柑橘SAR及其信号转导基因CsSABP2在黄龙病菌侵染中的响应特征

doi:10.3864/j.issn.0578-1752.2021.08.006

摘要/Abstract

摘要：

【背景】柑橘系统获得性抗性（systemic acquired resistance,SAR）在柑橘抗黄龙病（Huanglongbing,HLB）过程中起着重要作用。水杨酸（SA）和水杨酸甲酯（MeSA）信号互换是激活植物SAR的关键信号转导途径,但其在抗HLB危害中的作用依然不清楚。【目的】以柑橘HLB不同耐病品种为材料,研究柑橘SAR及其信号转导的关键酶基因CsSABP2（salicylic acid binding protein 2）在HLB 病原菌‘Candidatus Liberibacter asiaticus’（CLas）侵染中的响应特征,了解柑橘SAR及CsSABP2响应CLas侵染的作用。【方法】从SAR Marker基因CsPR1、CsPR2、CsPR5表达、活性氧H₂O₂水平和淀粉含量变化等方面分析CLas侵染、外施SA和MeSA调控柑橘SAR的特征。进一步,基于易感HLB锦橙（Citrus sinensis）和耐HLB酸柚（C. grandis）感染CLas转录组测序比较分析,筛选和克隆响应CLas侵染的CsSABP2差异表达基因;生物信息学分析预测差异基因的生物学功能;qRT-PCR分析差异表达基因在锦橙、酸柚和耐HLB马蜂柑（C. hystrix）中响应CLas感染的表达变化;以锦橙叶片为试材分析差异表达基因响应外源SA和MeSA诱导的表达特征。【结果】SAR Marker基因响应CLas表达分析表明,CsPR1、CsPR2和CsPR5均正向响应CLas侵染上调表达,CsPR2和CsPR5在酸柚和马蜂柑中表达水平高于锦橙,特别是叶肉中两个基因的表达水平均显著高于锦橙;相反,CsPR1在叶脉中上调表达水平明显高于叶肉,且在锦橙叶脉中表达水平显著高于酸柚和马蜂柑叶脉中。离体模拟SA和MeSA调控柑橘SAR反应显示,与SA处理相比,MeSA处理明显诱导CsPR1、CsPR2和CsPR5上调表达,同时非处理部位基因（特别是CsPR5）表达水平也明显上调。H₂O₂检测结果表明,外源MeSA诱导非处理部位H₂O₂积累显著强于SA。同时,通过对外施MeSA、SA的感病叶片进行连续5周的淀粉含量检测,发现MeSA能明显减少感病叶片中淀粉的积累。进一步转录组数据和生物信息学分析表明,4个SAR关键调控基因CsSABP2-1、CsSABP2-2、CsSABP2-3、CsSABP2-4显著响应CLas侵染而差异表达,且编码蛋白质均含SABP2水解活性必需的保守结构域。qRT-PCR结果表明,CsSABP2-1、CsSABP2-4在耐病品种酸柚和马蜂柑中受CLas诱导高水平表达且在叶脉中的表达水平高于叶肉;CsSABP2-2和CsSABP2-3表达水平变化不明显。激素诱导结果显示,CsSABP2主要响应MeSA的诱导表达,MeSA显著诱导CsSABP2-2高水平表达（>10倍）,且显著下调CsSABP2-1和CsSABP2-4的表达（下调至0 h表达量的15%—55%）。【结论】耐病品种酸柚和马蜂柑SAR响应CLas的反应明显强于易感病品种锦橙,且MeSA在介导柑橘SAR抗病反应中起正向调控作用。SA与MeSA信号转导的关键酶基因CsSABP2-1 和CsSABP2-4在柑橘SAR响应CLas侵染中起着重要作用,其高水平表达与柑橘HLB抗性紧密相关;而且CsSABP2-1 、CsSABP2-2 、CsSABP2-4在调控SAR应答柑橘CLas侵染中可能起着关键的协同作用。

关键词: 柑橘黄龙病, 韧皮部杆菌亚洲种, 系统获得性抗性, 水杨酸, 水杨酸甲酯, SABP2, 基因表达

Abstract:

