showed thatthe expression level of BmVMP23gene in l-emmutant was much lower than inwild-type, while BmEP80gene did not express in ovaries of the l-emmutantcompletely. The results of inverse PCR showed that the mutation was located betweenthe3′-UTR of BmVMP23and the forepart of BmEP80, and destroyed the structuralintegrity of the two genes.The results of the RNAi showed that the eggs laid by the individuals treated withBmEP80siRNAs appeared with a triangular shape on the egg surface, consistent withthe l-emmutation. However, the eggs laid by all individuals treated with BmVMP23siRNAs did not appear this phenomenon. So we concluded that BmEP80was mostlikely the gene causing the l-emmutation.3. Differential expression of the ovary proteins between WT and l-emmutant2-DE was used to investigate the differential expression of the egg proteins betweenthe WT and l-emmutant, and the result showed that the BmEP80protein wasexpressed in the ovary from the9thday of the pupal stage of the WT, but not wasexpressed at all in the ovary of the l-emmutant. However, there was no difference ofthe other proteins between the WT and l-emmutant except the BmEP80protein. Thisresult further indicated that the the defection of BmEP80resulted in the termination ofBmEP80protein expression, thereby triggering the l-emmutation.4. Analysis the expression regulation of the candidate gene BmVMP23The results of the sequence analysis have shown that the3′-UTR of BmVMP23wasdestroyed, and3′-UTR is an important regulatory site of the microRNAs. Toinvestigate the relationship between the miRNAs and the expression of BmVMP23,we downloaded all the known miRNAs of the silkworm and performed a BLASTsearch for the3′-UTR (WT) sequence of BmVMP23, and found a miRNA(bmo-miR-1a-3p) that highly matched the3′-UTR (WT) sequence of BmVMP23.The results of the qRT-PCR indicated that the expression level ofbmo-miR-1a-3p in the ovaries of WT was higher than that in the l-emmutant, and the expression level was contrary to the expression level of BmVMP23. We confirmedthat the bmo-miR-1a-3p can inhibit the expression of BmVMP23through transfectionin vitro.The above results confirmed that the mutation of BmEP80is responsible for thel-emmutant. This study laid a foundation for further study of the formationmechanism of the l-emmutant. It has an important scientific significance and value.