莲藕多酚氧化酶互作蛋白的筛选及验证

doi:10.3864/j.issn.0578-1752.2020.18.013

中国农业科学 ›› 2020, Vol. 53 ›› Issue (18): 3777-3791.doi: 10.3864/j.issn.0578-1752.2020.18.013

莲藕多酚氧化酶互作蛋白的筛选及验证

原新博¹(),程婷婷¹,惠小涵¹,陈章玉¹,王瑞红²,柯卫东³,郭宏波¹()

¹西北农林科技大学化学与药学院/中药指纹图谱国家地方联合工程研究中心/陕西省中药指纹图谱与天然产物库研究中心,陕西杨凌 712100
²西北农林科技大学生命科学学院,陕西杨凌 712100
³武汉市农业科学院蔬菜研究所,武汉430065

收稿日期:2020-02-22 接受日期:2020-07-13 出版日期:2020-09-16 发布日期:2020-09-25
通讯作者: 郭宏波
作者简介:原新博,E-mail： xinboyuan678@163.com。
基金资助:
国家重点研发计划(2016YFD0100204-29);国家杨凌农业高新技术产业示范区科技计划(2018SF-08)

Screening of Polyphenol Oxidase Interaction Proteins from Nelumbo nucifera and Their Verification

YUAN XinBo¹(),CHENG TingTing¹,XI XiaoHan¹,CHEN ZhangYu¹,WANG RuiHong²,KE WeiDong³,GUO HongBo¹()

¹College of Chemistry and Pharmacy, Northwest Agriculture and Forestry University/State Local Joint Research Center of TCM Fingerprint/Shaanxi Research Center of TCM Fingerprint and Natural Products Library, Yangling 712100, Shaanxi
²College of Life Sciences Northwest Agriculture and Forestry University, Yangling 712100, Shaanxi
³Institute of Vegetable, Wuhan Academy of Agricultural Sciences, Wuhan 430065

Received:2020-02-22 Accepted:2020-07-13 Online:2020-09-16 Published:2020-09-25
Contact: HongBo GUO

摘要/Abstract

摘要：

【目的】以中国莲（Nelumbo nucifera Gaertn.）的多酚氧化酶家族成员之一NnPPO1（GenBank: ADC92563.1）为研究对象,筛选、验证与其互作的蛋白,为深入研究莲藕PPO的分子作用机制、精准抑制其活性奠定基础。【方法】利用酵母双杂交（Y2H）技术,验证莲藕抗氧化酶是否与NnPPO1存在互作关系。通过转化酵母试验检测互作蛋白过氧化氢酶同工酶NnCAT1（GenBank:XP_010242894.1）的毒性和自激活活性。构建双分子荧光标记试验（BiFC）所用的35S-NnPPO1-SPYNER173、35S-NnCAT1-SPYCEM表达载体,通过农杆菌介导法,将重组质粒转化烟草,进一步验证NnPPO1与NnCAT1之间的互作关系。根据NnPPO1和NnCAT1的结构域截短蛋白,寻找互作关键结构域。利用PlantCARE分析NnPPO1与NnCAT1启动子相关作用元件,利用实时荧光定量PCR（qRT-PCR）分析NnPPO1与NnCAT1在莲藕不同组织中的表达水平。采用同源重组的方法构建35S-NnCAT1-GFP融合蛋白表达载体,用于亚细胞定位分析。运用DNAMAN软件对NnCAT1序列和其他物种序列进行多序列比对。运用生物信息学网站对NnCAT1进行理化性质与结构分析。【结果】NnCAT1与NnPPO1存在蛋白互作,且NnCAT1蛋白无自激活作用,对酵母菌株也没有毒性。在细胞膜和细胞核上观察到黄色荧光信号,进一步证实NnPPO1与NnCAT1之间存在互作。NnPPO1的酪氨酸酶结构域在蛋白互作中发挥主要作用。NnPPO1与NnCAT1启动子序列上存在多种顺式调控元件,如光响应元件Box 4、GT1-motif、TCT-motif,逆境响应元件ARE以及激素响应元件CGTCA-motif、TGACG-motif等。NnPPO1与NnCAT1的表达模式基本相同,在各组织中均有表达,均在叶中表达量最高,在茎尖中表达量最低。亚细胞定位结果显示NnCAT1定位于细胞膜和细胞核中。莲藕NnCAT1的相对分子质量约为57.0 kD,理论等电点（PI）为6.93;该蛋白为同源四聚体亲水性蛋白质;无跨膜结构和信号肽,存在叶绿体转运肽,前21 aa为转运肽区域;二级结构由27.64%的α-螺旋、15.65%延长链、6.30%β-转角和50.41%无规则卷曲构成。【结论】筛选出NnCAT1与NnPPO1之间存在蛋白互作,用BiFC进一步证实两者互作的真实性,NnPPO1保守的酪氨酸酶结构域在互作中发挥主要作用;NnPPO1与NnCAT1的表达模式基本相同,推测NnPPO1与NnCAT1协同互作导致莲藕等果蔬褐变。

