水稻泛素连接酶D3与抗病相关蛋白VOZ2的互作分析

doi:10.3969/j.issn.1004-1524.20221670

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (1): 9-17.DOI: 10.3969/j.issn.1004-1524.20221670

水稻泛素连接酶D3与抗病相关蛋白VOZ2的互作分析

罗英杰¹^,²(), 崔维军², 王忠华¹, 吴月燕¹, 林宏友², 周洁², 严成其¹^,³^,^*(), 王栩鸣²^,^*()

1.浙江万里学院生物与环境学院,浙江宁波 315100
2.浙江省农业科学院农产品质量安全危害因子与风险防控国家重点实验室,浙江杭州310021
3.宁波市农业科学研究院生物技术研究所,浙江宁波315100

收稿日期:2022-11-22 出版日期:2024-01-25 发布日期:2024-02-18
作者简介:严成其,E-mail: yanchengqi@163.com;
罗英杰(1997—),男,浙江金华人,硕士研究生,主要从事水稻抗病育种研究。E-mail: 652967273@qq.com
通讯作者: * 王栩鸣,E-mail: xmwang@zaas.ac.cn
基金资助:
宁波市科技重大项目(2019B10004);宁波市科技重大项目(2022Z175);浙江省重点研发计划(2021C02053)

Interaction analysis between rice ubiquitin ligase D3 and the disease resistance associated protein VOZ2

LUO Yingjie¹^,²(), CUI Weijun², WANG Zhonghua¹, WU Yueyan¹, LIN Hongyou², ZHOU Jie², YAN Chengqi¹^,³^,^*(), WANG Xuming²^,^*()

1. College of Biological & Enviromental Sciences, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China
2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
3. Institute of Biotechnology, Ningbo Academy of Agricultural Sciences, Ningbo 315100, Zhejiang, China

Received:2022-11-22 Online:2024-01-25 Published:2024-02-18

摘要/Abstract

摘要：

多蘖矮秆基因dwarf-3(D3)是水稻独脚金内酯信号转导过程中的重要节点基因,拟南芥中的MAX2基因与D3同源,且MAX2参与拟南芥的抗病防卫反应。本研究以水稻泛素连接酶D3为诱饵进行酵母双杂筛库,发现水稻抗病相关蛋白维管植物单锌指蛋白VOZ2与D3存在潜在的相互作用。通过酵母双杂交试验证实,D3与VOZ2存在互作。通过荧光定量PCR证实,接种水稻白叶枯病菌后,VOZ2基因在转录水平上的表达受到显著诱导。利用水稻原生质体开展的亚细胞共定位实验发现,D3与VOZ2共定位于细胞核。双分子荧光互补实验发现,D3与VOZ2在烟草叶肉细胞的细胞核和细胞质均产生较强的荧光,进一步证实了D3与VOZ2的相互作用。研究结果为进一步探究D3和VOZ2在水稻抗病防卫反应中的功能与分子机理奠定了基础。

关键词: 水稻, 泛素连接酶D3, 维管植物单锌指蛋白VOZ2, 蛋白互作, 酵母双杂交, 亚细胞共定位

Abstract:

The tiller dwarf gene dwarf-3 (D3) is an important node gene in strigolactone signal transduction in rice. It has been reported that the MAX2 gene in Arabidopsis thaliana is homologous to D3, and MAX2 is involved in the defense response of A. thaliana. In this study, the yeast two-hybrid library was screened with D3 protein as bait, and it was found that there was a potential interaction between rice disease resistance-related protein vascular plant one-zinc finger protein 2 (VOZ2) and D3. The interaction between D3 and VOZ2 was confirmed by yeast two-hybrid validation. Real-time quantitative PCR experiments showed that the expression of VOZ2 gene was significantly induced at the transcriptional level after inoculation with Xanthomonas oryzae pv. oryzae. Subcellular co-localization experiment using rice protoplasts showed that D3 and VOZ2 were co-localized in the nucleus. Bimolecular fluorescence complementation experiments showed that D3 and VOZ2 produced strong fluorescence in the nucleus and cytoplasm of tobacco mesophyll cells, further confirming the interaction between D3 and VOZ2. The study result laid a foundation for further exploring of the function and molecular mechanism of D3 and VOZ2 in rice disease resistance and defense response.

Key words: rice, ubiquitin ligase D3, vascular plant one-zinc finger protein VOZ2, protein interaction, yeast two-hybrid, subcellular co-localization

中图分类号:

S511

罗英杰, 崔维军, 王忠华, 吴月燕, 林宏友, 周洁, 严成其, 王栩鸣. 水稻泛素连接酶D3与抗病相关蛋白VOZ2的互作分析[J]. 浙江农业学报, 2024, 36(1): 9-17.

