金柑FcRGA1抗溃疡病机制初探

doi:10.16420/j.issn.0513-353x.2021-0940

摘要/Abstract

摘要：

NBS-LRR类抗病蛋白是植物中最丰富的一类R蛋白成员，该家族含有典型的NB-ARC结构域与LRR结构域，主要参与植物抵御病原物入侵的防御过程。从金柑（Fortunella crassifolia）中克隆了1个含有NB-ARC结构域的R基因，命名为FcRGA1。该基因含有3 954 bp的完整开放阅读框，编码1 317个氨基酸。qRT-PCR分析表明，FcRGA1在金柑的根、茎、叶中均有表达，叶片中最高，根中最低。溃疡病菌侵染可促进FcRGA1表达上调，接种后12 h表达量为对照的12.3倍。通过VIGS技术沉默金柑叶片FcRGA1后，溃疡病菌侵染的叶片失绿更为严重，病斑面积更大，菌落数与相对电导率显著增加，同时叶绿素含量与过氧化氢含量显著低于对照，表明FcRGA1可能通过对活性氧的调控影响金柑对溃疡病的抗性。

关键词: 金柑, 柑橘溃疡病, NBS-LRR, FcRGA1, 基因沉默, 抗性机制

Abstract:

NBS-LRR resistant proteins are the most abundant R protein members in plants. This family contains typical NB-ARC domains and LRR domains，which are mainly involved in the defense process of plants against pathogen’s invasion. In this study，an R gene containing the NB-ARC domain was cloned from kumquat（Fortunella crassifolia）and named FcRGA1. This gene contained a complete open reading frame（ORF）of 3 954 bp and encoded 1 317 amino acids. qRT-PCR analysis showed that the FcRGA1 gene was expressed in the roots，stems and leaves of kumquat，with the highest expression level in the leaves and the lowest in the roots. Furthermore，the expression of FcRGA1 gene was up-regulated by infection with canker bacteria and reached the highest value at 12 h，which was 12.3 times higher than that of the control. After silencing the FcRGA1 gene by VIGS（Virus-induced Gene Silencing），kumquat leaves in the silenced group showed more severe chlorosis symptom than those in control group after inoculated by Xcc（Xanthomonas citri subsp. citri），concurrent with significantly higher lesion area，bacterial growth and relative electrolytic leakage. Whereas，the chlorophyll content and hydrogen peroxide content were significantly lower in comparison with the control group，indicating that FcRGA1 might affect the resistance of kumquat to citrus canker through the regulation of reactive oxygen species.

Key words: kumquat, citrus canker, NBS-LRR, FcRGA1, gene silencing, resistance mechanism

中图分类号:

S666.1

戴文珊, 吴玥, 王敏. 金柑FcRGA1抗溃疡病机制初探[J]. 园艺学报, 2022, 49(11): 2325-233.

DAI Wenshan, WU Yue, WANG Min. Preliminary Study on Mechanisms of Resistance to Citrus Canker of Kumquat FcRGA1[J]. Acta Horticulturae Sinica, 2022, 49(11): 2325-233.

图/表 6

图1 FcRGA1的克隆与基因结构

Fig. 1 Cloning and gene structure analysis of FcRGA1

图2 金柑FcRGA1与拟南芥（At）含NB-ARC结构域R蛋白的氨基酸序列进化树横坐标为比例尺，代表序列间差异数值的单位长度，对应为每100个氨基酸位置有10个变化的距离，节点处数字代表亲缘关系的远近。

Fig. 2 Amino acid sequences phylogenetic tree of FcRGA1 and Arabidopsis（At）R protein containing NB-ARC domain The abscissa is a distance scale，representing the unit length of difference between sequences，corresponding a distance of 10 changes per 100 amino acid positions，and the numbers at node represent the distance of the kinship.

图3 金柑FcRGA1的表达模式星号表示显著性差异水平，**P < 0.01。

Fig. 3 Expression pattern analysis of FcRAG1 Asterisks indicate significant difference levels，**P < 0.01.

图4 基因沉默后FcRGA1在金柑叶片中的相对表达量以各时间点对照的基因表达量为1。星号表示显著性差异水平，**P < 0.01。

Fig. 4 Expression analysis of the FcRGA1 in the FcRGA1-silencing kumquat leaves The gene expression at each time of the control was taken as 1 for normalization. Asterisks indicate significant difference levels，**P < 0.01.

图5 沉默FcRGA1的金柑叶片接种溃疡病菌后的表型（a）、细菌荧光观察（b）和细菌计数（c） C：对照;R：FcRGA1-VIGS;dpi：接种后天数。星号表示FcRGA1-VIGS与对照在同一个时间点存在显著性差异，*P < 0.05，**P < 0.01。

Fig. 5 The phenotype（a），fluorescence observation（b）and bacterial count（c）of control and FcRGA1-silencing kumquat leaves inoculated with Xcc C：Control;R：FcRGA1-VIGS;dpi：Days post inoculation. Asterisks indicate significant difference between FcRGA1-VIGS and control at the same time point. *P < 0.05，**P < 0.01.

图6 沉默FcRGA1的金柑叶片接种溃疡病菌后的相对电导率、叶绿素含量和过氧化氢含量星号表示显著性差异水平，**P < 0.01。

Fig. 6 Electrolyte leakage，chlorophyll content and H2O2 content of control and FcRGA1-silencing kumquat leaves after inoculated with Xcc Asterisks indicate significant difference levels，**P < 0.01.

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