关键词: 莲藕, NnPPO1, NnCAT1, 蛋白互作, 亚细胞定位, 组织特异性表达

Abstract:

【Objective】The protein interacting with NnPPO1 (GenBank: ADC92563.1), a member of the polyphenol oxidase family of China Lotus (Nelumbo nucifera Gaertn.), was screened and verified, which laid the foundation for further studying of the molecular mechanism of PPO and precise inhibition of its activity in Nelumbo nucifera. 【Method】The interaction between Nelumbo nucifera antioxidant enzyme and NnPPO1 was verified by yeast double hybridization (Y2H). The toxicity and self-activation activity of the interacting protein catalase isoenzyme NnCAT1 (GenBank: XP_010242894.1) were detected by the transformed yeast experiment. The expression vectors of 35S-NnPPO1-SPYNER173 and 35S-NnCAT1-SPYCEM used in the BiFC experiment were constructed. The recombinant plasmid was transformed into tobacco by an agrobacteria-mediated method to further verify the interaction between NnPPO1 and NnCAT1. The proteins were truncated based on the domains of NnPPO1 and NnCAT1 to find the key domains of interaction. PlantCARE and real-time fluorescent quantitative PCR (qRT-PCR) were used to analyze the promoter-related components of NnPPO1 and NnCAT1 as well as their expression levels in different tissues of Nelumbo nucifera. The 35S-NnCAT1-GFP fusion protein expression vector was constructed by homologous recombination method to analyze subcellular localization. DNAMAN software was used to perform multi-sequence alignment between the NnCAT1 sequence and other species sequences. The basic character and structure of NnCAT1 were analyzed by using bioinformatics website. 【Result】NnCAT1 interacts with NnPPO1, had no self-activation or toxicity to yeast. The interaction between NnPPO1 and NnCAT1 was further confirmed by the yellow fluorescence signal observed on the cell membrane and nucleus. The tyrosinase domain of NnPPO1 played a major role in protein interactions. NnPPO1 and NnCAT1 promoter sequences contained a variety of cis-regulatory elements, such as photoresponse element (Box 4, GT1-motif, and TCT-motif), stress response element (ARE), and hormone response element (CGTCA-motif and TGACG-motif). The expression patterns of NnPPO1 and NnCAT1 were almost the same. They were expressed in all tissues, with the highest expression in leaf and lowest expression in stem tip. Subcellular localization showed that NnCAT1 was localized in the cell membrane and nucleus. NnCAT1 had a molecular weight of 57.0 kD and a theoretical isoelectric point (PI) of 6.93. This protein was a homologous tetramer hydrophilic protein without transmembrane structure and signal peptide, and its chloroplast transport peptide was located in the former 21 aa. The secondary structure was composed of 27.64% α-helix, 15.65% extended chain, 6.30% β-turn and 50.41% random coil. 【Conclusion】Firstly, NnCAT1 interacted with NnPPO1, and the authenticity of the interaction was further verified by the BiFC test. Secondly, the conserved tyrosinase domain of NnPPO1 played a major role in the interaction. NnPPO1 and NnCAT1 had almost the same expression pattern, and they might lead to browning of fruits and vegetables such as Nelumbo nucifera through a synergistic effect.

