The Development Of The HyCBE Gene Editing System And The Preliminary Exploration Of The Lily Gene Editing System

Lilium is a world-famous bulb flower which is popular among the people all over the world.Since its beautiful appearance,colorful variety and rare fragrance lily enjoys high ornamental value and economy value.However,as the native country of lilium genus,the breeding of lilium in our country was relative backwardness and the obtain of lilium germplasm resources mainly depended on foreign imports.Recent years,gene editing technology has been widely applied to plant molecular breeding because of its advantages such as simple operation and high efficiency.Although gene editing technology has been used in lily,high effiency and mature system should be developed in lily.On the other hand,base editors can edit single base without double-stranded DNA breaks thus provide a useful tool for creating point mutant plant materials.Since the discovery of base editing technology,how to generate high efficiency base editors have become the important issue for the scientists.In this paper,we explored the regeneration system of lily based on somatic embryogesis and established a high efficiency system for callus induction.We established the genetic transformation and gene editing system for lily based on lily callus.We fused the cytosine base editors(CBEs)with the single-stranded DNA binding domain(Rad51 ss DBD)for the purpose to achieve the CBEs with higher efficiency and wider editing window.Main results were as follow:1.We first focused on the Somatic Embryogenesis of the Lilium Oriental.And found that the most suitable plant growth regulators for lily callus induction was 2.0mg L^-1 PIC+0.2 mg L^-1 NAA.Besides,adding 0.5mg L^-16-BA was helpful for the seedling regeneration.2.We focused on several important horticultural traits of lily:pollen-free lily or lily with little pollen,double type lily and keeping period of cut flowers and found the corresponding key genes:Lo MYB80(corresponding to pollen content),LLA23(corresponding to pollen content),AG1(corresponding to double flower),AG2(corresponding to double flower),ACO(corresponding to double flower).The CRISPR/Cas9 vectors were constructed with these genes in lily.Agrobacterium tumefaciens EHA105 was used to infect the callus,with 20-25 mg L^-1hygromycin selection,477 plants were generated.Further more,we established the suspension cell culture system of lily.Agrobacterium tumefaciens-mediated transformation was also carried on above callus and cell,but no edited plants were detected.3.CBE system was optimized in rice,we fused a rice codon-optimized human Rad51 DBD and rice Rad51 DBD to several CBEs to form hy CBEs:hy Anc BE4max,hy Anc BE4max-NG,hyrice Anc BE4max,hyevo FERNY and hyriceevo FERNY.4.5 target sites were chosen to test the perform between Anc BE4max and hy Anc BE4max.Positive T0 plants were sequenced by Sanger sequencing,compared with Anc BE4max,the editing window of hy Anc BE4max was expended from C2-C10to C5-C16.The editing efficiency in the center window(C4-C8)had no big difference:4.5%?54.1%for Anc BE4max and?55%for hy Anc BE4max;For hy Anc BE4max,the C-to-T efficiency at positon C10 was 25%however only 10%for Anc BE4max.At position C14 and C16,hy Anc BE4max respectively had 10%and 5%editing efficiency,however Anc BE4max had no editing efficiency.Similarly,four target sites were choosen to test Anc BE4max-NG and hy Anc BE4max-NG.The editing window of Anc BE4max-NG was from C4-C10 while hy Anc BE4max-NG was from C5-C9.The average editing efficiency for hy Anc BE4max at C5 and C8 was 50%and 30%which was a little high than Anc BE4max(36.5%at C5,5%at C9).Besides,two target sites were choosen to test for hyrice Anc BE4max,hyevo FERNY and hyriceevo FERNY which also showed that the editing scope was expended.Compared with Anc BE4max,the editing window of hyrice Anc BE4max was expended from C6to C14.Compared with evo FERNY,the editing scope of hyevo FERNY and hyriceevo FERNY was respectively expended to C14 and C12.In summary,this paper explored the genetic transformation process in lily and has made a solid foundation for the application of lily and has successfully optimized the base editing system for expanding the editing window in rice,which helps to supply more genetic materials for agronomic breeding.