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作物学报 ›› 2025, Vol. 51 ›› Issue (5): 1156-1165.doi: 10.3724/SP.J.1006.2025.44161

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

黄麻U6启动子克隆及其转录活性分析

黄梦欣1,2(), 庄灵玲1,2, 程佩佩1,2, 李秦1,2, 徐建堂1,2, 陶爱芬1,2, 方平平1,2, 祁建民1,2, 张立武1,2,*()   

  1. 1福建农林大学作物遗传育种与综合利用教育部重点实验室 / 农业农村部闽台作物生物育种重点实验室 / 福建省作物设计育种重点实验室, 福建福州 350002
    2福建农林大学农业农村部东南黄红麻科学观测实验站 / 福建省麻类种质资源共享平台 / 福建省南方经济作物遗传育种与多用途开发国际科技合作基地, 福建福州 350002
  • 收稿日期:2024-09-25 接受日期:2025-01-23 出版日期:2025-05-12 网络出版日期:2025-02-07
  • 通讯作者: *张立武, E-mail: lwzhang@fafu.edu.com
  • 作者简介:E-mail: 2429616851@qq.com
  • 基金资助:
    国家自然科学基金项目(32472219);福建省自然科学基金项目(2023J01443);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-16);福建农林大学科技创新专项基金(KFB23001);福建农林大学科技创新专项基金(KFB24080)

Cloning and transcriptional activity analysis of U6 promoter in jute (Corchorus capsularis)

HUANG Meng-Xin1,2(), ZHUANG Ling-Ling1,2, CHENG Pei-Pei1,2, LI Qin1,2, XU Jian-Tang1,2, TAO Ai-Fen1,2, FANG Ping-Ping1,2, QI Jian-Min1,2, ZHANG Li-Wu1,2,*()   

  1. 1Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University / Key Laboratory of Ministry of Agriculture and Rural Affairs for Biological Breeding of Fujian and Taiwan Crops / Fujian Key Laboratory for Crop Breeding by Design, Fuzhou 350002, Fujian, China
    2Experiment Station of Ministry of Agriculture and Rural Affairs for Jute and Kenaf Scientific Observation in Southeast China, Fujian Agriculture and Forestry University / Public Platform of Fujian for Germplasm Resources of Bast Fiber Crops / Fujian International Science and Technology Cooperation Base for Genetics, Breeding and Multiple Utilization Development of Southern Economic Crops, Fuzhou 350002, Fujian, China
  • Received:2024-09-25 Accepted:2025-01-23 Published:2025-05-12 Published online:2025-02-07
  • Contact: *E-mail: lwzhang@fafu.edu.com
  • Supported by:
    National Natural Science Foundation of China(32472219);Fujian Provincial Natural Science Foundation(2023J01443);China Agriculture Research System of MOF and MARA(CARS-16);Science and Technology Innovation Project of Fujian Agriculture and Foresty University(KFB23001);Science and Technology Innovation Project of Fujian Agriculture and Foresty University(KFB24080)

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摘要：

U6启动子是CRISPR/Cas9体系中驱动单向导RNA (single guide RNA, sgRNA)转录的重要元件, 内源U6启动子相比外源U6启动子通常具有更高的启动效率。然而, 目前黄麻内源U6启动子的研究还尚未见报道。本研究利用拟南芥保守的sgRNA AtU6-26序列, 从黄麻“梅峰4号”基因组中克隆到相似性最高的CcU6.1与CcU6.3两个候选启动子。通过构建CcU6.1与CcU6.3分别驱动GUS报告基因的融合表达载体, 利用农杆菌介导的转化法分别转染本氏烟草叶片和黄麻毛状根, 通过GUS组织化学染色分析启动子的转录活性。同源比对结果显示, CcU6.1与CcU6.3启动子均具有影响U6启动子转录活性的2个必要元件USE和TATA box。GUS组织化学染色表明, 黄麻这2个U6启动子均具有转录活性, 但在烟草叶片和黄麻毛状根中CcU6.1启动子的转录活性均弱于CcU6.3启动子, 荧光定量PCR进一步验证了这一结果。考虑到过长的U6启动子可能会削弱其转录活性, 于是比较分析CcU6.3与AtU6-26启动子的顺式作用元件, 发现CcU6.3启动子5′端截短后的序列即从转录起始位点至-550 bp位置, 可能会进一步提高其转录活性。本研究率先在黄麻中克隆到具有较高转录活性的U6启动子CcU6.3, 为构建黄麻属CRISPR/Cas9基因编辑系统提供了应用潜力的启动子。