LUO Yingjie, CUI Weijun, WANG Zhonghua, WU Yueyan, LIN Hongyou, ZHOU Jie, YAN Chengqi, WANG Xuming. Interaction analysis between rice ubiquitin ligase D3 and the disease resistance associated protein VOZ2[J]. Acta Agriculturae Zhejiangensis, 2024, 36(1): 9-17.

图/表 7

表1 引物信息

Table 1 Primer information

名称Name	引物序列Primer sequence (5'→3')	用途Usage
D3-CDS-F	ATGGCGGAAGAGGAGGAGG	基因克隆
D3-CDS-R	CTAATCATCAATTTGCCGGCT	Gene cloning
VOZ2-CDS-F	AGTCCTCTTCTTCGCCCAC	基因克隆(含部分UTR)
VOZ2-CDS-R	AATATGCTATCTTCATGTCCCGT	Gene cloning (contains some UTR)
D3-pB2E-F	TCCGAATTCGCCCTTATGGCGGAAGAGGAGGAGG	D3-pB2E载体构建
D3-pB2E-R	GTCGAATTCGCCCTTCTAATCATCAATTTGCCGGCT	Construction of D3-pB2E vector
VOZ2-pB2E-F	TCCGAATTCGCCCTTATGGCCGGCGATCCGGCC	VOZ2-pB2E载体构建
VOZ2-pB2E-R	GTCGAATTCGCCCTTTCATGTCCCGTCACTAGGGTTCC	Construction of VOZ2-pB2E vector
D3-pGBKT7-F	AGGAGGACCTGCATATGGCGGAAGAGGAGGAGG	D3-pGBKT7载体构建
D3-pGBKT7-R	GCAGGTCGACGGATCCCTAATCATCAATTTGCCGGCTG	Construction of D3-pGBKT7 vector
VOZ2-pGBKT7-F	CAGATTACGCTCATATGGCCGGCGATCCGGCC	VOZ2-pGBKT7载体构建
VOZ2-pGBKT7-R	CGAGCTCGATGGATCCTCATGTCCCGTCACTAGGGTTCC	Construction of VOZ2-pGBKT7 vector
VOZ2-35S-Flag-F	CGGTACCCGGGGATCCATGGCCGGCGATCCGGCCG	VOZ2-3×flag载体构建
VOZ2-35S-Flag-R	GGGCGAATTGGTCGACTGTCCCGTCACTAGGGTTCCAGT	Construction of VOZ2-3×flag vector
VOZ2-RT-F	AGGGCGATTGCGGAGGAATG	实时荧光定量PCR
VOZ2-RT-R	ATTGGGGCCGTCTGGGTTTG	qRT-PCR

图1 D3自激活验证

Fig.1 Self-activation verification of D3

图2 酵母双杂交法验证D3与VOZ2的蛋白互作

Fig.2 The protein interaction between D3 and VOZ2 verified by yeast two-hybrid method

图3 酵母的β-半乳糖苷酶活性共转pGBKT7-D3和pGADT7-VOZ2的酵母,以及阳性对照(共转pGBKT7-53和pGADT7-T的酵母)、阴性对照(共转pGBKT7-Lam和pGADT7-T的酵母)β-半乳糖苷酶的米氏常数平均值分别为0.396、0.629、0.214。该实验共进行了3次生物学重复2次技术重复,并采用t检验对比各组数据差异。**表示与对照相比差异显著(P≤0.01)。下同。

Fig.3 β-Galactosidase activity of yeast The average Michaelis constant values of β-galactosidase in pGBKT7-D3 and pGADT7-VOZ2 co-transformed yeast, positive control yeast (con-transformed with pGBKT7-53 and pGADT7-T) and negative control yeast (co-transformed with pGBKT7-Lam and pGADT7-T) were 0.396,0.629 and 0.214, respectively. Three biological replicates and two technical replicates were carried out, and t-test was used to compare the data differences between the groups. ** indicated significant difference (P≤0.01) compared with the control. The same as below.

图4 Xoo侵染水稻后VOZ2基因的表达水平 ***表示P≤0.001。

Fig.4 The expression level of VOZ2 gene after Xoo infection in rice *** represented P≤0.001.

图5 D3与VOZ2的亚细胞定位 BF,明场;GFP,绿色荧光;RFP,红色荧光;Merge,混合通道。

Fig.5 Subcellular localization of D3 and VOZ2 BF, Bright field; GFP, Green fluorescent; RFP, Red fluorescent; Merge, Merge channel.

图6 双分子荧光互补法验证D3与VOZ2之间的蛋白互作 BF,明场;YFP,黄色荧光;Chl,叶绿体自发荧光;Merge, 混合通道。

Fig.6 The protein interaction between D3 and VOZ2 verified by bimolecular fluorescent complimentary method BF, Bright field; YFP, Yellow fluorescent; Chl, Chloroplast fluorescent; Merge, Merge channel.

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水稻泛素连接酶D3与抗病相关蛋白VOZ2的互作分析

Interaction analysis between rice ubiquitin ligase D3 and the disease resistance associated protein VOZ2

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