辣椒CaSYT1的鉴定及其在疫霉侵染过程中的功能初探

doi:10.16420/j.issn.0513-353x.2023-0113

摘要/Abstract

摘要：

为明确辣椒（Capsicum annuum）编码突触囊泡结合蛋白（Synaptotagmin，SYT）基因CaSYT1在辣椒应答疫霉侵染中可能发挥的作用，通过蛋白保守结构预测、亚细胞定位、疫霉及植物生长调节剂处理、瞬时表达、基因沉默等方法对CaSYT1的表达和功能进行了初步研究。结果表明：CaSYT1含有1个跨膜螺旋结构域和2个C2结构域，定位在细胞膜；CaSYT1的转录水平受疫霉侵染以及外源SA的诱导，且在疫霉侵染以及外源SA处理下CaSYT1启动子活性被激活并驱动了下游基因的转录表达；CaSYT1的瞬时表达能够诱发辣椒叶片内H₂O₂的大量积累，触发了明显的过敏性细胞坏死；CaSYT1沉默增强了辣椒应答疫霉侵染的抗病性，且伴随着CaPR1、NPR1、CaDEF1抗病相关基因的上调表达，暗示CaSYT1在辣椒应答疫霉侵染中起负调节作用，而其瞬时表达却能触发H₂O₂的积累和过敏性细胞坏死，表明CaSYT1以不依赖于细胞坏死和H₂O₂积累的方式在辣椒应答疫霉侵染中起着负调节作用。

关键词: 辣椒, CaSYT1, 疫霉, 基因鉴定, 信号通路, 表达分析

Abstract:

In order to clarify whether the gene CaSYT1 encoding Synaptotagmin（SYT）protein in pepper may play an important role in pepper’s response to Phytophthora capsici infection. The expression pattern and function of CaSYT1 were preliminarily studied by protein conserved structure prediction，subcellular localization，P. capsici and exogenous hormone treatments，transient expression and virus-induced gene silencing. The results showed that CaSYT1 contained one transmembrane helix domain and two C2 domains，which were located to the plasma membrane. The transcript levels of CaSYT1 were induced by P. capsici infection and exogenous hormone SA，and the CaSYT1 promoter activities were activated and trigger the expression of downstream genes under P. capsici infection and exogenous SA treatment. The transient expression of CaSYT1 can induce the accumulation of H₂O₂ in pepper leaves and trigger obvious hypersensitive response（HR）-like cell death. CaSYT1 silencing enhanced pepper resistance to P. capsici infection，accompanied by up- regulated expression of resistance-related genes，including CaPR1，NPR1，and CaDEF1，suggesting CaSYT1plays a negative regulatory role in pepper response to P. capsici infection. CaSYT1 transient expression can trigger H₂O₂ accumulation and cell death，indicating that CaSYT1 plays a negative regulatory role in pepper response to P. capsici infection in a manner independent of cell death and H₂O₂ accumulation.

Key words: Capsicum annuum, CaSYT1, Phytophthora capsici, gene identification, signal passage, expression analysis

刘艳艳, 丁颖, 刘兴华, 郑佳秋, 刘志钦. 辣椒CaSYT1的鉴定及其在疫霉侵染过程中的功能初探[J]. 园艺学报, 2024, 51(3): 533-544.

LIU Yanyan, DING Ying, LIU Xinghua, ZHENG Jiaqiu, LIU Zhiqin. Identification of Pepper CaSYT1 and Its Function in Phytophthora capsici Infection[J]. Acta Horticulturae Sinica, 2024, 51(3): 533-544.

