Application Of CRISPR/Cas9 And ABE Gene Editing System In Brassica Oleracea L

Gene-specific editing technology is one of important technical tool support for plant functional gene research and crop genetic breeding.CRISPR/Cas9 gene editing system is the most popular gene editing technology at present.Its working principle is mainly to target and combine sgRNA with the target DNA sequence,so as to activate the nuclease of Cas9 to cut the target DNA and produce the damage of DSB.Through the non-homologous end joining and homologous recombination,nucleotides insertion,deletion,substitution at the target site are produced,so as to achieve the knockout and editing of the target gene.However,the efficiency of homologous recombination is far lower than that of non-homologous end joining,resulting in uncontrollable repair results at the target site.The single base editing technology can transform the single base of the specific location point in the target gene without DSB.At present,CBE editor and ABE editor have been developed to achieve C/G?T/A conversion and A/T?G/C conversion in the target sequence.The establishment of this system makes the realization of single base editing no longer depend on DNA double strand break and can produce four forms of base conversion,which not only avoids the limitation of low efficiency of HR repair pathway,but also gets rid of the randomness of NHEJ repair pathway.The flowering process of plants is influenced by the external environment and regulated by the intricate internal genes.FT and CO regulate the flowering process of plants.Mutation of FT and CO gene will promote late flowering.The cabbage green keeping gene HG is highly homologous with the NYC1 gene in Arabidopsis,which is involved in the plant chlorophyll metabolism pathway.The mutant HG slows down the leaf aging process of the plant,keeps green for a long time,greatly prolongs the life cycle of the plant,and maximizes the edible value of vegetables.The mutant genes of BoFT,BoCO and BoHG can keep the Brassica oleracea L leaf ball green for a long time without ball breaking and flowering,which not only keeps the commercial value of Brassica oleracea L,but also extends the supply period of Brassica oleracea L to a certain extent,so as to improve the production efficiency of Brassica oleracea L.In this experiment,Brassica oleracea L were used as the research objects to knock out the BoFT and BoCO genes of Brassica oleracea L by the CRISPR/Cas9 gene editingsystem for the late flowering and bolting resistant Brassica oleracea L materials generation.ABE base editor was used to edit the PDS gene of Nicotiana tabacum in the first,then the BoFT,BoCO and BoHG genes,which regulate flowering and leaf green keeping of Brassica oleracea L were targeted for base editing to obtain green-keeping and bolting resistant varieties.The results of this study are as follows:1.The CRISPR/cas9 system was used to edit the BoFT and BoCO genes of Brassica oleracea L.A double genes knock-out vector p Cas-t Bo FT-ABC/t Bo CO-ABCD with Bar gene as the screening marker was constructed and transformed into Brassica oleracea L self-incompatibility line F416 by Agrobacterium mediated method.Twenty-one positive transgenic plants of(from a callus)were detected.Only one of the three mutation sites of the BoFT gene was detected mutation(C site),and the mutation efficiency was 100%.Among the four sites of BoCO gene,B and C sites were mutated,and the mutation efficiency was 100%.The results showed that the mutation frequency of C site of BoFT gene was 58.75%,and that of B site and C site of BoCO gene was 78.08% and 10.96%,respectively.Compared with the wild-type self-incompatibility line F416,there was no obvious change in the plant type of the mutant,but the development of flower organs was abnormal,such as stigma,stamen and so on.There was no significant difference in the flowering time of the double heterozygous mutant compared with the wild-type control.The flowering time of the double-gene homozygous mutant was significantly later than the wild-type control,and the flowering time of homozygous mutant only one of the gene mutations was in between the double-gene homozygous mutant and double-gene heterozygous mutant.However,it was found that it was difficult for mutant materials to produce seeds by self-pollination,and the reason is for further study.2.ABE editor for the Brassica oleracea L BoFT,BoCO and BoHG.We constructed pCas-t Nt PDS-ABCD gene editing vector and p ABE-t Nt PDS-ABCD base editing vector with Bar gene and MYB visual marker gene as screening marker.Both the vectors were transformed into the tobacco by Agrobacterium-mediated method,respectively.The results showed the ABE editing vector can achieve effective A/T?G/C conversion in tobacco.However,compared with pCas-tNtPDS-ABCD tobacco transgenic plants,there were obvious albino seedlings were observed,but no obvious albino seedlings were found in pABE-tNtPDS-ABCD single base editing vector transgenic plants.The expression vector p ABE-Bar MYB-t Bo FT-A /tBo CO-A/t Bo HG-AB was constructed to target the A/T bases at the junction of the intron andexon of the genes related to the regulation of flowering and green-keeping genes BoFT,BoCO,and BoHG in cabbage.The target genes in transgenic plants were sequenced.The results show that A/T?G/C conversion at the specified-window is successfully achieved in BoFT and BoHG genes in cabbage through the ABE base editor.The single base editing efficiency at the target site of BoFT was 7.69%,and the single base editing efficiency of BoHG was 76.92%.Two mutants were selected randomly to perform monoclonal sequencing on the BoHG gene.The results showed that the base substitution frequency of BoHG gene at target site A was 23.53%,and the base substitution frequency at site B was 100%.