Key words: Nelumbo nucifera, NnPPO1, NnCAT1, protein interaction, subcellular localization, tissue-specific expression

原新博,程婷婷,惠小涵,陈章玉,王瑞红,柯卫东,郭宏波. 莲藕多酚氧化酶互作蛋白的筛选及验证[J]. 中国农业科学, 2020, 53(18): 3777-3791.

YUAN XinBo,CHENG TingTing,XI XiaoHan,CHEN ZhangYu,WANG RuiHong,KE WeiDong,GUO HongBo. Screening of Polyphenol Oxidase Interaction Proteins from Nelumbo nucifera and Their Verification[J]. Scientia Agricultura Sinica, 2020, 53(18): 3777-3791.

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图/表 11

表1

PCR 引物列表"

引物名称Name	序列Sequence	用途Use
APX-EcoR I-F	ggaggccagtgaattcATGGCTTGTCTGGGCGGTGC	酵母双杂交 Yeast two hybrid
APX-Xho I-R	tcatctgcagctcgagTCATGTTGAGAAACCCTCAGGAGG
NnCAT1-EcoR I-F	ggaggccagtgaattcATGGATCCTTACAAGTATCGCCC
NnCAT1-Xho I-R	tcatctgcagctcgagTCACATGCTCGGCTTCAC
cytCuZnSOD-EcoR I-F	ggaggccagtgaattcATGGTGAAGGCTGTTGCGG
cytCuZnSOD-Xho I-R	tcatctgcagctcgagTTAACCCTGCAAGCCAATAATACC
PER1-EcoR I-F	ggaggccagtgaattcATGCCTGGACTGACGATCGG
PER1-Xho I-R	tcatctgcagctcgagTCAGACGTTGGTAAAACGGAG
POD3L-EcoR I-F	ggaggccagtgaattcATGAGGACTACTTATCTTCCGTT
POD3L-Xho I-R	tcatctgcagctcgagTTAAGGGTTCACAACCGCACACT
POD43L-EcoR I-F	ggaggccagtgaattcATGGCTCTGGTTTTTGCTC
POD43L-Xho I-R	tcatctgcagctcgagTCAATTGAAAGACCTGCAGGAC
PODP7L-EcoR I-F	ggaggccagtgaattcATGGCCTCCATCATCACCC
PODP7L-Xho I-R	tcatctgcagctcgagTCAGTTCACTCTCCTGCAATTC
NnCAT1^16-398-EcoR I-F	ggaggccagtgaattcATGTTCTGGAGCACAAATTCTGG
NnCAT1^16-398-Xho I-R	tcatctgcagctcgagTCAATCATACCTTGAAGGAAAGTAA
NnCAT1^16-493-EcoR I-F	ggaggccagtgaattcATGTTCTGGAGCACAAATTCTGG
NnCAT1^16-493-Xho I-R	tcatctgcagctcgagTCACATGCTCGGCTTCAC
NnPPO1^175-380-Nco I-F	ctgcatatggccatgATGCCACGTAACTTCACGCA
NnPPO1^175-380-BamH I-R	gcaggtcgacggatcTCATATTGTCCACATCCGGTCGAC
NnPPO1^381-468-NcoI-F	ctgcatatggccatgATGTGGAAAAAGCTGGGAGG
NnPPO1^381-468-BamH I-R	gcaggtcgacggatcTCACTCACTGACGGTTGGC
NnPPO1^469-597-Nco I-F	ctgcatatggccatgATGTTCCCCAAAGAACTTGATGCG
NnPPO1^469-597-BamH I-R	gcaggtcgacggatcTCACGAAGCGAACACTATCTTG
NnCAT1-BD-Nco I-F	ctgcatatggccatgATGGATCCTTACAAGTATCGCCC	自激活验证 Self-activated tast
NnCAT1-BD-BamH I-R	gcaggtcgacggatcTCACATGCTCGGCTTCAC	自激活验证 Self-activated tast
NnPPO1-BiFC-N-BamH I-F	cccaggcctactagtggatccATGGCGTCGCTGTCTCCC	双分子荧光标记 BiFC
NnPPO1-BiFC-N-Xho I-R	cccgggagcggtaccctcgagTCACGAAGCGAACACTATCTTGA
NnCAT1-BiFC-C-BamH I-F	tggcgcgccactagtggatccATGGATCCTTACAAGTATCGCCC
NnCAT1-BiFC-C-Xho I-R	cccgggagcggtaccctcgagCATGCTCGGCTTCACATTGA
NnCAT1-GFP-BamH I-F	ggactctagaggatccATGGATCCTTACAAGTATCGCCC	亚细胞定位 Subcellulcar locatization
NnCAT1-GFP-Kpn I-R	cccttgctcaccatggtaccCATGCTCGGCTTCACATTGAGAC	亚细胞定位 Subcellulcar locatization
β-actin-F	GCCATCCAGGCCGTTCTCTC	qRT-PCR
β-actin-R	GGGACAGTGTGGCTGACACC
NnPPO1-qPCR-F	CAAACTCCGCGATGCCAAGC
NnPPO1-qPCR-R	CGTCGAGCCATTGGACACCA
NnCAT1-qPCR-F	TTTGCCCTGGCGTTGTGGTC
NnCAT1-qPCR-R	GGTGAGCACACTTGGGAGCA

