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作物学报 ›› 2022, Vol. 48 ›› Issue (9): 2242-2254.doi: 10.3724/SP.J.1006.2022.11079

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦TaPIP1基因克隆及其在花药开裂中潜在功能分析

谭照国1,2(), 苑少华2(), 李艳梅2, 白建芳2, 岳洁茹2, 刘子涵2, 张天豹2, 赵福永1, 赵昌平2, 许本波1, 张胜全2,*(), 庞斌双2,*(), 张立平1,2,*()   

  1. 1.长江大学生命科学学院, 湖北荆州 434025
    2.北京市农林科学院杂交小麦所 / 北京杂交小麦分子遗传北京市重点实验室, 北京 100097
  • 收稿日期:2021-09-08 接受日期:2022-01-05 出版日期:2022-09-12 网络出版日期:2022-02-15
  • 通讯作者: 张胜全,庞斌双,张立平
  • 作者简介:谭照国, E-mail: tanzhaoguo@foxmail.com;
    苑少华, E-mail: keaidehuahua0830@126.com第一联系人：

    **同等贡献

  • 基金资助:
    国家自然科学基金项目(31872881);北京市农林科学院杰出科学家项目(JKZX201907);北京市农林科学院科技创新能力建设专项(KJCX20210439)

Cloning of TaPIP1 gene and its potential function in anther dehiscence in wheat

TAN Zhao-Guo1,2(), YUAN Shao-Hua2(), LI Yan-Mei2, BAI Jian-Fang2, YUE Jie-Ru2, LIU Zi-Han2, ZHANG Tian-Bao2, ZHAO Fu-Yong1, ZHAO Chang-Ping2, XU Ben-Bo1, ZHANG Sheng-Quan2,*(), PANG Bin-Shuang2,*(), ZHNAG Li-Ping1,2,*()   

  1. 1. College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China
    2. Beijing Academy of Agriculture and Forestry Science Research Institute of Hybrid Wheat / Beijing Key Laboratory of Molecular Genetics in Hybrid Wheat, Beijing 100097, China
  • Received:2021-09-08 Accepted:2022-01-05 Published:2022-09-12 Published online:2022-02-15
  • Contact: ZHANG Sheng-Quan,PANG Bin-Shuang,ZHNAG Li-Ping
  • About author:First author contact:

    ** Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(31872881);Beijing Excellent Talents Project and Outstanding Scientist Program of BAAFS(JKZX201907);Special Project of Science and Technology Innovation Ability Construction of BAAFS(KJCX20210439)

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摘要：

二系杂交小麦育种是小麦产量提高的重要途径之一。光温敏雄性不育小麦生殖生长过程中花药的发育及开裂情况直接影响杂交小麦的制种效率和产量。植物花药的开裂与脱水活动紧密相关。水通道蛋白(aquaporins, AQPs)是高效转运水分及特异小分子的膜内在蛋白, 其中质膜内在蛋白(plasma membrane intrinsic proteins, PIPs)在水分的吸收与外排中发挥着重要作用。为进一步了解水通道蛋白在小麦光温敏核雄性不育系花药开裂中的作用提供理论基础。本研究以不育系BS366花药的cDNA为模板, 克隆获得了TaPIP1基因。利用生物信息学软件对TaPIP1进行分析, 该基因包含一个879 bp的开放阅读框, 编码292个氨基酸。TaPIP1的启动子区存在赤霉素、脱落酸、茉莉酸和光等响应元件。TaPIP1属于MIP超家族, 具有典型的NPA保守结构域, 亚细胞定位于细胞质膜与核膜上。miRNA互作预测发现TaPIP1受tae-miR1131与tae-miR408的剪切抑制, 表明TaPIP1可能和与植物的抗氧化能力相关。通过蛋白互作预测及qPCR实验, 表明TaPIP1可与热激蛋白(heat shock protein 90, TaHSP90)相互作用, 在高温和干旱复合胁迫下, 参与花药细胞壁膨压调控, 进而调控花药的开裂。

