The Construction Of CRISPR/Cas9 Mediated Camellia Sinensis Genome Editing System

CRISPR/Cas9 technology?clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9?is a novel and powerful approach for targeted genome editing,such as targeted gene knock out or site-directed mutagenesis in a simple and easy way.The present work aimed to develope an efficient method to construct a CRISPR/Cas9 expression vector for genome editing the tea caffeine synthase?TCS?encoding gene using general PCR,overlapping PCR and golden gate cloning technology.At the same time,the genetic transformation and regeneration system for Camellia sinensis var.sinensis was optimized.Additionally,the TCS CRISPR/Cas9 gene editing vector was transformed into somatical embryos for knocking down TCS gene.The main results of this work are as follows:1.Construction of CRISPR/Cas9 vector for editing TCS gene in tea plantPrimers of the target sequences T1 and T2 were designed according to the sequence of TCS.The U3d promoter and T1 target sequence?T1-gRNA?were obtained by ordinary PCR amplification using pYLgRNA-AtU3d-LacZ as a template,and U3b promoter and the T2 target sequence?T2-gRNA?were obtained using the pYLgRNA-AtU3b as a template.The U3d promoter and the T1-gRNA were assembled together to form a T1sgRNA expression cassette using overlapping PCR technology.The same technique was used to assemble the U3b promoter and the T2-gRNA together into a T2sgRNA expression cassette.Furthermore,the Golden Gate Cloning technology was used to combine the T1sgRNA expression cassette,the T2sgRNA expression cassette and with the pYL CRISPR/Cas9P35S-H binary expression vector to form the CRISPR/Cas9 mediated TCS gene editing vector.Sequencing analysis confirmed that the T1 and T2 target sequences were detected in the constructed vector sequences.And the presence of conserved gRNA sequences was detected close to the target sequence.Moreover,U3d and U3b promoter sequences were detected upstream of the T1 and T2target sequences.Finally,the sequence of pYLCRISPR/Cas9P35S-H binary expression vector was detected downstream the T2sgRNA expression cassette and upstream the T1sgRNA expression cassette.The sequencing results verified that the T1sgRNA expression cassette and T2sgRNA expression cassette were successfully constructed and successfully assembled into the pYLCRISPR/Cas9P35S-H binary expression vector,indicating that the CRISPR/Cas9 mediated TCS gene editing vector was successfully constructed.2.Optimized the tea plant regeneration systemThe effect of tea cultivar,explants and inducing conditions on the tea plant cell dedifferentiation and re-differentiation were investigated and the suitable cultivar,explants and combination of different plant growth regulator for regeneration of tea plant were determined.Thus,the regeneration system of tea plant was improved.The results showed the leaves from cultivar“Bixiangzao”were suitable explant when it was cultured on1/2MS medium containing 0.1mg/L TDZ and 0.1mg/L IBA,which the reddish-brown callus induction rate was the highest,about 40.96%,within 4 weeks.When the cotyledons form cultivar“Xiangfeicui”were selected as explants that were induced on1/2MS medium containing 3.000mg/L 6-BA,0.100mg/L NAA,the induction rate of somatic embryo was the highest,about 13.58%,within 6 weeks.The appropriate medium for rooting was 1/2MS+1.0 mg/L 2,4-D+0.1 mg/L TDZ+0.1 mg/L IBA.For this treatment,the induction rate of adventitious buds was 18%.The appropriate induction medium for re-differentiation of somatic embryo was 1/2MS+1.0g/L Glutamine+0.1mg/L GA₃+0.15mg/L NAA.In this treatment,the root can be induced within 4weeks.3.Optimization of the tea plant genetic transformation systemThe optimal tea plant regeneration condition was developed by investigating the important factors which significantly affect the transformation efficiency of tea plant,including the explants,stress of transgenic selection,concentration of Agrobacterium and acetosyringone concentration and so on.These results revealed that somatic embryo was the most suitable for genetic transformation of tea plant.While,the callus turned to brown and to death after infection by Agobacterium.The optimal selection stress was determined and the best concentration of hygromycin in the selection medium is 50 mg/L.The optimum concentration of cefotoxine to inhibit the growth of Agrobacterium is 400 mg/L.In addition,the Agrobacterium tumefaciens in the early logarthmic growth phase showed highest infection ability.Acetosyringone solution at a concentration of 150?moL/L significantly improved the transformation effecience.Finally,the CRISPR/Cas9 gene editing vector for TCS in tea plant was successfully transformed into somatic embryos of tea plant through the genetic transformation system developed in this study and the mutation was found in the TCS gene in somatic embryos of tea plant.