小麦TaPP2-A13基因的表达响应逆境胁迫并与SCF复合体接头蛋白TaSKP1相互作用

doi:10.3724/SP.J.1006.2021.01042

作物学报 ›› 2021, Vol. 47 ›› Issue (2): 224-236.doi: 10.3724/SP.J.1006.2021.01042

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

小麦TaPP2-A13基因的表达响应逆境胁迫并与SCF复合体接头蛋白TaSKP1相互作用

孟钰玉¹(), 魏春茹¹, 范润侨¹, 于秀梅¹^,²^,^*(), 王逍冬², 赵伟全², 魏新燕², 康振生³, 刘大群²

¹河北农业大学生命科学学院 / 河北省植物生理与分子病理学重点实验室, 河北保定 071001
²河北省农作物病虫害生物防治技术创新中心, 河北保定 071001
³西北农林科技大学植物保护学院 / 旱区作物逆境生物学国家重点实验室, 陕西杨凌 712100

收稿日期:2020-05-12 接受日期:2020-09-13 出版日期:2021-02-12 网络出版日期:2020-09-30
通讯作者: 于秀梅
作者简介:E-mail: 183656991@qq.com
基金资助:
河北省高等学校科学技术研究项目(ZD2019086);河北省自然科学基金项目(C2020204050);国家自然科学基金项目(31301649);旱区作物逆境生物学国家重点实验室开放课题基金(CSBAAKF2018008)

TaPP2-A13 gene shows induced expression pattern in wheat responses to stresses and interacts with adaptor protein SKP1 from SCF complex

MENG Yu-Yu¹(), WEI Chun-Ru¹, FAN Run-Qiao¹, YU Xiu-Mei¹^,²^,^*(), WANG Xiao-Dong², ZHAO Wei-Quan², WEI Xin-Yan², KANG Zhen-Sheng³, LIU Da-Qun²

¹College of Life Sciences, Hebei Agricultural University / Hebei Key Laboratory of Plant Physiology and Molecular Pathology, Baoding 071001, Hebei, China
²Technological Innovation Center for Biological Control of Crop Diseases and Insect Pests of Hebei Province, Baoding 071001, Hebei, China
³College of Plant Protection, Northwest A&F University / State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi, China

Received:2020-05-12 Accepted:2020-09-13 Published:2021-02-12 Published online:2020-09-30
Contact: YU Xiu-Mei
Supported by:
Higher Education Science and Technology Research Project of Hebei Province(ZD2019086);Natural Science Foundation of Hebei Province(C2020204050);National Natural Science Foundation of China(31301649);Open Research Fund of State Key Laboratory of Crop Stress Biology for Arid Areas(CSBAAKF2018008)

摘要/Abstract

摘要：

为探究韧皮部蛋白2 (Phloem protein 2, PP2)基因在小麦(Triticum aestivum)响应逆境胁迫中的功能及作用机制, 本文以小麦抗叶锈病近等基因系TcLr15为材料获得一个小麦韧皮部蛋白2基因TaPP2-A13。该基因编码一个由298个氨基酸组成的亲水性蛋白质, 相对分子量为33.18 kD, 等电点为6.36。其N端为F-box结构域, C端具有典型的PP2结构域, 属于F-box/PP2 (FBP)亚家族成员。TaPP2-A13与禾本科植物中PP2-A13蛋白的亲缘关系较近。表达模式分析表明, TaPP2-A13的表达受叶锈菌(Puccinia triticina)侵染的诱导, 且感病组合中表达更强。用脱落酸(ABA)、水杨酸(SA)和甲基茉莉酸(MeJA)处理后, TaPP2-A13总体呈现上调表达趋势。利用聚乙二醇(PEG)和H₂O₂处理后, 小麦TaPP2-A13显著下调表达, 而NaCl处理后TaPP2-A13呈现先上调后下调的表达趋势。亚细胞定位结果表明TaPP2-A13在细胞核和细胞质均有分布。以构建的重组载体BD-TaPP2-A13为诱饵筛选酵母文库, 获得11种可能与TaPP2-A13互作的蛋白, 酵母双杂交(Yeast 2 Hybrid, Y2H)确定其中的5种蛋白TaPP2C5、TaSLY1、TaCHI、TaRbcS和TaSKP1分别与TaPP2-A13存在相互作用。进一步利用BiFC和Co-IP确认TaSKP1与TaPP2-A13两种蛋白质之间的相互作用关系, 推测TaPP2-A13可能为SCF复合体成员。本研究结果为深入解析TaPP2-A13蛋白的功能, 并探究其调控网络奠定了基础。

关键词: 韧皮部蛋白2, 小麦叶锈病, 非生物逆境, 激素, 表达模式, 蛋白互作

Abstract:

