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

Abstract

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

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

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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