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植物学报 >

2020 , Vol. 55 >Issue 3: 299 - 307

DOI: https://doi.org/10.11983/CBB19217

研究报告

毛竹不同截短U3启动子的克隆及表达分析

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  • 1中国林业科学研究院亚热带林业研究所, 杭州 311400
    2南京林业大学, 南京 210037

收稿日期: 2019-11-07

  录用日期: 2020-03-24

  网络出版日期: 2020-03-24

基金资助

中国林业科学研究院中央级公益性科研院所基本科研业务费专项资金(CAFYBB2020ZB004);浙江省基础公益研究计划(LGN18C160007)

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Cloning and Expression Analysis of Different Truncated U3 Promoters in Phyllostachys edulis

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  • 1The Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China
    2Nanjing Forestry University, Nanjing 210037, China

Received date: 2019-11-07

  Accepted date: 2020-03-24

  Online published: 2020-03-24

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摘要

具有明确转录起始位点的U3和U6启动子是CRISPR/Cas9技术中驱动sgRNA转录的重要元件。从毛竹(Phyllostachys edulis)中克隆了2个PeU3启动子, 均进行了3个不同长度的截短, 长度分别为550、397和149 bp及561、392和152 bp; 并分别构建6个启动子驱动的GUS及LUC植物表达载体, 再利用农杆菌(Agrobacterium tumefaciens)介导法分别转化麻竹(Dendrocalamus latiflorus)愈伤组织和烟草(Nicotiana benthamiana)叶片。结果显示, 这些PeU3启动子总体都具有转录活性, 不同PeU3启动子以及同一PeU3启动子不同截短时其转录活性不同, 其中长度为397 bp的PeU3-1-2pro启动子活性最强, 可为构建竹子CRISPR/Cas9基因组编辑体系提供更多理想的内源启动子。

关键词： 基因组编辑; LUC活性; PeU3启动子; 毛竹

本文引用格式

凡惠金, 金康鸣, 卓仁英, 乔桂荣 . 毛竹不同截短U3启动子的克隆及表达分析[J]. 植物学报, 2020 , 55(3) : 299 -307 . DOI: 10.11983/CBB19217

Abstract

The U3 and U6 promoters with well-defined transcription initiation sites are important elements driving sgRNA transcription in the CRISPR/Cas9 genome editing system. According to the two sequences of PeU3 promoter cloned from Phyllostachys edulis, six different truncated U3 promoters were successfully cloned and were 550 bp, 397 bp, 149 bp, and 561 bp, 392 bp, 152 bp, respectively in length. GUS and LUC expression vectors were constructed by corresponding truncated promoter and transformed into the callus of Dendrocalamus latiflorus and tobacco leaf by the Agrobacterium-mediated method, respectively. Our results indicate that, all of these U3 promoters have different transcriptional activity, and the Peu3-1-2pro promoter with a length of 397 bp has the strongest activity. It provides more ideal endogenous promoters for constructing CRISPR/Cas9 genome editing system of P. edulis.

Key words： genome editing; LUC activity; PeU3 promoter; Phyllostachys edulis

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