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核桃JrTT1-1启动子不同长度片段响应干旱的活性分析

谢牧洪 李文凯 张昭龙 崔茂凯 王艺 孙雅薇 杨桂燕

谢牧洪, 李文凯, 张昭龙, 崔茂凯, 王艺, 孙雅薇, 杨桂燕. 核桃JrTT1-1启动子不同长度片段响应干旱的活性分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210126
引用本文: 谢牧洪, 李文凯, 张昭龙, 崔茂凯, 王艺, 孙雅薇, 杨桂燕. 核桃JrTT1-1启动子不同长度片段响应干旱的活性分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20210126
Xie Muhong, Li Weikai, Zhang Zhaolong, Cui Maokai, Wang Yi, Sun Yawei, Yang Guiyan. Activity analysis of different length fragments of walnut JrTT1-1 promoter in response to drought stress[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210126
Citation: Xie Muhong, Li Weikai, Zhang Zhaolong, Cui Maokai, Wang Yi, Sun Yawei, Yang Guiyan. Activity analysis of different length fragments of walnut JrTT1-1 promoter in response to drought stress[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20210126

核桃JrTT1-1启动子不同长度片段响应干旱的活性分析

doi: 10.12171/j.1000-1522.20210126
基金项目: 国家自然科学基金(32171804、31800510),西北农林科技大学卓越农林创新实验计划项目(2018011117)
详细信息
    作者简介:

    谢牧洪。主要研究方向:林木逆境生理与分子生物学。Email:643929078@qq.com 地址:712100 陕西省杨凌示范区西北农林科技大学林学院

    责任作者:

    杨桂燕,博士,副教授。主要研究方向:林木遗传育种。Email:yangguiyan@nwsuaf.edu.cn 地址:同上

  • 中图分类号: S664.1

Activity analysis of different length fragments of walnut JrTT1-1 promoter in response to drought stress

  • 摘要:   目的  TTl是C2H2-ZFP(WIP型锌指结构)类转录因子调控蛋白,核桃JrTT1-1基因启动子含有干旱响应元件,具有调控干旱胁迫的功能。本研究通过分离JrTT1-1基因不同长度启动子片段并对其受干旱胁迫后的表达活性进行分析,探讨JrTT1-1基因响应干旱胁迫的机制。  方法  根据WRKY顺式作用元件的分布,将JrTT1-1基因启动子分为1 002 bp(−1 ~ −1 002)、720 bp(−1 ~ −720)、448 bp(−1 ~ −448)、174 bp(−1 ~ −174)、149 bp(−1 ~ −149)5个片段,分别记为S1、S2、S3、S4、S5。用S1、S2、S3、S4、S5分别替换pCAMBIA1301载体的CaMV35S启动子构建重组载体,通过农杆菌介导的蘸花法转化拟南芥,经潮霉素筛选、PCR验证及GUS基因表达分析确定后培养至T3代。对不同生长期不同组织进行GUS酶活性测定,评价不同片段的时空表达活性。将S1、S2、S3、S4、S5转基因植株种子萌发生长30 d进行干旱处理(50 mmol/L甘露醇),未干旱处理的设为对照(CK),分析整株、根及地上部分GUS酶活性,评价不同片段响应干旱的差异。  结果  正常生长条件下,S1、S2、S3、S4、S5转基因拟南芥在不同生长时期、不同组织器官中均能检测出GUS酶活性,但不同片段GUS活性具有差异,且随着片段变短,活性降低;但S1和S2之间的差异不显著。比较成熟种子、鲜种子、35 d根、茎、叶、花的GUS活性,发现不同组织之间也有区别,体现了5个片段的组织表达特异性。与CK相比,干旱胁迫下,5个片段整株、根和地上部分的GUS活性均显著提高,其中干旱胁迫后S1、S2、S3、S4、S5全株的GUS活性分别为CK的1.50、1.46、1.47、1.46、2.23倍,根GUS活性分别为CK的1.29、1.29、1.28、1.53、1.36倍,地上部分GUS活性分别为CK的1.62、1.59、1.57、1.59、2.30倍。  结论  JrTT1-1基因启动子片段的表达活性与其长度呈正相关性,每个长度启动子片段活性具有根、茎、叶、花、种子等组织特异性;WRKY元件及其数量可能与干旱胁迫调节作用相关,且JrTT1-1启动子在干旱胁迫下的表达也具有组织差异性。

