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作物学报 ›› 2022, Vol. 48 ›› Issue (4): 851-859.doi: 10.3724/SP.J.1006.2022.13013

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

过表达ZmCIPKHT基因增强植物耐热性

许静1(), 高景阳1, 李程成2, 宋云霞1, 董朝沛1, 王昭1, 李云梦1, 栾一凡1, 陈甲法2, 周子键2,*(), 吴建宇1,2,*()   

  1. 1河南农业大学农学院, 河南郑州 450002
    2河南农业大学生命科学学院, 河南郑州 450002
  • 收稿日期:2021-02-03 接受日期:2021-07-12 出版日期:2022-04-12 网络出版日期:2021-08-10
  • 通讯作者: 周子键,吴建宇
  • 作者简介:E-mail: 709823684@qq.com
  • 基金资助:
    河南省自然科学基金重点项目资助(162300410130)

Overexpression of ZmCIPKHT enhances heat tolerance in plant

XU Jing1(), GAO Jing-Yang1, LI Cheng-Cheng2, SONG Yun-Xia1, DONG Chao-Pei1, WANG Zhao1, LI Yun-Meng1, LUAN Yi-Fan1, CHEN Jia-Fa2, ZHOU Zi-Jian2,*(), WU Jian-Yu1,2,*()   

  1. 1College of Agriculture, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2College of Life Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2021-02-03 Accepted:2021-07-12 Published:2022-04-12 Published online:2021-08-10
  • Contact: ZHOU Zi-Jian,WU Jian-Yu
  • Supported by:
    Key Project of Henan Natural Science Foundation(162300410130)

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摘要：

高温胁迫对植物正常生长发育及产量的影响越来越显著。为了适应外界环境的变化, 植物进化出了一系列应对高温胁迫的分子遗传机制。类钙调磷酸酶B蛋白(CBL)互作蛋白激酶(CIPK), 在ABA信号转导途径上积极参与植物对高温胁迫的响应。在前期全基因组关联分析的基础上, 本实验克隆了一个与玉米耐高温性状相关的候选基因ZmCIPKHT, qRT-PCR结果表明ZmCIPKHT基因受高温胁迫的显著诱导。室内表型鉴定的实验发现过表达ZmCIPKHT的转基因拟南芥植株在高温胁迫下, 比野生型的存活率显著提高, 生长状态更好。玉米原生质体瞬时转化实验证明ZmCIPKHT蛋白定位于细胞核中。酵母双杂交实验验证了ZmCIPKHT蛋白与玉米CBLs家族中的ZmCBL4蛋白存在互作关系。同时, ZmCIPKHT转基因拟南芥在高温胁迫条件下, 脱落酸(ABA)通路相关基因的表达水平有其相应的变化, 揭示了ZmCIPKHT可能依赖于ABA信号转导通路来增强植物的耐热性。这些结果为解析玉米CBL-CIPK信号通路依赖于ABA途径对植物非生物胁迫响应的分子机制提供了新的实验根据。

关键词: 玉米, 耐热性, 蛋白激酶, CBL-CIPK, ABA信号通路

Abstract:

The effects of high temperature stress on the normal growth and yield of plants is more and more serious. To adapt the changes of external environment, plants have evolved a series of molecular genetic mechanisms to respond to high temperature stress. The calcineurin B-like protein (CBL) interacting protein kinase (CIPK) is actively involved in response to high temperature stress depended on ABA signal transduction pathway in plants. Based on previous genome-wide association analysis, a candidate gene ZmCIPKHT related to maize high temperature tolerance was cloned in this study. Real-time quantitative PCR results showed that ZmCIPKHT gene was significantly induced by high temperature stress. Transient transformation of maize protoplasts revealed that ZmCIPKHT was localized in the nucleus. Overexpressing ZmCIPKHT plants of transgenic Arabidopsis thaliana had significantly higher survival rate and better growth status than wild type under high temperature stress. The yeast two-hybrid experiment confirmed that the interaction between ZmCIPKHT protein and ZmCBL4 protein in maize CBLs family. The relative expression levels of genes related to abscisic acid (ABA) pathway in transgenic Arabidopsis thaliana with ZmCIPKHT under high temperature stress were changed accordingly, indicating that the regulations of ZmCIPKHT genes under high temperature stress were in the ABA-dependent pathway. These results provide a new experimental basis for elucidating the molecular mechanism of maize CBL-CIPK signaling pathway dependent on ABA pathway to abiotic stress in plants.

Key words: maize, heat tolerance, protein kinase, CBL-CIPK, ABA signal pathway

表1

本实验中所用到的引物"

引物
Primer ID		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
qZmCIPKHT-F/R CCATAACCAGATCACGTCAAAA GGTAACCTGAAACACAATTCCG
EF1α-F/R TGGGCCTACTGGTCTTACTACTGA ACATACCCACGCTTCAGATCCT
ZmCIPKHT-F/R CGGGGGACTCTTGACCATGGTAATGGACGAGAGGAGGACTA TACTAGTCAGATCTACCATGGTCTGCTGCGTCGGCAAA
PGBKT7-ZmCIPKHT-F/R ATGGCCATGGAGGCCGAATTCATGGACGAGAGGAGGACTATTTTG CCGCTGCAGGTCGACGGATCCCTACTGCTGCGTCGGCAAA
qPYL2-F/R GTCAGAGAAGTGACCGTAATCT CGACGTCACTGATTTCTAGTTC
qFOA1-F/R GCTTACATCGTTGGAGAAG GCACAGAACTTGGAACATAA
qHAI2-F/R TACATCCGTCTTTTGTACGGAA CGTGAAGTCTCTCTTTACACCT
qSnRK2.3-F/R ATTTCAAGAATCTTCGTGGCTG CTGTGTTGCTCTCGTTCATTAG

图1

ZmCIPKHT的蛋白结构域分析和植物中CIPK蛋白的系统进化树"

图2

ZmCIPKHT基因的表达分析"

图3

ZmCIPKHT过表达拟南芥株系的分子检测 A: ZmCIPKHT过表达株的检测; B: ZmCIPKHT的转录检测; WT: 野生型; OE: 过表达株系。"

图4

高温胁迫处理后转基因拟南芥的耐热鉴定及存活率统计 A: 高温胁迫后转基因拟南芥的表型鉴定; B: 高温胁迫后转基因拟南芥存活率统计; WT: 野生型拟南芥; OE: 过表达拟南芥, 数据为3次生物学重复±标准差。"

图5

正常条件和高温胁迫条件下野生型WT和过表达转基因拟南芥OE-4、OE-6株系ABA通路相关基因的表达分析 将野生型材料WT和过表达拟南芥株系OE-4、OE-6进行37℃ 1周的高温胁迫处理, 检测ABA通路相关基因表达量, 数据为3次生物学重复±标准差。"

图6

玉米ZmCIPKHT蛋白在原生质体中的亚细胞定位 GFP: GFP标记的ZmCIPKHT在玉米原生质体的亚细胞定位; Bright: 同一视野光镜下的原生质体; Merged: 光镜及荧光照片的叠加; 35S:GFP: 转入空载体的原生质体; 35S:ZmCIPKHT:GFP: 转入目的载体的原生质体。标尺为10 μm。"

图7

酵母双杂交实验检测ZmCIPKHT与ZmCBLs的互作"

图8

ZmCIPKHT介导的Ca2+途径依赖ABA信号通路调控玉米耐热性的模式图"

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