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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (9): 2242-2254.doi: 10.3724/SP.J.1006.2022.11079

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2022-09-12 Published:2022-02-15
  • Contact: ZHANG Sheng-Quan,PANG Bin-Shuang,ZHNAG Li-Ping E-mail:tanzhaoguo@foxmail.com;keaidehuahua0830@126.com;lpzhang8@126.com;cp_zhao@vip.sohu.com;zsq8200@126.com
  • 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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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

Table S1

Primer sequences used in this study"

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

Table S2

Database and software used for bioinformatics analysis in this study"

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/

Fig. 1

PCR amplification and sequence analysis of TaPIP1 gene A: cloning of TaPIP1 gene; M: DL2000 marker; 1: TaPIP1 PCR product. B: CDS sequence analysis of TaPIP1 gene."

Fig. 2

Prediction of signal peptide and domain of TaPIP1 A: signal peptide; B-C: tertiary structure of the protein; D: conservative domain; E: exon regions."

Table 1

Analysis of promoter elements"

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

Fig. 3

Amino acid sequence alignment of TaPIP1 and PIP in other plants LOC100839000: Brachypodium distachyon (L.) Beauv.; LOC100839000: Hordeum vulgare L.; LOC112879877: Panicum hallii var. hallii; LOC8059708: Sorghum bicolor (L.) Moench; LOC542014: Zea mays L.; LOC101785185: Setaria italica (L.) Beauv.; LOC102719100: Oryza brachyantha."

Fig. 4

Phylogenetic tree of TaPIP1 and PIP in other species and their corresponding motif prediction LOC109743575: Aegilops tauschii subsp. strangulata; LOC119325066: Triticum dicoccoides; KAE8779834: Hordeum vulgare L.; AHX84137: Leymus chinensis; 778372 Hordeum vulgare L.; LOC100845597: Brachypodium distachyon (L.) Beauv.; LOC8059708: Sorghum bicolor (L.) Moench; LOC112879877: Panicum hallii var. halli; LOC542014: Zea mays L.; LOC101785185: Setaria italica (L.) Beauv.; LOC109708083: Ananas comosus (Linn.) Merr.; THU54116: Musa balbisiana; LOC102719100: Oryza brachyantha; LOC100839000: Brachypodium distachyon (L.) Beauv.; ASF57558: Paspalum vaginatum."

Table 2

Motif sequences of TaPIP1"

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

Fig. 5

Interaction analysis of TaPIP1 with miRNA A: TaPIP1 and miRNA interaction; B: miRNA hairpin of tae-miR1131; C: miRNA hairpin of tae-miR408."

Fig. 6

Protein interaction prediction and heatmap of TaPIP1 A: network diagram of TaPIP1 protein interaction; B: cluster heat maps of protein interactions under different stress (Blue point: TaHSP90; Green point: TaPIP1)."

Fig. 7

Subcellular localization of TaPIP1 in wheat protoplasts"

Fig. 8

Relative expression of TaPIP1 and TaHSP90 in different anther development stages and tissues in wheat A: FPKM values of TaPIP1 and HSP90; B: relative expression of TaPIP1 and TaHSP90 during the development of different flower medicines in wheat under different breeding environments; C: relative expression of TaPIP1 in different tissues of wheat. “a, b, c, and d”: significant different at P = 0.005 level for the relative expression of TaPIP1; “α, β, χ, δ, and ε”: significant different at P = 0.005 level for the relative expression of TaHSP90."

Fig. 9

Relative expression of TaPIP1, TaHSP90, and tae-miR1131 under high and low temperature treatment A: relative expression of TaPIP1, HSP90, and tae-miR1131 in three anther development stages under different treatments; B: relative expression of TaPIP1, HSP90 and tae-miR1131 in leaf, stem and gume under different treatments. “a, b, c, d, and e”: significant different at P=0.005 level for the relative expression of TaPIP1; “α, β, χ, δ, and ε”: significant different at P = 0.005 level for the relative expression of TaHSP90; “1, 2, 3, 4, 5, and 6”: significant different at P = 0.005 level for the relative expression of tae-miR1131."

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