Functional Reconstruction Of Myb Mutant Gene And Editing Of The CENH3 Gene In Cabbage Based On Prime Editing

Cabbage is a green leafy vegetable of the Cruciferous Brassica genus.It is widely planted all over the world,and it is also one of the commonly cultivated vegetable crops in China.It plays an irreplaceable role to meet balanced supply of vegetables annual in China.Cabbage is a typical cross-pollinated crop with obvious heterosis,and hybrid cultivars breeding is the main way of cabbage breeding.Cabbage is a typical biennial crop with the characteristics of self-incompatibility.Low-temperature vernalization is required strictly for flowering.Breeding of homozygous inbred lines by traditional methods is time-consuming,labor-intensive,and space-consuming.Therefore,it is imminent to develop fast,convenient,accurate and reliable new technologies for cabbage breeding.Haploid inducible lines have been found or created in some crops.Therefore,the development of haploid induction lines in cabbage is the great value to promote the breeding of hybrids varieties.Mutations in the centromeric histone CENH3 gene have been shown to have haploid-inducibility in a variety of plants.Therefore,it is possible to directly obtain haploid inducible lines of cabbage by using the Prime editing(PE)editor that can realize arbitrary changes of specific bases to mutate specific amino acids of CENH3 gene in cabbage,thereby promoting the rapid breeding of inbred lines.In this study,we constructed a Prime editor by fusing n Cas9(H840A)and reverse transcriptase quintuple mutant(MLV)from Moloney murine leukemia virus with codon-optimized of cabbage.And the Prime editor was imported into tobacco and cabbage to verify its editing ability and editing efficiency.In addition,we designed an enrichment system for PE editing try to overcome the chimeras of editing plants and improve the editing efficiency.1.Verify the editing ability and editing efficiency of the PE editing system in tobacco.First,an exogenous MYB gene with nonsense mutation was inserted into the expression cassette of the p BI525 intermediate vector,and then the expression cassette was loaded into the p CAMBIA2300 vector to construct the p CA2300-MYB(TAG)editing instruction vector.Then,peg RNA and g RNA were designed according to the nonsense mutation site,and loaded into the PE editing vector to construct the PE-MYB targeted editing vector.Finally,p CA2300-MYB(TAG)and PE-MYB were cotransformed into tobacco leaves disc by Agrobacterium-mediated method.Total 75 callus were finally obtained through Kan and PPT screening in culture medium,only one of callus show purple-red spots,so the editing efficiency of PE editor for MYB gene in tobacco was 1.3%(1/75).In order to further confirm the reliability of the editing results,we subculture the purplish red callus and 11 regenerated plants were obtained,10 of which contained sporadic purplish-red spots on the leaves and 1 plant with complete purplish-red leave.The DNA of all regenerated plants was extracted by CTAB method,and the MYB gene was cloned for sanger sequencing.No mutation was detected in 10 plants with purple-red spots,while the expected mutation was detected in plants with complete purple-red leaves.The experimental results show that the PE editing system we constructed can perform accurate editing normally.2.The editing of CENH3 gene in cabbage by PE system.According to previous study,four mutations,including G83 E,Δ83G84T,E89 K,and R124 C in the CENH3 gene of cabbage were designed for PE editing,and the corresponding peg RNA and g RNA for PE editing were obtained by GG assemble,respectively,and loaded into PE vector to generated PE-G83 E,PE-83G84 T,PE-E89 K,PE-R124 C vectors.The hypocotyls of To1000 cabbage were infected by Agrobacterium-mediated transformation,and regenerated plants were obtained by PPT screening.Total of 82 regenerated plants from 17 calluses of PE-G83 E were obtained.All plants contained Cas and MLV gene by PCR identification.However,no edited plants were found of CENH3 gene target site in all plants by Sanger sequencing analysis.Total of 91 regenerated plants from 25 calluses of PE-83G84 T transformation were obtained,no edited plants were found although all plants contained Cas and MLV gene.Total of 135 regenerated plants with Cas and MLV gene positive from 30 calluses of PE-E89 K transformation were obtained,except the No.107 plant had double peaks at the g RNA target site,and no editing was found in other plants.The PE-R124 C transformation generated 120 Cas and MLV gene positive plants from 15 calluses,and the No.1 and No.9 plants showed double peaks peaks at peg RNA target site.In summary,the PE editor failed to obtain the expected editing of CENH3 gene in cabbage.But the editing by-product appeared at the target of the g RNA of PE-E89 K regenerated plants,with an editing efficiency of 0.7%(1/135).Editing by-product appeared at the peg RNA targets of PE-R124 C regenerated plants,and the editing efficiency was 1.7%(2/120).3.The enrichment of PE editing in tobacco.We fused the NPTⅡ gene to the 3’ end of the nonsense mutation of the MYB gene,inserted it into the p BI525 intermediate vector expression cassette,and then loaded the expression cassette into the p CAMBIA1300 vector to construct the p CA1300-MYB(TAG)-NPTⅡ vector.The p CA1300-MYB(TAG)-NPTⅡ vector and PE-MYB vector were co-transformed into tobacco leaves disc screening by Kan and PPT,total 32 calluses were obtained,of which 6 calluses showed purple-red spots,and the editing efficiency was as high as 18.8 %(6/32).Sanger sequencing was performed on the shoots formed by purple callus,and expected editing was found.The results show that the PE editing efficiency can be improved using the enrichment system,but more effective measures needed to develop to solve the chimeras’ plants problem that lack of mutation enrichment methods.