中国农业科学2018，Vol.51Issue(2)：315-326,12.DOI:10.3864/j.issn.0578-1752.2018.02.011
番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立
Different S1U6 Promoters Cloning and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Tomato
摘要
Abstract
[Objective] U6 promoter is a vital element for the transcription of sgRNA in the CRISPR/Cas9 system.It is necessary to clone some endogenous U6 promoters with high transcription activity and construct CRISPR/Cas9 vector,which could provide a strong technical basis for functional genomics and molecular breeding in tomato.[Method] Four different tomato U6 promoters were cloned by first round of PCR amplification from tomato cultivar Zhongshu 4.Each U6 promoter with two different length was truncated and used to construct plant expression vector carried S1U6 promoter::GUS,respectively.The Eight GUS fusion expression vectors were transformed into tomato leaves by agroinfiltration.According to the degree of GUS staining,the promoter with high transcription activity was selected to construct the CRISPR/Cas9 gene editing vector with target sequence from powdery mildew-related gene MLO1 and EDR1.These gene editing vectors were transformed into tomato protoplast by PEG method.The mutation of endogenous target genes in each transformed tomato protoplast was analyzed by a restriction enzyme PCR (RE-PCR) assay.Finally,the types of endogenous gene mutation were analyzed by sequencing.The efficiency of the CRISPR/Cas9 system based on tomato endogenous U6 promoter was verified by the frequency distribution map of mutant loci.[Result] 4 kinds and 8different lengths of tomato U6 promoters were obtained by two rounds of PCR.Their length was 452,202,448,206,433,190,448 and 218 bp,respectively.After sequences analysis,results showed that the four tomato U6 promoters also contained the USE motif and TATA box which were found in Arabidopsis U6 promoters.The construction of GUS fusion expression vectors driven by corresponding truncated tomato U6 promoters were done and transformed into tomato leaves.The GUS histochemical staining showed that the transformed tomato leaves were dyed blue,which indicated that all 8 SlU6 promoters have transcription activity.The SlU6-2P4 promoter was chose to drive sgRNA transcription and construct the CRISPR/Cas9 system with target sequence from MLO1 and EDR1 respectively.The result showed that endogenous SlU6-2P4 promoter could drive sgRNA transcription and gene MLO1 was edited successfully in tomato.Sequence analysis revealed that all types of gene mutations are base substitution and the hotspot of mutation only exists in the target region of endogenous gene.[Conclusion] 4 kinds of SlU6 promoters with high transcription efficiency were obtained from tomato.The established CRISPR/Cas9 system based on SIU6-2 promoter could successfully achieve the editing of endogenous genes in tomato.关键词
CRISPR/Cas9/S1U6启动子/克隆/番茄/基因编辑Key words
CRISPR/Cas9/S1U6 promoter/clone/tomato/gene editing引用本文复制引用
蒲艳,刘超,李继洋,阿尔祖古丽·塔什,胡燕,刘晓东..番茄U6启动子的克隆及CRISPR/Cas9基因编辑体系的建立[J].中国农业科学,2018,51(2):315-326,12.基金项目
国家自然科学基金(31560534) （31560534）
