中国农业科学 ›› 2020, Vol. 53 ›› Issue (18): 3777-3791.doi: 10.3864/j.issn.0578-1752.2020.18.013
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(
)
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: 基金资助:
YUAN XinBo1(),CHENG TingTing1,XI XiaoHan1,CHEN ZhangYu1,WANG RuiHong2,KE WeiDong3,GUO HongBo1(
)
Received:
2020-02-22
Accepted:
2020-07-13
Online:
2020-09-16
Published:
2020-09-25
Contact:
HongBo GUO
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。
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.
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 | |
NnCAT116-398-EcoR I-F | ggaggccagtgaattcATGTTCTGGAGCACAAATTCTGG | |
NnCAT116-398-Xho I-R | tcatctgcagctcgagTCAATCATACCTTGAAGGAAAGTAA | |
NnCAT116-493-EcoR I-F | ggaggccagtgaattcATGTTCTGGAGCACAAATTCTGG | |
NnCAT116-493-Xho I-R | tcatctgcagctcgagTCACATGCTCGGCTTCAC | |
NnPPO1175-380-Nco I-F | ctgcatatggccatgATGCCACGTAACTTCACGCA | |
NnPPO1175-380-BamH I-R | gcaggtcgacggatcTCATATTGTCCACATCCGGTCGAC | |
NnPPO1381-468-NcoI-F | ctgcatatggccatgATGTGGAAAAAGCTGGGAGG | |
NnPPO1381-468-BamH I-R | gcaggtcgacggatcTCACTCACTGACGGTTGGC | |
NnPPO1469-597-Nco I-F | ctgcatatggccatgATGTTCCCCAAAGAACTTGATGCG | |
NnPPO1469-597-BamH I-R | gcaggtcgacggatcTCACGAAGCGAACACTATCTTG | |
NnCAT1-BD-Nco I-F | ctgcatatggccatgATGGATCCTTACAAGTATCGCCC | 自激活验证 Self-activated tast |
NnCAT1-BD-BamH I-R | gcaggtcgacggatcTCACATGCTCGGCTTCAC | |
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 | |
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2
NnPPO1与NnCAT1启动子响应元件"
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 |
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