GST pull-down联合LC-MS/MS筛选柑橘抗溃疡病转录因子CsBZIP40的互作蛋白

doi:10.3864/j.issn.0578-1752.2019.13.005

摘要/Abstract

摘要：

【目的】CsBZIP40是一个与溃疡病抗性相关的转录因子,本研究旨在筛选转录因子CsBZIP40在响应柑橘溃疡病菌（Xanthomonas citri subsp. citri,Xcc）侵染过程中的互作蛋白,对CsBZIP40的互作网络进行分析,为柑橘抗溃疡病的分子育种提供理论依据。【方法】采用GST pull-down技术筛选柑橘在溃疡病菌侵染过程中CsBZIP40的互作蛋白。首先,构建带有GST标签的CsBZIP40蛋白融合表达载体,经IPTG（异丙基硫代半乳糖苷）诱导表达、纯化后获得GST-CsBZIP40融合蛋白作为诱饵蛋白;然后,将GST-CsBZIP40融合蛋白固定在谷胱甘肽亲和磁珠上,用固定在亲和磁珠的GST-CsBZIP40诱饵蛋白与接种溃疡病菌或LB培养基后柑橘叶片总蛋白进行孵育,与GST-CsBZIP40诱饵蛋白结合的蛋白复合物洗脱收集后进行SDS-PAGE凝胶电泳验证。将验证成功的样品洗脱液进行液相色谱串联质谱（LC-MS/MS）检测,鉴定出未侵染和侵染状态下CsBZIP40的互作蛋白,将检测到的蛋白利用甜橙基因组数据库进行注释,筛选出在溃疡病菌侵染过程中与CsBZIP40特异结合的蛋白,并进行GO、KEGG和互作网络分析。【结果】过表达CsBZIP40的转基因植株表型正常,与对照植株无明显差异。过表达CsBZIP40的转基因植株溃疡病抗性评价中病斑面积、病情指数均显著小于野生型植株,分别为野生型的45%和54%。以该转基因植株为材料成功提取出侵染溃疡病菌后和未接种溃疡病菌状态下的柑橘叶片总蛋白。成功构建出GST-CsBZIP40诱饵蛋白表达载体,诱导表达纯化出GST-BZIP40诱饵蛋白。利用GST-CsBZIP40诱饵蛋白从侵染溃疡病菌后柑橘总蛋白和未接菌柑橘总蛋白中成功钓取蛋白,并用LC-MS/MS检测。经过比对、注释和筛选,在柑橘溃疡病菌侵染过程中与GST-BZIP40特异结合的蛋白有53个,这些蛋白参与多个分子功能和通路。在这53个蛋白中,有6个蛋白（Cs1g02310、Cs3g05280、Cs3g23950、Cs6g13880、Cs7g12130、orange1.1t04973）可能与植物抗病性密切相关。数据库中已经证明53个蛋白中44个与CsBZIP40有直接或间接的互作关系。【结论】溃疡病菌侵染过程中有53个蛋白与CsBZIP40互作,根据注释6个蛋白与植物抗病性密切相关,这些蛋白可能在提高柑橘生物胁迫抗逆性方面发挥着重要的作用。

关键词: 柑橘, 柑橘溃疡病, 柑橘溃疡病菌, BZIP, GST pull-down, 互作蛋白

Abstract:

【Objective】 CsBZIP40 is a citrus canker-related transcription factor. The objective of this study is to screen the interacting proteins of CsBZIP40 in response to Xanthomonas citri subsp. citri (Xcc) infection, and to analyze the interaction network of CsBZIP40, so as to provide a theoretical basis for molecular breeding of citrus canker resistance. 【Method】 The GST pull-down strategy was used to screen the interacting proteins of CsBZIP40 in the infection process of Xcc. Firstly, the vector of GST-CsBZIP40 was constructed and induced by IPTG (isopropyl β-D-thiogalactoside). The fusion protein of GST-CsBZIP40 was purified for the next GST pull-down. Then GST-CsBZIP40 was fixed on the GST-beads and incubated with total proteins extracted from CsBZIP40 over-expression plants infected by Xcc or LB medium as control. The protein complex bound to GST-CsBZIP40 bait protein was eluted and collected, and then verified by SDS-PAGE gel electrophoresis. The interacting proteins of CsBZIP40 were detected by LC-MS/MS and then annotated based on the genomic database of Citrus sinensis. The GO, KEGG pathways and the interaction network of the interacting proteins were also analyzed. 【Result】 The phenotype of transgenic plants over-expression CsBZIP40 was normal, and there was no significant difference between the transgenic plants and the control plants. The lesion area on the over-expression plant was significantly smaller compared to that on the wild-type (WT) (45%) and the disease index of CsBZIP40 over-expression plant was significantly lower than that of WT (54%). The total proteins were successfully extracted from CsBZIP40 over-expression plant infected by Xcc or LB medium, and the GST-CsBZIP40 was expressed and purified for the GST pull-down. GST-CsBZIP40 bait protein was used to catch the protein from the total proteins of Xcc-infected and uninfected citrus, then the protein was detected by LC-MS/MS. After comparison, annotation and screening, there are 53 interacting proteins specifically binding to GST-BZIP40 in the process of Xcc infection. These proteins are involved in many molecular functions and pathways. Among the 53 proteins, 6 proteins (Cs1g02310, Cs3g05280, Cs3g23950, Cs6g13880, Cs7g12130, orange1.1t04973) may be closely related to the plant disease resistance, 44 of the 53 proteins have been proven to interact directly or indirectly with CsBZIP40 in the database. 【Conclusion】 In the infection of Xcc, 53 proteins interacted with CsBZIP40 were detected. According to the annotation, 6 proteins are closely related to plant disease resistance. These proteins may play an important role in improving the stress resistance of citrus under biological stress.

Key words: Citrus sinensis, citrus bacteria canker, Xanthomonas citri subsp. citri (Xcc), BZIP, GST pull-down, interacting protein

窦万福,祁静静,胡安华,陈善春,彭爱红,许兰珍,雷天刚,姚利晓,何永睿,李强. GST pull-down联合LC-MS/MS筛选柑橘抗溃疡病转录因子CsBZIP40的互作蛋白[J]. 中国农业科学, 2019, 52(13): 2243-2255.

DOU WanFu,QI JingJing,HU AnHua,CHEN ShanChun,PENG AiHong,XU LanZhen,LEI TianGang,YAO LiXiao,HE YongRui,LI Qiang. Screening of Interacting Proteins of Anti-Canker Transcription Factor CsBZIP40 in Citrus by GST Pull-Down Combined with LC-MS/MS[J]. Scientia Agricultura Sinica, 2019, 52(13): 2243-2255.

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基因 ID Gene ID	功能预测 Predicted protein function	等电点 PI	分子量 Molecular weight (kD)	染色体位置 Chromosomal loci
Cs1g02310	TGA1 resistance (SAR) via its interaction with NPR1	7.07	40.83	chr1:1,637,432..1,643,794
Cs3g05280	Disease resistance protein	5.83	130.03	chr3:6,932,350..6,936,297
Cs3g23950	Transcription factor MYB	8.75	28.06	chr3:26,088,190..26,089,510
Cs6g13880	Peroxiredoxin-2	8.49	29.50	chr6:15,280,950..15,284,180
Cs7g12130	Heat shock protein 83	5.34	90.12	chr7:8,048,239..8,054,656
orange1.1t04973	Death-associated protein kinase 1	5.85	35.14	chrUn:40,320,115..40,324,317