Genetic Regulation Of Hyphantria Cunea Using Genome Editing And Transgenic Technology

Hyphantria cunea is an important international quarantine pest that poses a great threat to the economy and ecosystem of agriculture and forestry in our country.At present,the control of fall webworm mainly relies on chemical pesticides.Although these efforts have obtained some effect in control of population density to a certain extend,it has huge impacts on the environment,ecology,sericulture and beekeeping industries in a long-term use.Thus,the environmentally friendly and species-specific new method is urgently needed.As insect population genetic regulation is a new approach in fall webworm,the research on this insect is almost blank.Therefore,this study is aimed to establish an efficient genetic regulation strain for better control of H.cunea population through the genome editing technology?CRISPR/Cas9 system?and the piggy Bac-mediated transgenic technology.The major results are demonstrated below:1. We established an embryonic micro-injection platform and a CRISPR/Cas9-mediated gene knockout technology system in H.cunea.Referring to the embryonic micro-injection method of Bombyx mori,a micro-injection platform with stable hatch rate was established in H.cunea.Then,through this system,a highly efficient CRISPR/Cas9 gene knockout system was established in the H.cunea,and we generated a clear and stable phenotype of the Hcyellow gene knockout mutant.The mutation of this gene proved the effectiveness of the embryonic micro-injection platform and the CRISPR/Cas9 gene knockout system in H.cunea.2. A piggy Bac-mediated genetic manipulation technology system was established in H.cunea.In view of the subsequent functional research and screening of target genes for genetic regulation of H.cunea populations,we identified the Hcu6 and Hcnos promoters.Based on the micro-injection platform,we charactered a highly active Hcu62 promoter with the Hcyellow gene as the target gene through Bm N cell assay in vitro and directly injection experiment of plasmids in vivo.Then,we successfully generated the transgenic Hcyellow RNAi mutant strain Ysh2 with more obvious and stable phenotype driven by Hcu62promoter through micro-injection and fluorescent screening.The transgenic technology system was first established in H.cunea,providing important technical supports for its genetic regulation research.3. A CRISPR/Cas9-mediated Hcdsx heritable sterility mutant strain was established in H.cunea.In the functional analysis of Hcdsx gene,we identfied the sex-biased expression pattern of Hcdsx through cloning and expression analysis,and then obtained three exon-specific knockout Hcdsx mutants,which was similar to common region-,female-and male-specifically Bmdsx gene knockout mutants in the silkworm.In Hcdsx mutants,there were serveral sexually dimorphic mutational phenotypes,including larval gonads,pupal genopore,adult external genitalia,abdominal segements,body size,antenna and downstream genes,of which some was more serious in the sex-specific mutants,dsx^e3and dsx^e5,and became incompletely sex reversal comparing to the dsx^e1mutant.As a result,it caused some abnormalities in reproduction-related phenotypes,including mating behavior,oviposition and fertilization,resulting in sex-specific sterilities.Remarkably,we further obtained an inheritable mutant of dsx^e3through genetic crosses,which had shown potential application value in the genetic regulation of the H.cunea.4. The Hcmasc gene was demonstrated to be invovled in sex determination and dose compensation pathway of both male and female in H.cunea.In the functional analysis of the Hcmasc gene,we obtained two mutants,Hcmasc^e4and Hcmasc^C,from different sites knockout experiments.In the Hcmasc^e4mutant,only males generated abnormal phenotypes and we also identifie its masculinization site through Bm N expression assay,two conserved cysteine Cys-249 and Cys-252 in exon 4 of Hcmasc gene.In the Hcmasc^Cmutant,some defects in external genitalia of both sexes and imbalanced sex ratios,indicating that it did participate in both male and female sex determination and dosage compensation in H.cunea.And piggy Bac-mediated transgenic overexpression of Hcmasc1 gene resulted in MC1 lethal,further suggesting that it affected growth and development of MC1 insects likely through the dosage compensation.5. A piggy Bac-mediated transgenic Hcser2 RNAi male sterility mutant strain was established in H.cunea.In the functional analysis of the Hcser2 gene,CRISPR/Cas9-mediated knockout mutant and piggy Bac-mediated transgenic RNAi mutant showed that mutation of Hcser2 gene could induce male-specific sterility.More importantly,the mating behavior of the transgenic mutant was not affected,and the sterile phenotype could be transmitted to the offspring through genetic crosses of female mutants,which is of great significance for the genetic regulation of H.cunea.In conclusion,a stable embryonic micro-injection platform,an efficient CRISPR/Cas9-mediated gene knockout technology system and a piggy Bac-mediated transgenic system were established in H.cunea.Based on the established genetic manipulation technologies,we obtained two target genes,Hcdsx and Hcser2,for genetic regulation in the H.cunea,and established a CRISPR/Cas9-based inheritable Hcdsx gene mutant and a piggy Bac-mediated transgenic Hcser2 RNAi strain,which laid a solid theoretical basis for genetic regulation of the H.cunea.