【Background】Plant systemic acquired resistance (SAR) plays an important role in citrus against Huanglongbing (HLB). The signal exchange between salicylic acid (SA) and methyl salicylate (MeSA) is a key signaling for activating SAR, but its roles in HLB is still unclear. 【Objective】In order to understand regulation mechanisms of citrus SAR in HLB, response characteristics of SAR and its key enzyme gene CsSABP2 (salicylic acid binding protein 2) in ‘Candidatus Liberibacter asiaticus’ (CLas) infection were compared among citrus varieties with different HLB disease-tolerance. 【Method】The response characteristics of citrus SAR in CLas, SA, and MeSA inoculation were determined based on the expression of SAR Marker gene CsPR1, CsPR2, CsPR5, levels of reactive oxygen species (H₂O₂) and starch. Furthermore, according to comparative transcriptome data between HLB-susceptible variety Jincheng orange (JC, Citrus sinensis) and HLB-tolerant variety Sour pomelo (SP, C. grandis), the differentially expressed genes of CsSABP2s were screened and cloned. The biological function of selected genes was predicted by bioinformatics analysis. qRT-PCR was further used to analyze expression profiles of CsSABP2s induced by Clas infection in JC, SP and other HLB-tolerant variety Kaffir lime (KL, C. hystrix) and induced by exogenous SA and MeSA in JC variety. 【Result】qRT-PCR analysis showed that CsPR1, CsPR2 and CsPR5 were up-regulated in response to CLas infection, and the expression level of CsPR2 and CsPR5 in SP and KL, especially in mesophyll, was significantly higher than that in JC. On the contrast, the expression level of CsPR1 in vein was significantly higher than that in mesophyll, and its expression level in JC vein was significantly higher than that in SP and KL vein. Hormone treatment showed that MeSA treatment obviously induced up-regulated expressions of CsPR1, CsPR2 and CsPR5 in treated and non-treated sites compared to SA treatment. Exogenous MeSA induced H₂O₂accumulation in non-treated sites, which was stronger than that of SA treatment. MeSA significantly reduced the accumulation of starch in Clas-infected leaves during five weeks of hormone treatment. Transcriptome data and bioinformatics analysis showed that CsSABP2-1, CsSABP2-2, CsSABP2-3, and CsSABP2-4 had significantly different expressions in response to CLas infection, and their encoded proteins contained conserved domains necessary for SABP2 hydrolysis activity. qRT-PCR showed CsSABP2-1 and CsSABP2-4 were significantly up-regulated by CLas in the HLB-resistant varieties SP and KL, and the expression level in vein was higher than that in mesophyll. The expression levels of CsSABP2-2 and CsSABP2-3 did not change significantly. Hormone induction experiments show that CsSABP2 was mainly induced by MeSA, and MeSA significantly up-regulated CsSABP2-2 expression (>10 times), but significantly down-regulated expressions of CsSABP2-1 and CsSABP2-4 (down to 15%-55% of the expression at 0 h). 【Conclusion】The SAR response to CLas infection in the HLB-tolerant varieties Sour pomelo and Kaffir lime is significantly stronger than that in HLB-susceptible variety Jincheng orange, and MeSA plays a positive role in regulating citrus SAR against HLB. Its key enzyme genes CsSABP2-1 and CsSABP2-4 play an important role in SA and MeSA signal transduction responding to CLas infection, and their high-level expressions are closely related to citrus HLB tolerance; and CsSABP2-1, CsSABP2- 2, CsSABP2-4 may play a key synergistic role in the signal conversion between SA and MeSA responding to CLas infection.

Key words: citrus Huanglongbing, ‘Candidatus Liberibacter asiaticus’ (CLas), systemic acquired resistance (SAR), salicylic acid (SA), methyl salicylate (MeSA), SABP2, gene expression

赵珂,郑林,杜美霞,龙俊宏,何永睿,陈善春,邹修平. 柑橘SAR及其信号转导基因CsSABP2在黄龙病菌侵染中的响应特征[J]. 中国农业科学, 2021, 54(8): 1638-1652.

ZHAO Ke,ZHENG Lin,DU MeiXia,LONG JunHong,HE YongRui,CHEN ShanChun,ZOU XiuPing. Response Characteristics of Plant SAR and Its Signaling Gene CsSABP2 to Huanglongbing Infection in Citrus[J]. Scientia Agricultura Sinica, 2021, 54(8): 1638-1652.

