辣椒与本氏烟中模式识别受体FLS2-BAK1的互作分析

doi:10.16420/j.issn.0513-353x.2024-0461

摘要/Abstract

摘要：

位于植物细胞膜的受体激酶通过与共受体激酶互作结合，在免疫反应的激活过程中发挥重要作用。拟南芥模式识别受体模块FLS2-BAK1的互作机制已被解析，而辣椒与本氏烟中FLS2-BAK1功能结构域之间的互作关系尚不清楚。本研究中克隆了辣椒CaFLS2、CaBAK1与本氏烟NbFLS2、NbBAK1，并对它们的序列结构特征、亚细胞定位和功能结构域之间的互作关系进行分析，结果表明它们均具有LRR型受体激酶的典型结构域，本氏烟NbFLS2基因编码区可能存在多种序列类型；CaFLS2、CaBAK1、NbFLS2s与NbBAK1均定位于细胞膜；双分子荧光互补实验结果表明CaFLS2-CaBAK1、NbFLS2s-NbBAK1的胞外结构域之间及胞内结构域之间可能存在互作结合，此外NbFLS2-1、NbFLS2-2可能与NbBAK1相互作用，且在本氏烟中共表达NbFLS2s-NbBAK1可诱导产生活性氧。研究结果有助于增进对辣椒与本氏烟FLS2-BAK1功能模块的认识，为解析茄科作物模式识别受体介导的免疫信号网络奠定基础。

关键词: 辣椒, 受体激酶, 互作关系, 功能结构域, 双分子荧光互补

Abstract:

Through interacting with co-receptor kinase，plant receptor kinases play an important role in the activating of immune response. The interaction mechanism of pattern recognition receptor module FLS2-BAK1 in Arabidopsis has been revealed. While the interaction relationship between the function domain of FLS2 and BAK1 in pepper and tobacco is still unclear. In the present study，CaFLS2-CaBAK1 and NbFLS2-NbBAK1 were cloned from Capsicum annuum and Nicotiana benthamiana respectively. Then structure character analysis，subcellular location and interaction relationship of them were analyzed. Research results showed that CaFLS2，CaBAK1，NbFLS2s and NbBAK1 contain the character domain of LRR-type receptor kinase and NbFLS2 may has different type coding sequence. All of the receptor kinases are localized on the cell membrane. The BiFC experiment results indicated that there is an interaction relationship between the extracellular domain，intracellular domain of CaFLS2 and CaBAK1，and also NbFLS2s and NbBAK1. What’s more，the interaction between the full-length protein of NbFLS2s and NbBAK1 was detected with BiFC assay，and their co-expression could induce ROS production in N. benthamiana. These results not only increase the knowledge of FLS2-BAK1 module in pepper and N. benthamiana，but also provide a basis for revealing the immunity signal pathway medicated by receptor kinase in Solanaceae crops.

Key words: pepper, receptor kinase, interaction analysis, function domain, bimolecular fluorescence complementation

靳晶豪, 苏超, 陈意, 孙世超, 陈新新, 陈孝仁. 辣椒与本氏烟中模式识别受体FLS2-BAK1的互作分析[J]. 园艺学报, 2025, 52(10): 2613-2624.

JIN Jinghao, SU Chao, CHEN Yi, SUN Shichao, CHEN Xinxin, CHEN Xiaoren. Interaction Analysis of the Pattern Recognition Receptor FLS2-BAK1 in Pepper and Nicotiana benthmiana[J]. Acta Horticulturae Sinica, 2025, 52(10): 2613-2624.

