CRISPR/Cas9 Mediated The Establishment Of Gene Editing System In Tomato

Gene editing technology have been as one of the most important science progresses in 2012,which could achieve gene editing anywhere you want accurately and it has good prospect of functional genomic and molecular design breeding on plant.U6 promoter is a vital element for the transcription of sgDNA in the CRISPR/Cas9 system.There were kinds of U6 promoters in the tomato genome,whether they had transcriptional function in tomato leaves were not clear.Screeing a high transcription activity U6 promoter could improve specificity in CRISPR/Cas9.There hasn't any report on endogenous U6 promoter and whether it has difference between endogenous and extraneous U6 promoter to transcribe sgDNA on tomato.This research takes two aspect of works.In one respect,cloning and identifying the function of U6 and U3 promoters,Which could provide desirable promoters for the construction of a CRISPR/Cas9 system.On the other side,we choose the SlU6-2P4::GUS,leaves stained with deep blue,to construct the CRISPR/Cas9 system vector and powdery mildew correlative gene MLO1 and EDR1 as the target,Which contains three target sequences respectively.To compare the efficiency of tomato endogenous U6 promoters with Arabidopsis U6 promoters,we also construct the CRISPR/Cas9 system vector with Arabidopsis U6 and U6-26 promoters.(1).The different truncated SlU3-1P,SlU3-3P and SlU3-4P promoters from tomato variety Zhongsh4 were performed to clone by two rounds of PCR amplification,altogether there were six different promoters.Lengths were 489 bp?318 bp?450 bp?248 bp?457 bp and 48 bp respectively.Six GUS fusion expression vectors were constructed and transformed into tomato leaves.After sequences comparison between Arabidopsis and tomato U3 promoters,results showed that the tomato U3 promoters contained the conserved USE motif and TATA box and the distance between two elements was fixed just like Arabidopsis U3 promoter.GUS fusion expression vectors driven by corresponding truncated promoter were constructed and transformed in tomato leaves by the vacuum in filtration transformation method.Results of GUS histochemical staining showed that the six truncated U3 promoters could drive GUS expression in tomato leaves and tomato leaves could be stained blue,Which could provide ideal endogenetic promoters for the construction of CRISPR/Cas9 system for tomato functional genomics research.(2).CRISPR/Cas9 gene editing system is completely a fresh technology compared with TALENs and ZFNs,which has greatly prospect in agriculture and genetic engineering in plant.U6 promoter is a vital element for the transcription of sgRNA in the CRISPR/Cas9 system,but there are few reporter on this research.It is necessary for us to clone some endogenous promoters with high transcription activity.Four tomato U6 promoters had cloned by two rounds of PCR amplification from tomato variety Zhongsh4,altogether there were eight different promoters.And the length were 452 bp,202 bp,448 bp,206 bp,433 bp,190 bp,448 bp and 218 bp respectively.Eight GUS fusion expression vectors were constructed and transformed into tomato leaves.After sequences comparison of U6 promoter between Arabidopsis and tomato,the results showed that the tomato U6 promoters also contained the USE motif and TATA box,the distance between two elements was fixed just like Arabidopsis U6 promoter.GUS fusion expression vectors driven by corresponding truncated promoters and transformed into tomato leaves by the vacuum in filtration transformation method.The GUS histochemical staining results showed that the eight truncated U6 promoters could drive GUS expression in tomato leaves.We choose the SlU6-2P4::GUS,leaves stained with deep blue,to construct the CRISPR/Cas9 gene editing vector and powdery mildew correlative gene MLO1 and EDR1 as the target,Which contains three target sequences respectively.To compare the efficiency of tomato endogenous U6 promoters with Arabidopsis U6 promoters,we also construct the CRISPR/Cas9 gene editing vector with Arabidopsis U6 and U6-26 promoters.The result showed that endogenous SlU6-2P4 promoter and Arabidopsis U6 promoter could achieve gene editing in tomato respectively.Additionally,we found that there were same result after gene editing by CRISPR/Cas9 system that sgRNA has driven by two different U6 promoters in tomato protoplasts.