Genetic Improvement Of Important Economic Traits Of Bombyx Mori Using Genome Editing

The silkworm,Bombyx mori(B.mori),is an economically important insect.The yield,quality of silk and the increment of added value of subsequent products are determined by the traits of silkworm varieties.Therefore,cultivating excellent varieties has always been the goal of sericulture workers.Traditionally,the expected traits are obtained by mutation screening,and then the target traits are integrated into various production varieties through genetic hybridization.However,this method is limited by many factors,which affects the breeding cycle.Recently,the advancement of genome sequencing and the application of gene editing technology in silkworm has settled a well foundation for directional breeding and breeding progress.The research aimed to focus on two important aspect of silkworm yield and quality traits.Firstly,the transgenic and genome editing techniques was used to study the function of the key genes in the juvenile hormone(JH)and 20-hydroxyecdysone(20E)signaling pathways,and to elucidate the mechanism of JH and 20 E in regulating the development of silkworm,and to construct silkworm strains with high yield.Secondly,the transgenesis,gene knock out and gene knock in were integrated to establish a female-specific,embryonic lethal system,and to achieve male only rearing and the advance of silk quality.The research results will provide the theoretical basis for the improvement of economic traits of silkworm.The main results contain the following aspects: 1.Functional analysis of BmJHE involved in JH metabolism pathway in silkwormJuvenile hormone esterase(JHE)is the primary JH-specific degradation enzyme in most insects.The transgenic CRISPR/Cas9 system was applied to knock out BmJHE and further analyze the effect of JH on the development of silkworm in vivo.Depletion of BmJHE resulted in the extension of growth during larval stages.Extension of the feeding stages resulted in larger body size.Female mutant whole cocoons weight and was 47% heavier than WT.Male mutant whole cocoons weight was 58% heavier than WT.The Quantitative Real-time PCR(qRT-PCR)results showed that the transcript level of the JH inducible gene,Krüppel homolog 1(Kr-h1),as the indicator of JH titer was upregulated,which indicated depletion of JHE delayed JH metabolism and further affected body size through the regulation of the larval growth period.2.Functional analysis of BmTorso involved in 20 E biosynthesis pathway in silkwormProthoracicotropic hormone(PTTH)receptor,Torso,is a receptor-tyrosine-kinase,which locates in the middle position of ecdysone biosynthesis pathway.Activation of Torso by PTTH triggers the biosynthesis of 20-hydroxyecdysone.In this research,BmTorso was disrupted.BmTorso mutants showed a severe developmental delay in both the larval and pupal stages.Female mutant cocoon shell weight was 162% heavier than WT.Male mutant cocoon shell weight was 167% heavier than WT.The 20 E titers of BmTorso mutant in the hemolymph declined sharply not only at wandering stage,but also at pupal stage.The qRT-PCR results showed that that mRNA levels of genes involved in 20 E biosynthesis and 20 E signalling pathways were significantly reduced in BmTorso mutants.The transcriptome analysis revealed that genes involved in longevity pathway were affected by BmTorso deletion,which indicated that BmTorso is required to coordinate developmental timing and body size by regulating the biosynthesis of 20 E and longevity pathways.3.Establishment of a female-specific,embryonic lethal system in silkwormBreeding of male silkworm varieties and achievement of male only rearing can improve the silk quality due to the advantages of strong vitality,high raw silk rate and superior silk quality by male only rearing.In the silkworm,males are homomorphic sex chromosome(ZZ).Females possess heteromorphic sex chromosomes(ZW).The research established a female-specific,embryonic lethal system in silkworm.The Cas9 expression cassettes was integrated into W chromosome using transcriptional activator-like effector nuclease(TALEN)–mediated genome knock in.And one strain with W-specific Cas9 expression driven by the silkworm germ cell-specific Nanos(Nos)promoter was established.As the same time,another transgenic strains with small-guide RNA(sgRNA)expression targeting B.mori transformer 2(BmTra2),an embryonic lethal gene,was established.Transgenic strains were crossed with the strain with W-specific Cas9 expression.Approximately 50% of the embryos in the filial 1(F1)progeny failed to hatch.Scoring sex ratios revealed that all females died during the embryonic stage,whereas males hatched normally and developed well.