表1

图1

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图3

图4

图5

图6

表2

图7

图8

图9

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元件名称Elements name	功能Function	所属序列Owned sequence
GT1-motif	光响应元件Light responsive element	NnPPO1、NnCAT1
ARE	厌氧诱导必不可少的顺式作用调节元件 Cis-acting regulatory element essential for the anaerobic induction	NnPPO1、NnCAT1
CGTCA-motif、TGACG-motif	MeJA反应性中涉及的顺式作用调节元件 Cis-acting regulatory element involved in the MeJA-responsiveness	NnPPO1、NnCAT1
CAAT-box	启动子和增强子区域常见的顺式作用元件 Common cis-acting element in promoter and enhancer regions	NnPPO1、NnCAT1
Box 4	涉及光响应性的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness	NnPPO1、NnCAT1
TCT-motif	光响应元件的一部分Part of a light responsive element	NnPPO1、NnCAT1
TATA-box	转录启动子周围-30个核心启动子 Core promoter element around -30 of transcription start	NnPPO1、NnCAT1
P-box	赤霉素反应元件Gibberellin-responsive element	NnPPO1
A-box	顺式作用调节元件Cis-acting regulatory element	NnCAT1
ABRE	脱落酸反应性涉及的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness	NnCAT1
MBS	MYB结合位点参与干旱诱导MYB binding site involved in drought-inducibility	NnCAT1
TC-rich repeats	参与防御和应激反应的顺式作用元件 Cis-acting element involved in defense and stress responsiveness	NnCAT1
TCA-element	水杨酸反应性涉及的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness	NnCAT1
G-box	参与光响应的顺式作用调节元件 Cis-acting regulatory element involved in light responsiveness	NnCAT1

莲藕多酚氧化酶互作蛋白的筛选及验证

Screening of Polyphenol Oxidase Interaction Proteins from Nelumbo nucifera and Their Verification

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