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作物学报 ›› 2021, Vol. 47 ›› Issue (3): 472-480.doi: 10.3724/SP.J.1006.2021.03027

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

质膜内在蛋白ZmPIP1;1参与玉米耐旱性和光合作用的功能分析

周练, 刘朝显, 熊雨涵, 周京, 蔡一林*()   

  1. 西南大学玉米研究所 / 农业科学研究院 / 南方山地作物逆境生物学国家级培育基地, 重庆 400715
  • 收稿日期:2020-05-14 接受日期:2020-09-13 出版日期:2021-03-12 网络出版日期:2020-10-06
  • 通讯作者: 蔡一林
  • 作者简介:E-mail: zhoulianjojo@swu.edu.cn
  • 基金资助:
    国家自然科学基金项目资助(31601312)

Functional analysis of plasma membrane intrinsic protein ZmPIP1;1 involved in drought tolerance and photosynthesis in maize

ZHOU Lian, LIU Chao-Xian, XIONG Yu-Han, ZHOU Jing, CAI Yi-Lin*()   

  1. Maize Research Institute / Academy of Agricultural Sciences / State Cultivation Base of Crop Stress Biology for Southern Mountainous Land, Southwest University, Chongqing 400715, China
  • Received:2020-05-14 Accepted:2020-09-13 Published:2021-03-12 Published online:2020-10-06
  • Contact: CAI Yi-Lin
  • Supported by:
    National Natural Science Foundation of China(31601312)

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摘要：

质膜内在蛋白(plasma membrane intrinsic proteins, PIPs)是水通道蛋白主要亚家族成员之一, 在植物生长发育过程中具有重要调节功能。前期研究表明, 玉米ZmPIP1;1基因表达受到渗透和盐胁迫的强烈诱导, 但其在玉米中的生物学功能尚不明确。本研究通过玉米遗传转化获得了ZmPIP1;1超表达转基因株系, 干旱胁迫实验揭示了ZmPIP1;1超表达转基因株系较野生型具有较低的水分散失率及较强的干旱胁迫耐性。转录组测序结果表明参与ABA生物合成及其信号通路相关基因的表达水平发生了显著变化。在田间正常生长条件下, ZmPIP1;1超表达转基因植株与野生型在生长发育过程中没有明显差异, 但转基因玉米株系具有较高的光合效率, 粒宽和百粒重增加, 玉米单果穗的产量提高。此外, 通过荧光双分子互补实验观察到ZmPIP1;1和ZmPIP2;6蛋白在玉米叶肉细胞原生质体的细胞质膜和叶绿体膜上存在互作, 并且可能导致了ZmPIP蛋白的重定位。该研究为ZmPIP1;1分子机制的解析奠定了重要基础, 为玉米高光效分子设计育种开辟了新的途径。

关键词: 玉米, ZmPIP, 干旱胁迫, 光合作用, 重定位

Abstract:

Plasma membrane intrinsic protein (PIP) is one of the main subfamily of aquaporin regulates diverse physiology functions during plant growth and development. In previous research, the expression of ZmPIP1;1 was induced by osmosis or salt stress. However, the biological function of ZmPIP1;1 was still unclear in maize. In this study, ZmPIP1;1 overexpressed transgenic plants were obtained and exhibited less water loss rate and enhanced drought tolerance compared to wild type. Transcriptome sequencing indicated significant changes in the expression levels of genes involved in ABA biosynthesis and its signaling pathways. Photosynthetic activity, kernel width and kernel weight was increased in ZmPIP1;1 overexpression maize, but not growth under normal condition compared with wild type. Moreover, interaction between ZmPIP1;1 and ZmPIP2;6 was observed by bimolecular fluorescence complementation (BiFC) experiment, resulting in re-localized on plasma membrane and chloroplast in maize mesophyll protoplast. Our study laid an important foundation for understanding the molecular mechanism of ZmPIP1;1, and provided a new method of molecular breeding for high photosynthetic efficiency.

Key words: maize, ZmPIP, drought stress, photosynthesis, re-localization

图1

ZmPIP1;1超表达转基因玉米的检测 A: ZmPIP1;1超表达载体的T-DNA序列示意图。B: ZmPIP1;1超表达T1代转基因株系的PCR检测; 1~5: 独立的转基因株系; MK: DNA marker; PC: 质粒正对照; WT: 野生型。C: 4个独立的ZmPIP1;1超表达T3代转基因株系的qRT-PCR分析。"

图2

ZmPIP1;1超表达转基因植株在干旱处理下的表型以及水分散失率 A: 野生型和ZmPIP1;1超表达转基因玉米干旱处理15 d的表型。B: 土培干旱处理后不同基因型植株与正常条件下相比的相对地上部分生物量, 数值为4个生物学重复的平均值, 误差线为标准误。C: 野生型和ZmPIP1;1超表达转基因株系叶片水分散失率的检测。*表示转基因玉米叶片的水分散失率相比野生型在P < 0.01水平差异显著。"

图3

保守序列的聚类同源组(COG)的功能分类 T和G柱分别代表“信号传导机制”和“碳水化合物运输和代谢”。"

图4

玉米ABA生物合成与信号通路的关键基因的表达分析 正常和干旱胁迫条件下野生型和ZmPIP1;1超表达株系叶中(A)参与ABA合成的ZmNCED9-1、ZmNCED9-2、ZmABA1和ZmAAO3基因; (B)参与ABA信号通路的ZmABF1、ZmPYL2、ZmABI5和ZmSnRK3基因的表达。所有数据为3个生物学重复, 误差线代表SD。*表示转基因株系与野生型的基因表达水平在P < 0.01时差异显著。ZmACT1的表达作为内参。"

图5

野生型和ZmPIP1;1超表达转基因玉米植株的AN和Ci 所有数据为平均值±标准误(n = 4); *表示转基因玉米植株的AN和Ci相比野生型在P < 0.01水平差异显著。"

图6

ZmPIP1;1超表达转基因玉米籽粒的表型鉴定 野生型和ZmPIP1;1转基因玉米籽粒表型测定(A)和照片(B)。*表示转基因玉米籽粒的十粒宽、每穗粒重和百粒重相比野生型在P < 0.01水平差异显著。标尺为1 cm。"

图7

玉米原生质体中ZmPIP1;1与ZmPIP2;6的BiFC实验观察 pCAMBIA-ZmPIP2;6-GFP、pDOE-ZmPIP1;1-ZmPIP2;6和pDOE- ZmPIP2;6-ZmPIP1;1载体与mCherry标记的质膜标记(mCherry- PM; CD3-1007)共定位。标尺为20 μm。"

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