关键词: 黄麻, U6启动子, CRISPR/Cas9, 本氏烟草, 毛状根, 转录活性

Abstract:

The U6 promoter is a critical element for driving the transcription of single guide RNA (sgRNA) in the CRISPR/Cas9 system, with endogenous U6 promoters often exhibiting higher efficiency than exogenous ones. However, no studies to date have focused on endogenous U6 promoters in jute (Corchorus L.). In this study, two candidate U6 promoters, CcU6.1 and CcU6.3, were cloned from the genome of the jute cultivar “Meifeng 4” using conserved sequences from the Arabidopsis thaliana U6-26 sgRNA promoter (AtU6-26). Fusion expression vectors carrying GUS reporter genes driven by the CcU6.1 and CcU6.3 promoters were constructed, and the transcriptional activities of these promoters were evaluated through Agrobacterium-mediated transformation of tobacco (Nicotiana benthamiana) leaves and jute hairy roots. Promoter activity was determined based on GUS histochemical staining. Homology analysis revealed that both CcU6.1 and CcU6.3 promoters contained two essential elements for U6 promoter activity: the USE and TATA boxes. GUS staining demonstrated that both jute U6 promoters exhibited transcriptional activity, although the CcU6.1 promoter showed weaker activity compared to the CcU6.3 promoter in both Nicotiana benthamiana leaves and jute hairy roots. Quantitative PCR further confirmed these findings. Since excessively long U6 promoters may reduce transcriptional efficiency, a comparative cis-regulatory element analysis of the CcU6.3 promoter and the AtU6-26 promoter were conducted. This analysis suggested that a truncated version of the CcU6.3 promoter, spanning from the transcriptional start site to the -550 bp region, could enhance transcriptional activity. This study is the first to identify and characterize the CcU6.3 promoter, which exhibits relatively high transcriptional activity in jute. The CcU6.3 promoter holds significant potential as a strong and efficient promoter for constructing CRISPR/Cas9 gene-editing systems in Corchorus species.

Key words: jute, U6 promoter, CRISPR/Cas9, Nicotiana benthamiana, hairy roots, transcriptional activity

表1

黄麻U6启动子克隆的引物序列"

引物名称Primer name 引物序列Primer sequence (5'-3')
Cc.U6.1-F ggccgcccccttcaccCTAACAGAACAAGCTTCTTATGTTTGC
Cc.U6.1-R cggcgcgcccacccttATCTTGAGTTGCTACTTTCTTATATAAAAAGGC
Cc.U6.3-F ggccgcccccttcaccCTATATGCAGCAAATGTTTTCCTCC
Cc.U6.3-R cggcgcgcccacccttACCCTCTTTGGTACTTTCTGATATATAAAC

图1

黄麻CcU6与拟南芥AtU6-26启动子序列的比较"

图2

黄麻U6启动子PCR扩增及中间载体鉴定 a: 黄麻U6启动子PCR扩增产物; M: DL2000 DNA marker; 1: CcU6.1; 2: CcU6.3。b: CcU6.1连接到中间载体阳性鉴定。c: CcU6.3连接到中间载体阳性鉴定。"

图3

黄麻U6启动子表达载体的PCR鉴定 M: DL2000 DNA marker。a: 表达载体在大肠杆菌TOP10中阳性鉴定; b: 表达载体在根癌农杆菌GV3101中阳性鉴定; c: 表达载体在发根农杆菌K599中阳性鉴定; d: PRNTR载体示意图; e: PGWB533载体示意图。"

图4

黄麻U6启动子在本氏烟草叶片中的GUS组织化学染色"

图5

黄麻U6启动子在黄麻毛状根中的GUS组织化学染色"

图6

黄麻2个U6启动子驱动GUS基因荧光定量PCR检测与比较 数据为3个启动子的转录活性。**表示在0.01概率水平差异显著, ***表示在0.001概率水平差异显著。"

图7

CcU6.3顺式作用元件预测 TATA-Box: 转录起始位点的核心启动子元件; CAAT-box: 启动子和增强子区域共有的顺式作用元件; Box 4: 参与光响应的顺式作用调控元件保守DNA模块的一部分; ARE: 厌氧性的顺式调控元件; AuxRR-core: 参与生长素响应的顺式作用调控元件。"

图8

CcU6.3与AtU6-26转录相关元件比较"

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