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作物杂志,2020, 第4期: 114–120 doi: 10.16035/j.issn.1001-7283.2020.04.016

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

玉米早期籽粒中强表达启动子的筛选

王莉1,2(), 王作平2(), 张中保2, 白玲1(), 吴忠义2()   

  1. 1河南大学省部共建作物逆境适应与改良国家重点实验室,475004,河南开封
    2北京市农林科学院北京农业生物技术研究中心/北京市农业基因资源和生物技术重点实验室,100097,北京
  • 收稿日期:2020-01-02 修回日期:2020-03-05 出版日期:2020-08-15 发布日期:2020-08-11
  • 通讯作者: 白玲,吴忠义
  • 作者简介:王莉,研究方向为细胞生物学,E-mail： 15537870386@163.com;|王作平为并列第一作者,研究方向为玉米分子育种,E-mail： wangzuoping@baafs.net.cn
  • 基金资助:
    北京市科委项目(Z171100001517001);北京市农林科学院储备性项目(KJCX20200407);北京市农林科学院储备性项目(KJCX20200104)

Screening of Strongly Expressed Promoters in Immature Maize Kernels

Wang Li1,2(), Wang Zuoping2(), Zhang Zhongbao2, Bai Ling1(), Wu Zhongyi2()   

  1. 1State Key Laboratory of Crop Stress Adaption and Improvement, Henan University, Kaifeng 475004, Henan, China
    2Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences/Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2020-01-02 Revised:2020-03-05 Online:2020-08-15 Published:2020-08-11
  • Contact: Bai Ling,Wu Zhongyi

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摘要：

在植物基因表达调控的过程中,启动子作为调控基因表达的顺式元件起着重要作用。为了筛选在玉米早期籽粒中强表达的启动子,选取6个启动子pCaMV35SD、pUbiquitin、pZmActin1、pZmSTK2、pZm66589和pZmbHLH148,分别构建EGFP表达载体并转染玉米原生质体,快速验证载体功能构建的正确性;同时采用花粉磁转染法将6个表达载体导入玉米自交系郑58中,并对不同启动子驱动的EGFP载体在授粉后48h玉米籽粒中的荧光强度和荧光检出率进行观察和统计分析。结果表明,6个启动子驱动EGFP表达载体构建正确;pCaMV35SD启动子驱动EGFP表达载体的荧光最强,6个启动子驱动EGFP表达载体由强到弱依次为pCaMV35SD>pZmSTK2>pZm66589>pZmbHLH148>pUbiquitin>pZmActin1,其荧光检出率分别为27.17%、27.17%、29.83%、23.84%、13.40%和30.57%。

关键词: 启动子, 花粉磁转染法, 玉米籽粒, 绿色荧光蛋白, 玉米原生质体

Abstract:

Promoters play an important role in regulating gene expression. To screen strongly expressed promoters in immature maize kernels, six promoters, pCaMV35SD, pUbiquitin, pZmActin1, pZmSTK2, pZm66589, and pZmbHLH148, were for EGFP expression in this study. Maize protoplasts were transfected to quickly verify the vector function. At the same time, six expression vectors were introduced into Zheng58 by pollen magnetic transfection. Later, the expression of EGFP driven by different promoters in immature maize kernels were observed 48h after pollination. The results showed that the expression of EGFP was detected in case of all six promoters but the fluorescence of EGFP driven by pCaMV35SD promoter was the strongest, and the order of EGFP fluorescence strength driven by six promoters was pCaMV35SD > pZmSTK2 >pZm66589 > pZmbHLH148 > pUbiquitin > pZmActin1, with the fluorescence detection rate of 27.17%, 27.17%, 29.83%, 23.84%, 13.40% and 30.57%, respectively.

Key words: Promoter, Pollen magnetofection, Maize kernel, Green fluorescent protein, Maize protoplast

表1

试验中启动子基本信息

启动子
Promoter		 长度
Length (bp)		 表达类型
Expression type		 参考文献
Reference
pCaMV35SD 770 组成型表达 [5]
pZmActin1 1 210 组成型表达 [6]
pUbiquitin 2 014 组成型表达 [7]
pZmSTK2 2 067 花粉特异性表达 [14]
pZm66589 2 000 胚特异性表达 [20]
pZmbHLH148 1 512 胚特异性表达 [21]

表2

试验中所用的引物

引物Primer 引物序列（5′-3′）Primer sequence (5′-3′)
Bar-F CTCTAGAAATGAGCCCAGAACGACGC (XbaⅠ)
Bar-R CGGATCCAGATCTTGGCAGGATATA (BamHⅠ)
pUbiquitin-EGFP-F ATCCTGCCAAGATCTGGATCCCGGTCGTGCCCCTCTCTAG
pUbiquitin-EGFP-R GCCCTTGCTCACCATGGTACCCTGCAGAAGTAACACCAAACAACAG
EGFP-F CCGGCATGCAAGCTGATAACG
EGFP-R GACACGCTGAACTTGTGGCC

图1

6个启动子-EGFP表达载体构建流程图 a. pYBA1132的T-DNA区;b. pYBA1132-Bar-EGFP的T-DNA区;c. pYBA1132-Bar-EGFP(-NptII)的T-DNA区;d. pCaMV35SD驱动EGFP的T-DNA区;e. pZmActin1、pZmSTK2、pZm66589、pZmbHLH148和pUbiquitin分别代表其他5个启动子的序列,可驱动EGFP表达,其T-DNA区与图1d中pCaMV35SD类似

图2

6个启动子-EGFP表达载体酶切鉴定 Marker：Trans 15 000bp DNA标记条带

图3

不同启动子驱动EGFP在原生质体中的表达 EGFP：绿色荧光场;Bright：明场;Merged：明场及荧光场的叠加;CK：未转染质粒的郑58原生质体;pCaMV35S：转入pYBA1132的原生质体;pCaMV35SD、pZmActin1、pZmSTK2、pZm66589、pZmbHLH148和pUbiquitin：6个启动子驱动EGFP重组载体转化的玉米原生质体;Bar=5μm,下同

图4

不同启动子驱动EGFP在郑58早期籽粒中的瞬时表达情况 CK：授粉48h后的空白对照早期籽粒;MNP：仅MNP处理的早期籽粒;pCaMV35SD、pZmActin1、pUbiquitin、pZmSTK2、pZmbHLH148和pZm66589：分别转入相对应启动子质粒的早期籽粒;Bar=200μm

表3

分别转染6个EGFP载体的郑58籽粒在授粉后48h的荧光检出率

启动子Promoter 荧光检出率Fluorescence detection rate (%)
pZmActin1 30.57±12.55a
pZm66589 29.83±9.16a
pCaMV35SD 27.17±7.96ab
pZmSTK2 27.17±7.96ab
pZmbHLH148 23.84±8.27ab
pUbiquitin 13.40±5.80b
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