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作物学报 ›› 2016, Vol. 42 ›› Issue (05): 675-683.doi: 10.3724/SP.J.1006.2016.00675

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花不同GbU6启动子截短克隆及功能鉴定

雷建峰1,李月1,徐新霞2,阿尔祖古丽•塔什1,蒲艳1,张巨松1,刘晓东1,*   

  1. College of Agronomy, Xinjiang Agricultural University, Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi 830052, China;
    Center of Bazhou Agricultural Technology Promotion, Korla 841000, China
  • 收稿日期:2015-10-25 修回日期:2016-01-11 出版日期:2016-05-12 网络出版日期:2016-02-18
  • 通讯作者: 刘晓东, E-mail: xiaodongliu75@aliyun.com;张巨松, E-mail: xjndzjs@163.com
  • 基金资助:

    本研究由国家自然科学基金项目(31560534)和新疆维吾尔自治区研究生科研创新项目(XJGRI2015084)资助。

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 Published:2016-05-12 Published online:2016-02-18
  • Contact: Liu Xiaodong, E-mail: xiaodongliu75@aliyun.com;Zhang Jusong, E-mail: xjndzjs@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).

摘要:

U6启动子是CRISPR/Cas9基因组编辑载体系统中驱动sgRNA转录的重要元件, 而使用较短序列的启动子也是构建CRISPR/Cas9基因组编辑载体的基本要求之一。将已经克隆的海岛棉GbU6-5P启动子(长度为1166bp), 采用Transfer PCR方法成功地截出6个长度不同的U6启动子, 其长度分别为672、468、358、280、202和105bp, 并分别构建了6个启动子驱动的GUS融合植物表达载体。将构建好的6个GbU6-5Ps::GUS-pCAMBIA1300与初始克隆的GbU6-5P::GUS-pCAMBIA1300植物表达载体一起利用农杆菌真空渗透转化法分别转染棉花花粉。GUS组织化学染色显示, 克隆到的7个不同截短大小的GbU6-5Ps启动子均能驱动GUS基因在棉花花粉中转录, 棉花花粉被染成蓝色但颜色深浅存在显著差异。结果显示启动子长度越短, 其转录活性越高。而且另外两种棉花U6启动子GbU6-1P和GbU6-7P也表现出类似的结果。本研究克隆了3个短小的、在棉花花粉细胞中具有转录功能的GbU6启动子。结果显示更短的U6启动子具有更高的转录活性, 而且这一特点在不同U6启动子上具有共性。这预示着使用更短U6启动子不仅符合构建CRISPR/Cas9基因组编辑载体的要求, 而且会提高sgRNA的转录水平, 进而可能提高基因组编辑效率。

关键词: 棉花, GbU6启动子, 截短克隆, 真空渗透, 棉花花粉

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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