关键词: 小麦, 水通道蛋白, 热激蛋白, miRNA, 花药开裂

Abstract:

Hybrid wheat breeding is one of the important ways to improve wheat yield. The development and dehiscence of anthers in photoperiod-temperature sensitive genic male sterile (PTGMS) wheat affect the seed production efficiency and yield of hybrid wheat directly. Anther dehiscence is closely related to dehydration. Aquaporins (AQPs) are membrane intrinsic proteins that efficiently transport water and specific small molecules. Plasma membrane intrinsic proteins (PIPs) play an important role in water absorption and efflux in plant cells. In order to understand the roles of PIP in anther dehiscence of PTGMS. In this study, TaPIP1 was cloned from the anther of PTGMS line BS366. The gene contained an open reading frame (ORF) of 879 bp, encoding a total of 292 amino acids. There were cis-responsive elements such as gibberellin, abscisic acid, jasmonic acid, and light in the promoter region of TaPIP1. The promoter region of TaPIP1 contained elements of gibberellin, abscisic acid, jasmonic acid, and light. TaPIP1 belonged to MIP superfamily, and had a typical NPA conserved domain, subcellular localication in the plasma membrane and nuclear membrane. The interaction between miRNAs and TaPIP1 was predicted by bioinformatics analysis, and the results showed that TaPIP1 may be regulated by tae-miR1131 and tae-miR408 that related to the antioxidant capacity of plants. Protein-protein interaction networks (PPI) and qRT-PCR experiments revealed that TaPIP1 could interact with heat shock protein 90 (TaHSP90), participating in the regulation of anther cell wall turgor under the combined stress of high temperature and drought, thus regulating anther dehiscence.

Key words: wheat, aquaporins, heat shock protein, miRNA, anther dehiscence

附表1

引物序列及用途"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Usage
TaPIP1-F CACCTCTCTCAACCAAGCCAA 基因扩增
Gene amplification
TaPIP1-R TTGCACGCGGTTTAATGGAG
TaPIP1-DF TATCTCTAGAGGATCCATGGAGGGCAAGGAGGA 亚细胞定位
Subcellular localization
TaPIP1-DR TGCTCACCATGGATCCCTTCAAGAGCCGCGAC
TaPIP1-QF TTCCAGACCACGCTGTACCA 实时荧光定量PCR
qPCR
TaPIP1-QR TGTTGGGACGTTCGTGCTGGTG
TaHSP90-QF TGAGTCCTAGTGGTCGCTGC
TaHSP90-QR CAGGGAACAAACTCCCTCAGT
tae-miR1131-F TACCGGTTCGTGGCTAACCAA
TaActin1-QF CCTACATTGCCCTGGACTACGAC 内参基因
Reference gene
TaActin1-QR GCAACGGAAACGCTCAGAACCA
TaU6-F GGAACGATACAGAGAAGATTAGC

附表2

生物信息分析使用的数据库及软件"

工具Tool 网址Website
Ensembl plants http://plants.ensembl.org/
ExPaSy http://web.expasy.org/
Prabi https://npsa-prabi.ibcp.fr/
SWISSMODEL http://swissmodel.expasy.org/
NCBI conserved domains https://www.ncbi.nlm.nih.gov/
SignalP 4.1 Server http://www.cbs.dtu/
ProtScale https://web.expasy.org/
NetPhos http://www.cbs.dtu/
Plant CARE http://bioinformatics.psb.ugent.be/
MEME http://meme-suite.org/
PsRNA Target http://www.zhaolab.org/
STRING https://version11.string-db.org/
plantgrn http://plantgrn.noble.org/
Cell-PLoc 2.0 http://www.csbio.sjtu.edu.cn/
expvip http://www.wheat-expression.com/

图1

TaPIP1的PCR扩增及其CDS序列分析 A: TaPIP1的克隆; M: DL2000 marker; 1: TaPIP1扩增产物。B: TaPIP1的编码序列分析。"

