Different SlU6 Promoters Cloning and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Tomato

PU Yan1 LIU Chao1 LI JiYang1 AERZU GULI·TaShi1 HU Yan1 LIU XiaoDong1

(1.College of Agronomy, Xinjiang Agricultural University/Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi 830052)

【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 transcriptional activity and construct CRISPR/Cas9 vector, which can 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 SlU6 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 transcriptional 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 8 different lengths of tomato U6 promoters were obtained by two rounds of PCR. Their lengths were 452, 202, 448, 206, 433, 190, 448 and 218 bp, respectively. After sequences analysis, the 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 had transcriptional activity. The SlU6-2P4 promoter was chosen 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. The sequence analysis revealed that all types of gene mutations were base substitution and the hotspot of mutation only existed in the target region of endogenous gene. [Conclusion] Four kinds of SlU6 promoters with high transcription efficiency were obtained from tomato. The established CRISPR/Cas9 system based on SlU6-2 promoter could successfully achieve the editing of endogenous genes in tomato.

【Keywords】 CRISPR/Cas9; SlU6 promoter; clone; tomato; gene editing;

【DOI】

【Funds】 National Natural Science Foundation of China (31560534)

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(Translated by CHEN T)

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This Article

ISSN:0578-1752

CN: 11-1328/S

Vol 51, No. 02, Pages 315-326

January 2018

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Article Outline

Abstract

  • 0 Introduction
  • 1 Materials and methods
  • 2 Results
  • 3 Discussion
  • 4 Conclusion
  • References