Application Of CRISPR/Cas9 System In Cabbage And Preliminary Study Of Single Base Editor VQR-BE3 In Tobacco

Brassica oleracea is a biennial vegetable crop of Brassica in the family cruciferae,which is one of the important vegetable crops in autumn and winter in China.At present,almost unique ogura CMS cytoplasmic sterility germplasm is used for Brassica oleracea hybrids breeding by male sterile approach,a potential genetic risk might be caused due to genetic vulnerability of cytoplasmic components.In addition,the restorer lines of ogura CMS in Brassica oleracea have been established by means of genetic engineering or distant hybridization,which broken the technical blockade of important germplasm in Brassica oleracea through ogura CMS,and the loss of excellent germplasm will be inevitable.Therefore,in order to solve the above problems,it is necessary to establish new CMS cytoplasmic male sterility resources for Brassica oleracea heterosis breeding.In addition,downy mildew,soft rot,sclerotinia,gray mold and other diseases occur frequently in the production of Brassica oleracea,and the diseases will further deteriorate with the aging of leaves.Therefore,it will be feasible to achieve the horizontal resistance to diseases in Brassica oleracea by the collaborative regulation of genes involved in disease defense system and leaf senescence.The cytoplasmic male sterility is mostly due to the abnormal mitochondrial genome.MSH1 gene is an important gene to maintain the stability of mitochondrial genome.In arabidopsis,tobacco and tomato,the deletion of MSH1 gene leads to the disorder of mitochondrial genome and male sterility.DMR6(downy mildew resistance 6)gene is a pathogen susceptibility related gene,and the protein it encodes is involved in the pathogen-plant compatibility mechanism and has a negative effect on plant pathogen defense.The absence of DMR6 gene gave plants horizontal resistance to a variety of pathogens in arabidopsis thaliana and tomato.SGR/HG gene is related to the regulation of chlorophyll degradation process,magnesium chelatase coded by the SGR/HG gene can extract magnesium from free chlorophyll and chlorophyll complex,which is an important step in chlorophyll degradation process.The defective SGR gene blocked the chlorophyll decomposition pathway,leading to sluggish green and anti-aging phenotypes in rice and Chinese cabbage.The CRISPR/Cas9 gene editing technology can cause bases deletion,replacement and insertion in a gene by NHEJ repair pathway activated by sgRNA guiding Cas9 protein cleavage at the target site of the gene.The CRISPR/Cas9 system is an ideal tool for mutant generation,but this system cannot achieve the precise editing of the target genes.By introducing a deaminase in the CRISPR/Cas9 system,single-base editing technique was developed to precisely replace a single base at a specified location in the gene.VQR-BE3 is a single-base editing system derivatived from BE3 bearing additional VQR amino acid substitutions in original SpCas9.The VQR-BE3 can target NGA PAMs to replace base C in the editing window with base T,so it is a valuble tool for plant gene editing and multi-trait stacking.In this study,the CRISPR/Cas9 system was used to knock out the gene BoMSH1 related to mitochondrial gene stability and the gene BoSRK gene related to self-incompatibility in cabbage.And a CRISPR/Cas9 gene editing system with phenotypic assisted screening marker was established to knock out BoDMR6,a gene related to disease resistance in cabbage,and BoHG,a staggered-green gene in leaf.In addition,a VQR-BE3 base editor that successfully implemented base editing in human cells is directly applied to model plant tobacco to test its single-base editing efficiency in the absence of plant codon optimization,as to lay the foundation for whether this single-base editor can be directly applied to Brassica oleracea..The results of this experiment are as follows:1.The CRISPR/Cas9 double gene knockout vector pCas-t BoMSH1-ABCD/ tBoSRK-ABCD of cabbage Bo MSH1 and BoSRK genes was constructed,and the self-incompatibilty line F416 of cabbage was transformed by agrobacterium-mediated transformation,and 34 transgenic plants were obtained finally.According to the mutation analysis,the mutation frequency of the BoMSH1 gene is 2.94%,while that of the BoSRK gene is 81.82%,and the mutation frequency of both genes is 2.41%.In this study,only one msh/srk mutant was obtained.Monoclonal sequencing showed that the BoMSH1 gene and BoSRK gene of this plant were heterozygous mutated,while 4 srk mutant were homozygous mutated.Compared with the wild-type F416,the msh1/srk mutant had serrated leaf margins.The results of pollen viability test showed that the pollen activity of msh1/srk mutant plants was basically the same as that of wild-type plants.Siliques can normally elongate and grow in the early stage of the msh1/srk mutant plant after bud and flowering stage self-crossing,however,the siliques shrivelled with dead embryo in the later stage,and no offspring seeds was obtained finally,the reason of which remains to be further studied.2.Vector pCas-MC-tBoDMR6-ABC/tBoHG-ABCD was constructed for BoDMR6 and BoHG genes knockout,for transgenic plant identification,MYB and mCherry phenotypic assistant screening genes were introduced into the vector.Twelve transgenic cabbage seedlings with purplish red phenotype were obtained from by agrobacterium-mediated transformation.The mutation frequency of BoDMR6 gene was found to be 0%,while that of Bo HG gene was 25%.Only three hg mutants were obtained,among which one was homozygous mutated and the rest were heterozygous mutated.3.A VQR-BE3 single base editing vector pCA-VQR-BE3-tNtPDS-ABCD that target tobacco NtPDS gene was constructed,41 transgenic tobacco plants were obtained by agrobacterium-mediated transformation.The sequencing results showed that there was no C-T base substitution in all target sites of NtPDS gene of all transgenic tobacco plants.To detect whether the target sites having affects on the C-T base replacement of the VQR-BE3,3 extra target sites in NtPDS gene were selected to constructed a new base editing vector pCA-VQR-BE3-MYB-tNtPDS-EFG with a visual selection marker MYB gene linked,the transgenic positive plants can be screened out easily by anthocyanin accumulation.Total 23 transgenic tobacco plants with purple color were obtained through agrobacterium-mediated transformation and visual marker assisted screening.Sequencing showed that the NtPDS gene in 23 transgenic tobacco plants did not occur C-T base substitution at any target.It is speculated that the reason why the VQR-BE3 single-base editing system used in this study failed to successfully achieve C-T base replacement in tobacco may be related to the lack of plant codon preference optimization or the absence of a nuclear localization signal at the 5 'end of the VQR gene.Certainly,a further investigation needs to be performed to figure out the reason behind.