图/表 7

表1 本试验中用于基因克隆与载体构建的引物信息

Table 1 Primers used for gene cloning and vector construction in this study

引物名称Prime name	引物序列（5′-3′）Primer sequence
NbFLS2-F	GGGGTACCATGTCACAGACAGTTTTATATGCA
NbFLS2-ECD-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACGGTTTTCTTGGAAAACCCGTGAGT
NbFLS2-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACAATTGCATCTTTTACCAAACAAGC
NbFLS2-ID-F	CGCCATATGGGTACCATGGAGGATATGATTCCAAAGTAT
NbBAK1-F	GGGGTACCATGGATCAATGGCTATTGGGGATC
NbBAK1-ECD-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACTCCTCCAGCAATCGCCCCGGT
NbBAK1-R	TACGATTCATCTGCAGCTCGAGTTAGTCGACTCTTGGCCCTGATAACTCATC
NbBAK1-ID-F	GGCCATGGAGGCCAGTGAATTCGGTACCATGCACTTCTTTGATGTACCTGCT
CaFLS2-F	CGGGGTACCATGATGTCAAACACAGTTGCA
CaFLS2-ECD-R	ACGCGTCGACAGTTTTCTTGGAAAAACCATGAGA
CaFLS2-ID-F	CGGGGTACCATGACAGAGGATACAAATCCAAAT
CaFLS2-R	ACGCGTCGACATTGAGATTTGATGATACTCC
CaBAK1-F	CGGGGTACCATGATGGATCAATGGGTGTTG
CaBAK1-ECD-R	ACGCGTCGACACCACCAGCAATCGCCCCGGT
CaBAK1-ID-F	CGGGGTACCATGCCAGAAGACCACTTCTTTGAT
CaBAK1-R	ACGCGTCGACTCTTGGCCCTGATAACTCATC

图1 辣椒与本氏烟中FLS2和BAK1的序列结构分析 A：CaFLS2与NbFLS2s的序列结构分析；B：CaBAK1与NbBAK1的序列结构分析。黑色三角形表示AtFLS2-AtBAK1互作的关键氨基酸位点

Fig. 1 Sequence structure characteristics analysis of FLS2s and BAK1 in pepper and tobacco A：Sequence structure analysis of NbFLS2s and CaFLS2；B：Sequence structure analysis of NbBAK1 and CaBAK1. Key residues in the interaction between AtFLS2 and AtBAK1 are indicated by black triangle

图2 NbFLS2-1、NbFLS2-2与NbBAK1的亚细胞定位 A：NbFLS2-1、NbFLS2-2和NbBAK1定位在细胞膜，拟南芥AtPIP2A-RFP为细胞膜标记蛋白；B：NbFLS2-1、NbFLS2-2和NbBAK1的蛋白检测

Fig. 2 Subcellular location of NbFLS2-1，NbFLS2-2 and NbBAK1 in tobacco A：NbFLS2-1，NbFLS2-2 and NbBAK1 were localized in the cell membrane，AtPIP2A-RFP was used as cell membrane marker；B：Protein detection of NbFLS2-1，NbFLS2-2 and NbBAK1

图3 CaFLS2与CaBAK1的亚细胞定位 A：CaFLS2和CaBAK1定位在细胞膜；B：CaFLS2和CaBAK1的蛋白检测

Fig. 3 Subcellular location of CaFLS2 and CaBAK1 in tobacco A：CaFLS2 and CaBAK1 were localized in the cell membrane；B：Protein detection of CaFLS2 and CaBAK1

图4 CaFLS2和CaBAK1的互作分析 A：LRR-RLK受体激酶的结构示意图；B：本氏烟叶片细胞的黄色荧光蛋白观察；C：本氏烟叶片中目的蛋白的检测。红色箭头所指为nYFP融合蛋白，蓝色箭头所指为cYFP融合蛋白。下同

Fig. 4 Interaction analysis between CaFLS2 and CaBAK1 with BiFC assay A：Structure diagram of LRR-RLK；B：Observation of yellow fluorescent protein in tobacco leaves；C：Detection of target protein in tobacco leaves. nYFP fusion protein and cYFP fusion protein were indicated with red and blue arrow，respectively. The same below

图5 NbFLS2s和NbBAK1的互作分析 A：本氏烟叶片细胞的黄色荧光蛋白观察；B：本氏烟叶片中目的蛋白的检测

Fig. 5 Interaction analysis of NbFLS2s and NbBAK1 with BiFC assay A：Observation of yellow fluorescent protein in tobacco leaves；B：Detection of target protein in tobacco leaves

图6 共表达NbFLS2和NbBAK1产生活性氧

Fig. 6 The co-expression of NbFLS2 and NbBAK1 could induce ROS production in tobacco leaves

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