To explore the function and molecular mechanism of Phloem protein 2 (PP2) gene in wheat (Triticum aestivum L.) response to stresses, a TaPP2-A13 putatively encoding a PP2 protein was obtained from TcLr15, a wheat near isogenic line against leaf rust pathogen, in the present study. The complete coding region of TaPP2-A13 encodes a hydrophilic polypeptide with molecular weight of 33.18 kD, and theoretical isoelectric point is 6.36. There is an F-box domain at N-terminal and a PP2 domain at C-terminal of the TaPP2-A13 protein sequence, which indicates that wheat TaPP2-A13 belongs to F-box/PP2 (FBP) subfamily. Wheat TaPP2-A13 shared relatively higher sequence similarity with PP2-A13 from Gramineae. Quantitative real-time PCR (qRT-PCR) results indicated that TaPP2-A13 was induced by infection of leaf rust pathogen (Puccinia triticina), and showed stronger expression in susceptible combination than that in resistant one. An obvious up-regulation of TaPP2-A13 was observed after treatment with abscisic acid (ABA), salicylic acid (SA) and methyl jasmonate (MeJA) in wheat. TaPP2-A13 was significantly down-regulated after treatment with PEG and H₂O₂, while TaPP2-A13 striking increased first, then fell down after NaCl treatment in wheat. Subcellular localization result indicated that TaPP2-A13 distributed in both of the nucleus and cytoplasm. The recombinant vector BD-TaPP2-A13 was used as the bait to screen Yeast 2 Hybrid (Y2H) library, 11 kinds of proteins were finally obtained. Further Y2H assays identified that TaPP2-A13 physically interacted with five kinds of proteins including TaPP2C5, TaSLY1, TaCHI, TaRbcS, and TaSKP1. BiFC and Co-IP results further confirmed that TaPP2-A13 interacted with TaSKP1, an adaptor protein from SKP1-Cullin-F-box (SCF) complex, which made us to speculate that TaPP2-A13 functions as a member of SCF complex by binding with TaSKP1. These findings laid some foundation for further analyzing the function of TaPP2-A13 and exploring its regulatory network.

Key words: phloem protein 2, wheat leaf rust, abiotic stresses, hormone, expression pattern, protein interaction

孟钰玉, 魏春茹, 范润侨, 于秀梅, 王逍冬, 赵伟全, 魏新燕, 康振生, 刘大群. 小麦TaPP2-A13基因的表达响应逆境胁迫并与SCF复合体接头蛋白TaSKP1相互作用[J]. 作物学报, 2021, 47(2): 224-236.

MENG Yu-Yu, WEI Chun-Ru, FAN Run-Qiao, YU Xiu-Mei, WANG Xiao-Dong, ZHAO Wei-Quan, WEI Xin-Yan, KANG Zhen-Sheng, LIU Da-Qun. TaPP2-A13 gene shows induced expression pattern in wheat responses to stresses and interacts with adaptor protein SKP1 from SCF complex[J]. Acta Agronomica Sinica, 2021, 47(2): 224-236.

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引物名称 Primer name	上游引物序列 Forward primer sequence (5'-3')	下游引物序列 Reverse primer sequence (5'-3')	退火温度 Annealing temperature (℃)
TaPP2-A13-F/R	AATGGCGGAATCCCTCGTG	CGGGTTTTGGACAAGAATGG	58.0
TaPP2-A13-qPCR-F/R	ATCGATGATCGGCGGTATTG	AGTGGAGCCGGAAGAGAAGG	59.5
TaGAPDH-qPCR-F/R	CTGCCTTGCTCCTCTTGCTAA	CTTGATGGAAGGACCAGCAAC	59.5
TaPP2-A13-His-F/R	gctgatatcggatccgaattcATGGGGGC GGGGGCTTCG	tgcggccgcaagcttgtcgacTTACTTGCGTATGC ACTCCTCG	60.0
TaPP2-A13-GFP-F/R	atacaccaaatcgactctagaATGGGGGC GGGGGCTTCG	catggtaccggatccactagtCTTGCGTATGCACT CCTCGG	60.0
TaPP2-A13-BD-F/R	CGgaatccATGGGGGC	ggatccTTACTTGCGTATGCACT	60.0
AD-F/R	TAATACGACTCACTATAGGGCG	AGATGGTGCACGATGCACAG	56.0
TaPP2-A13-NE-F/R	cccaggcctactagtggatccATGGGGGC GGGGGCTTCG	agcggtaccctcgaggtcgacCTTGCGTATGCACT CCTCGG	60.0
TaSKP1-FLAG-F/R	gagaacacgggggactctagaATGGCGG CCGCGGGAGAC	cgtcctaggcttaagtctagaCTCAAAGGCCCACT GGTTCTC	60.0
TaPP2-A13-HA-F/R	agacttaagcctaggacgcgtATGGGGGC GGGGGCTTCG	AtcgtatgggtacatacgcgtCTTGCGTATGCACT CCTCGG	60.0

登录号 Accession No.	蛋白名称 Protein name	克隆数 Number of clones
XP_020153404.1	Protein phosphatase 2C 5 (PP2C5)	1
KU857044.1	S-phase kinase-associated protein 1 (SKP1)	1
XM_020332687.1	UDP-glucose flavonoid 3-O-glucosyl transferase 7-like	3
EMS45698.1	SEC1 family transport protein SLY1	1
XM_020338494.1	3-ketoacyl-CoA thiolase 2, peroxisomal-like	2
LM992844.1	Rca1_beta gene for RUBISCO activase beta (TaRca1_beta)	3
KC776912.1	Ribulose activase A	2
AK330458.1	Ribulose-bisphosphate carboxylase small chain (RbcS)	1
AF251264.1	Ribulose bisphosphate carboxylase activase B (RcaB)	3
AY123222.1	Putative Fe-S precursor protein	2
BAB82472.1	Chitinase 2	1

小麦TaPP2-A13基因的表达响应逆境胁迫并与SCF复合体接头蛋白TaSKP1相互作用

TaPP2-A13 gene shows induced expression pattern in wheat responses to stresses and interacts with adaptor protein SKP1 from SCF complex

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