图/表 9

表1 本研究中所用的引物序列

Table 1 Primer sequences used in this study

用途 Usage	基因 Gene	正向序列（5′-3′） Forward sequence	反向序列（5′-3′） Reverse sequence
实时荧光定量 qRT-PCR	CaSYT1	ATGGGTTTTGTGAGTACGAT	AATTGGTGCCGCAATATCTT
	CaPR1（Rezaul et al.，2019）	CAGGATGCAACACTCTGGTGG	ATCAAAGGCCGGTTGGTC
	CaHIR1（Choi et al.，2012）	GACATGGTCCTGGTAACCCA	CCCAACAGAAGCCTGAAGAA
	CaNPR1（Dong，2004）	ACTTCTTCGCCGACGCCAAG	GCCAACACATTCACCAGAGCATC
	CaDEF1（Do et al.，2004）	GAGGAAGAAGTTTGAAAG	GAAATACACCACATGAAG
亚细胞定位载体构建Construction of subcellular localization vectors	CaSYT1	AAAAAGCAGGCTACATGGGTTTTGTGAGTACGAT	AGAAAGCTGGGTCGCTGCAATGGCGAACTGCATCT
瞬时表达载体构建Construction of transient overexpression vectors	CaSYT1	AAAAAGCAGGCTACATGGGTTTTGTGAGTACGAT	AGAAAGCTGGGTCTCAAGATGCAGTTCGCCATT
沉默载体构建 Construction of silent vectors	CaSYT1	AAAAAGCAGGCTACATGGGTTTTGTGAGTACGAT	AGAAAGCTGGGTCAATTGGTGCCGCAATATCTT

图1 CaSYT1和其他植物SYT1系统发育分析数字表示可信度。

Fig. 1 Phylogenetic analysisof CaSYT1 and SYT1 from other plants The number represents credibility.

图2 CaSYT1的亚细胞定位（A）和Western Blot检测（B）

Fig. 2 Subcellular localization（A）of CaSYT1 and Western Blot（B）

图3 辣椒CaSYT1在疫霉侵染和不同外源植物激素处理后的转录水平 *、**分别表示在P < 0.05、P < 0.01水平上差异显著。下同。

Fig. 3 The transcriptional expression levels of CaSYT1 in pepper plant leaves inoculated with Phytophthora capsica and treated with different exogenous phytohormones * and ** indicate that there are significant difference at the levels of P < 0.05 and P < 0.01. The same below.

图4 辣椒CaSYT1启动子顺式作用元件

Fig. 4 CaSYT1 promoter cis-acting element

图5 辣椒接种疫霉和不同植物生长调节剂处理后CaSYT1启动子驱动的GUS酶活性

Fig. 5 GUS enzyme activity driven by CaSYT1 after inoculation with Phytophthora capsici and treatment with different exogenous plant hormones

图6 辣椒瞬时表达CaSYT1对由类似过敏反应引起的细胞死亡（A）及电导率（B）的影响

Fig. 6 The effect of transient overexpression of CaSYT1 on hypersensitive response mimicked cell death（A）and conductivity（B）

图7 辣椒瞬时表达CaSYT1对防御相关基因表达的影响

Fig. 7 Effect of transient expression of CaSYT1 on expression of defense-related genes

图8 CaSYT1干扰增强辣椒植株应答疫霉的抗病性 A：阳性对照CaPDS沉默3周后出现漂白现象。B：接种疫霉24 h后CaSYT1相对表达量的Real-time RT-PCR检测。C：接种疫霉孢子后的辣椒幼苗表型。D：辣椒离体叶片接种疫霉菌块后的病斑。E：辣椒叶片相关抗性基因转录水平的real-time RT-PCR检测。

Fig. 8 CaSYT1-silencing enhances pepper’s resistance to Phytophthora capsici inoculation A：The phenotype of CaPDS-silenced pepper plant three weeks post agro-infiltration. B：The relative expression of CaSYT1 was detected by real-time RT-PCR 24h after inoculation with Phytophthora capsici. C：Phenotype of pepper seedlings after inoculation with Phytophthora capsici. D：Diseased spots on isolated leaves of pepper after inoculation of Phytophthora capsici. E：Real-time RT-PCR detection of transcriptional levels of related resistance genes in pepper leaves.

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