     

  • 图  1  依据WRKY识别元件划分的5个不同长度启动子片段

    Figure  1.  Situation of 5 promoter fragments of different length divided according to WRKY recognition motif

    图  2  启动子片段插入pCAMBIA1301的重组载体示意图

    Figure  2.  Schematic diagram of the recombinant vector with the promoter fragment inserted into pCAMBIA1301

    图  3  5个片段转入拟南芥各株系的GUS基因表达量

    S11 ~ S18、S21 ~ S28、S31 ~ S38、S41 ~ S48、S51 ~ S58分别为转S1、S2、S3、S4、S5片段的8个株系。S11 − S18, S21 − S28, S31 − S38, S41 − S48, S51 − S58 are 8 lines transformed with S1, S2, S3, S4 and S5 fragments, respectively.

    Figure  3.  GUS gene expression levels of 5 fragments transferred Arabidopsis lines

    图  4  不同启动子片段转基因拟南芥株系不同生长期(A)和不同组织(B)的GUS活性

    图上小写字母表示同生长阶段或同组织5个启动子片段之间的差异显著性(P < 0.05)。Different lowercase letters above the bars indicate significant difference between 5 promoter segments in the same growth stage or tissue (P < 0.05).

    Figure  4.  GUS activity in different growth stages (A) and tissues (B) of transgenic Arabidopsis lines with different promoter fragments

    图  5  不同启动子片段转基因植株种子萌发不同天数(A)及不同组织(B)GUS活性与片段长度的相关性Fig. 5 Correlations between GUS activity and fragment length of transgenic plants with different promoter fragments for different days of seed germination (A) and different tissues (B)

    图  6  不同启动子片段转基因拟南芥株系在干旱胁迫下的GUS活性

    A ~ C分别为30 d大植株的整株、根、地上部分的GUS活性。*表示同一个株系在甘露醇处理下的GUS活性与对照比差异显著(P < 0.05)。A−C are the GUS activity of the whole plant, roots and aerial parts of 30-day-old plants, respectively. * indicates that the GUS activity of the same line under mannitol treatment is significantly different from the control (P < 0.05).

    Figure  6.  GUS activity of transgenic Arabidopsis lines with different promoter fragments under drought stress

    表  1  不同片段启动子插入pCAMBIA1301的引物

    Table  1.   Primers used for different promoter fragments inserted into pCAMBIA1301

    片段
    Fragment
    上游引物
    Forward primer
    下游引物
    Reverse primer
    S1 5′-ATGCGGTACCTTCACTGATCAGTACAATAT-3′ 5′-TAGCGGATCCGGTTAAAGATTTGATAGGAAG-3′
    S2 5′-ATGCGGTACCAAGTGACTCAAGTTACATAT-3′
    S3 5′-ATGCGGTACCTTGACGGTGGCAAGTGAAAAAG-3′
    S4 5′-ATGCGGTACCGTCTGCAGGGGGTAATTTCT-3′
    S5 5′-ATGCGGTACCTTCCATGCTTGCTTTCAGCT-3′
    注:上游引物中下划线为酶切位点Kpn1,下游引物中下划线为酶切位点BamH1。Notes: underline in the forward primer is Kpn1, and the underline in the reverse primer is BamH1.
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  • 收稿日期:  2021-04-02
  • 录用日期:  2022-07-08
  • 修回日期:  2021-04-26
  • 网络出版日期:  2022-07-12

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