图2

TaPIP1的信号肽与结构预测结果 A: 信号肽; B~C: 三级结构; D: 保守结构域; E: 外显子区域。"

表1

启动子元件分析"

上游启动子元件
Upstream promoter element		 功能
Function
TATA-box 核心启动子元件 Core promoter element
ABRE 脱落酸响应元件 Abscisic acid responsiveness element
CGTCA-motif, TGACG-motif 茉莉酸甲酯响应元件 MeJA-responsiveness element
GARE-motif 赤霉素响应元件 Gibberellin responsive element
MRE, ACE, TCT-motif, GATA-motif, I-box 光响应元件 Light responsiveness element

图3

TaPIP1与其他植物PIP氨基酸序列比对 LOC100839000: 二穗短柄草; KAE8779834: 大麦; LOC112879877: 黍; LOC8059708: 高粱; LOC542014: 玉米; LOC101785185: 小米; LOC102719100: 水稻。"

图4

TaPIP1与其他物种PIP的系统进化树分析和motif预测 LOC109743575: 节节麦; LOC119325066: 拟二粒小麦; KAE8779834: 大麦; AHX84137: 羊草; 778372: 大麦; LOC100845597: 二穗短柄草; LOC8059708: 高粱; LOC112879877: 黍; LOC542014: 玉米; LOC101785185: 小米; LOC109708083: 菠萝; THU54116: 芭蕉; LOC102719100: 水稻; LOC100839000: 二穗短柄草; ASF57558: 海稗草。"

表2

基序序列"

基序Motif 基序序列Motif sequence
Motif 1 WSFGGMIFVLVYCTAGISGGHINPAVTFGLFLARKLSLTRAVFYIVMQCL
Motif 2 PIGFAVFLVHLATIPITGTGINPARSLGAAIIYNKKQAWDDHWIFWVGPF
Motif 3 DEKDYKEPPPAPLFEAGELTSWSFYRAGIAEFLATFLFLYISVLTVMGVV
Motif 4 GANSVAPGYTKGDGLGAEIVGTFVLVYTVFSATDAKRSARDSHVPILAPL
Motif 5 MEGKEEDVRLGANRYSERQPI
Motif 6 AALAAIYHVVVIRAIPFKSRD
Motif 7 GAICGAGVVKGFQTTLYQGNG
Motif 8 PSGSKCGTVGIQGIA
Motif 9 GTAAQG

图5

TaPIP与miRNA的互作分析 A: TaPIP1与miRNA互作图; B: tae-miR1131的发卡结构; C: tae-miR408的发卡结构。"

图6

TaPIP1的蛋白互作预测与热图分析 A: TaPIP1的蛋白互作预测; B: 不同处理下互作蛋白的聚类热图(蓝点: TaHSP90; 绿点: TaPIP1)。"

图7

TaPIP1的小麦原生质体亚细胞定位"

图8

TaPIP1及TaHSP90在小麦不同花药发育时期及组织中的表达 A: TaPIP1与TaHSP90的FPKM值; B: 不同育性环境下小麦不同花药发育时期TaPIP1与TaHSP90的相对表达量; C: TaPIP1在小麦不同组织中的相对表达量。a、b、c、d: TaPIP1的表达在P = 0.005显著性差异水平; α、β、χ、δ、ε: TaHSP90的表达在P = 0.005显著性差异水平。"

图9

高温与低温处理下TaPIP1与TaHSP90、tae-miR1131的相对表达量 A: 不同处理条件下3个花药发育时期TaPIP1、TaHSP90和tae-miR1131在花药中的相对表达量; B: 不同处理条件下TaPIP1、TaHSP90和tae-miR1131在叶片、茎和颖壳中的相对表达量。a、b、c、d、e: TaPIP1的表达在P=0.005显著性差异水平; α、β、χ、δ、ε: TaHSP90的表达在P = 0.005显著性差异水平; 1、2、3、4、5、6: tae-miR1131的表达在P = 0.005显著性差异水平。"

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doi: 10.1007/s11676-019-01082-w
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