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Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (08): 1160-1167.doi: 10.3724/SP.J.1006.2016.01160

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Construction of tgw6 Mutants in Rice Based on CRISPR/Cas9 Technology

WANG Jia-Feng,ZHENG Cai-Min,LIU Wei,LUO Wen-Long,WANG Hui,CHEN Zhi-Qiang*,GUO Tao*   

  1. National Engineering Research Center of Plant Space Breeding, South China Agricultural University, Guangzhou 510642, China?
  • Received:2015-12-17 Revised:2016-05-09 Online:2016-08-12 Published:2016-05-23
  • Contact: 郭涛,E-mail: guoguot@scau.edu.cn, Tel: 020-38604903; 陈志强,E-mail: chenlin@scau.edu.cn, Tel: 020-85283237 E-mail:bcjfwang@gmail.com
  • Supported by:

    This study was supported by Public Welfare Research and Capacity Building Transformation Funds in Guangdong (20150209), the National High Technology Research and Development Program of China (863 Program) (2011AA10A101) and Special Funds for the Construction of Modern Agricultural Industry Technology System (CARS-01-12).

Abstract:

A set of tgw6 (Thousand grain weight 6) mutants were constructed using CRISPR/Cas9 technology in this study. Three sites of 20 nt guide RNA (gRNA) targeted to the exon of TGW6 were designed and transcribed from the U3, U6a and U6b promoters, respectively. The three target sites of gRNA were then ligated to the vector pYLCRISPR/Cas9-MT(I) based on golden gate cloning strategy. The recombinant plasmid was transferred to a rice cultivar, H447 (R819/Yuzhenxiang//R819 BC3F6) by Agrobacterium-mediated transformation. Sequencing for the genomic DNA of TGW6 locusinT0 rice showed the mutagenesis frequency for TGW6 was more than 90%, including 51% of the homozygous deletion mutations. Further analysis for the T1 mutants showed almost all the homozygous deletion mutants improved the thousand grain weight significantly (more than 5%). The successful tgw6 editing not only provided a series of tgw6 mutants for high and stable yield of rice but also proved that CRISPR/Cas9 is a facile and powerful means of rice genetic engineering for scientific and agricultural applications, which has important theoretical and practical significance for rice breeding.

Key words: Rice, Genome editing, CRISPR/Cas9, TGW6, Thousand grain weight

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