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作物学报 ›› 2018, Vol. 44 ›› Issue (7): 1000-1009.doi: 10.3724/SP.J.1006.2018.01000

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米低磷胁迫诱导型强启动子P1502-ZmPHR1的克隆与表达分析

杨瑞娟1,2,白建荣2,*(),闫蕾2,苏亮2,王秀红3,李锐2,张丛卓2   

  1. 1 山西大学生物工程学院, 山西太原 030006
    2 山西省农业科学院作物科学研究所, 山西太原 030031
    3 山西省农业科学院现代农业研究中心, 山西太原 030031
  • 收稿日期:2018-01-02 接受日期:2018-03-26 出版日期:2018-07-10 网络出版日期:2018-04-10
  • 通讯作者: 白建荣
  • 基金资助:
    本研究由山西省农业科学院农业科技创新项目(ZDSYS1507)资助

Cloning and Expression Analysis of Strong Inducible Promoter P1502-ZmPHR1 Responding to Low Phosphorus Stress in Maize

Rui-Juan YANG1,2,Jian-Rong BAI2,*(),Lei YAN2,Liang SU2,Xiu-Hong WANG3,Rui LI2,Cong-Zhuo ZHANG2   

  1. 1 College of Biology Engineering, Shanxi University, Taiyuan 030006, Shanxi, China
    2 Institute of Crop Sciences, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
    3 Research Center of Modern Agriculture, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
  • Received:2018-01-02 Accepted:2018-03-26 Published:2018-07-10 Published online:2018-04-10
  • Contact: Jian-Rong BAI
  • Supported by:
    This study was supported by the Agricultural Science and Technology Innovation Program of Shanxi Academy of Agricultural Sciences (ZDSY1507).

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摘要：

玉米ZmPHR1基因是转录因子MYB-CC家族成员, 过量表达该基因可促进低磷环境中植物对磷的吸收并改善植物的生长。本研究从磷高效利用型玉米自交系478中获得了ZmPHR1基因的5°上游序列P2205-ZmPHR1, 序列长度2205 bp。将P2205-ZmPHR1及它的4种5°端缺失启动子片段与报告基因GUS连接后转化拟南芥。GUS组织化学染色与GUS定量分析结果表明, 在0~1502 bp的片段范围内, 随着片段长度的增加, 它对外界低磷环境的响应逐步增加, 在-1502 bp时, 响应时间短, 表达活性高, 持续时间长; 超过1502 bp其活性不但没有增高, 反而变为下降或无变化。推测0至-972 bp为启动子功能区段, -972 bp至-1502 bp为启动功能增强区段。转P1502-ZmPHR1拟南芥的实时定量PCR结果显示, P1502-ZmPHR1既受外界信号的刺激, 也受体内基因产物的影响, 有反馈调节的作用, 是一个受低磷胁迫诱导的强诱导型启动子, 它诱导基因表达的功能受体内外磷浓度的调节。该研究为在低磷环境改良作物提供了理想的启动子和应用依据。

关键词: 玉米, 低磷胁迫, 强诱导型启动子, P1502-ZmPHR1

Abstract:

ZmPHR1, a member of transcription factor MYB-CC family, enhanced phosphate absorption and improved plant growth when it was overexpressed under low-phosphate conditions. In order to elucidate the transcriptional regulatory mechanism of the promoter of ZmPHR1, its 5′ upstream sequence P2205-ZmPHR1 was cloned from P-efficient maize inbred line 478. The full length of P2205-ZmPHR1 and four sequence deletion fragments in different lengths fused with the reporter gene GUS were transformed into Arabidopsis, respectively. The results of histochemical and quantitative fluorometric GUS assay showed that in the interval from 0 to -1502 bp, the response time and activity of sequence deletion fragments were increased with increasing fragment length. However, the activities were decreased or unchanged when the length of fragments were longer than 1502 bp. It was considered that the interval from 0 to -972 bp was function region and that from -972 bp to -1502 bp was function enhancing region. The results of qPCR analysis in transgenic plants at different time points (0, 1, 2, 3, and 4 days) after P-stress treatment showed that the activities of P1502-ZmPHR1 were influenced by both external signals and internal gene products, having feedback regulation. This study indicates that P1502-ZmPHR1 is a strong inducible promoter responding to low phosphorus stress and its functional activity is regulated by both external and internal phosphate concentration. These fundings provide an ideal promoter for crop improvement under low phosphate conditions and a basis for its application.

Key words: maize, low phosphorus stress, strong inducible promoter, P1502-ZmPHR1

表1

PCR所用引物"

引物名称
Primer name		 序列
Sequence (5'-3')
SF1 GTCGACTTGTGATCGCAGTACGAAAA
SF2 GTCGACCATCACACCTGTACTTGGC
SF3 GTCGACTGCTGGGCTTTTGATTCC
SF4 GTCGACCAGGTGGACCCACGTATC
SF5 GTCGACCCTGTCGTGTTGGACTTG
SR AGATCTacCATTCAGAGCAGTGAACAAGCTC
qGUS-F CCGTCCCAAGCAGTTACAAT
qGUS-R CGATACGCTGATCCTTCAGATAG
AtACT2-qRT-F GGAATCGTTCACAGAAAATG
AtACT2-qRT-R CAAACAAATGGAGAAGCAAA

附图1

玉米478与B73 ZmPHR1基因5'上游序列比对"

图1

P2205-ZmPHR1扩增片段"

图2

P2205-ZmPHR1顺式作用元件分析"

图3

P2205-ZmPHR1 5°端缺失片段顺式作用元件示意图"

图4

P2205-ZmPHR1 5°端缺失片段扩增图"

图5

T1转基因拟南芥的PCR检测"

图6

低磷胁迫条件下转基因拟南芥GUS染色分析"

图7

低磷胁迫条件下转基因拟南芥GUS定量分析*和**分别表示低磷胁迫处理与对照(0 h)在0.05和0.01概率水平差异显著(t检验)。"

图8

低磷胁迫条件下转基因拟南芥TP1502-ZmPHR1表达量分析"

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