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Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (05): 675-683.doi: 10.3724/SP.J.1006.2016.00675

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

Cloning and Functional Analysis of Different TruncatedGbU6Promoters in Cotton

LEI Jian-Feng1,LI Yue1,XU Xin-Xia2,AERZUGULI•Tashi1,PU Yan1,ZHANG Ju-Song1,LIU Xiao-Dong1,*   

  1. 刘晓东, E-mail: xiaodongliu75@aliyun.com;张巨松, E-mail: xjndzjs@163.com
  • Received:2015-10-25 Revised:2016-01-11 Online:2016-05-12 Published:2016-02-18
  • Contact: Liu Xiaodong, E-mail: xiaodongliu75@aliyun.com;Zhang Jusong, E-mail: xjndzjs@163.com E-mail:15299175640@163.com
  • Supported by:

    This study was supported by the National Natural Science Foundation of China(31560534) and the Xinjiang Uygur Autonomous Region Graduate Science and Technology Innovation Projects for Graduate Students (XJGRI2015084).

Abstract:

U6 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system. Using a short promoter is also one of the basic requirements for the construction of CRISPR/Cas9 genomeediting vector. According to the sequence of GbU6-5Ppromoter cloned, six different truncated U6promoterswere successfully cloned using Transfer PCR method and their length were 672, 468, 358, 280, 202 and 105 bp, respectively.Together with GbU6-5P::GUS-pCAMBIA1300, GUS fusion expression vectors driven bycorresponding truncated promoter were constructed and transformed into cotton pollen by the vacuum infiltrationtransformation method.Results of GUS histochemical staining showed that the cloned seven truncatedGbU6-5Ppromoters could drive GUS expression in cotton pollen and all corresponding cottonpollen could be stained blue, but there exist different shades among them. Results showed that the shorter promoter, the stronger transcription activity, and so did the two other GbU6 promoter. In this study three short GbU6 promoters withtranscription activity in cotton pollen were cloned. GUS staining results showed that the shorter U6 promoter had higher transcriptional activity, which was the common characteristics in different U6 promoter.The above results indicated that using shorter U6 promoter not onlyconform to the requirement of constructing CRISPR/Cas9 genome editing vector,but also improve the transcription of sgRNA, which mayenhance the efficiency of genome editing finally.

Key words: Cotton, GbU6 promoter, Truncated cloning, Vacuum infiltration, Cotton pollen

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