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引物名称 Primer name	用途 Amplification	引物序列 Primer sequence (5′-3′)
OI1-F	CLas普通PCR检测 CLas common PCR detection	GCGCGTATGCAATACGAGCGGCA
OI2C-R	CLas普通PCR检测 CLas common PCR detection	GCCTCGCGACTTCGCAACCCAT
CsSABP2-1-F	基因克隆 Gene cloning	TCCCCCGGGATGGAAGAAGTAGTAGGCAT
CsSABP2-1-R	基因克隆 Gene cloning	CCGCTCGAGTTATGCATACTTAAGAGAAA
CsSABP2-2-F	基因克隆 Gene cloning	TCCCCCGGGATGGCAGAAGCCAAGAAACAGAA
CsSABP2-2-R	基因克隆 Gene cloning	CCGCTCGAGTTAAGCATACTTATGAGCAA
CsSABP2-3-F	基因克隆 Gene cloning	TCCCCCGGGATGAAACCAACGGAGAAAAT
CsSABP2-3-R	基因克隆 Gene cloning	CCGCTCGAGTTAAGCATACTTTTGAGCAA
CsSABP2-4-F	基因克隆 Gene cloning	TCCCCCGGGATGGGAGAAGAGATTAACAT
CsSABP2-4-R	基因克隆 Gene cloning	CCGCTCGAGCTAGTTGCAGCCAACAGAAG

引物名称 Primer name	用途 Amplification	引物序列 Primer sequence (5′-3′)
CsPR1-F	检测CsPR1的表达量 To detect the expression of CsPR1	AAATGTGGGTGAATGAGAAAGC
CsPR1-R	检测CsPR1的表达量 To detect the expression of CsPR1	ATTATTGTTGCACGTCACCTTG
CsPR2-F	检测CsPR2的表达量 To detect the expression of CsPR2	TTCCACTGCCATCGAAACTG
CsPR2-R	检测CsPR2的表达量 To detect the expression of CsPR2	GTAATCTTGTTTAAATGAGCCTCTTG
CsPR5-F	检测CsPR5的表达量 To detect the expression of CsPR5	CACCATTGCCAATAACCCTAATG
CsPR5-R	检测CsPR5的表达量 To detect the expression of CsPR5	GGGACAGTTACCGTTAAGATCAG
CsSABP2-1-F	检测CsSABP2-1的表达量 To detect the expression of CsSABP2-1	ATCTCCGTGGCTGTTTTCGT
CsSABP2-1-R	检测CsSABP2-1的表达量 To detect the expression of CsSABP2-1	CTGCCGTCCTCTTTTCCCAT
CsSABP2-2-F	检测CsSABP2-2的表达量 To detect the expression of CsSABP2-2	GCCAGACACCAAACACCAAC
CsSABP2-2-R	检测CsSABP2-2的表达量 To detect the expression of CsSABP2-2	ACATCTTTCCCAGCTCCACG
CsSABP2-3-F	检测CsSABP2-3的表达量 To detect the expression of CsSABP2-3	AGCATTCATGCCAGACACCA
CsSABP2-3-R	检测CsSABP2-3的表达量 To detect the expression of CsSABP2-3	GTGTCCAACCATTCCCCACT
CsSABP2-4-F	检测CsSABP2-4的表达量 To detect the expression of CsSABP2-4	TGACTTATCGGGATTCGGCG
CsSABP2-4-R	检测CsSABP2-4的表达量 To detect the expression of CsSABP2-4	TTGCGCTGCAATTCTTGCTT
actin-F	检测actin的表达量 To detect the expression of actin	CATCCCTCAGCACCTTCC
actin-R	检测actin的表达量 To detect the expression of actin	CCAACCTTAGCACTTCTCC

基因ID Gene ID	基因名称 Gene name	SP CLas vs SP mock		JC CLas vs JC mock		关键氨基酸分析 Analysis of key amino acids
基因ID Gene ID	基因名称 Gene name	log₂ fold change	校正值padj	log₂ fold change	校正值padj	关键氨基酸分析 Analysis of key amino acids
Cs1g23200	CsSABP2-1	3.44	1.88E-14	2.67	3.87E-16	有MeSA脂酶活性 It has MeSA lipase activity
Cs7g24820	CsSABP2-2	1.09	8.70E-05	-2.44	4.33E-35	有MeSA脂酶活性 It has MeSA lipase activity
Cs7g24830	CsSABP2-3	-2.05	3.75E-18	3.44	1.07E-12	有MeSA脂酶活性 It has MeSA lipase activity
Cs7g29470	CsSABP2-4	1.13	1.47E-03	2.17	5.33E-32	有MeSA脂酶活性 It has MeSA lipase activity
Cs2g16450	CsSABP2-5	—	—	1.29	5.14E-04	Ser突变,可能无MeSA脂酶活性 Serine is mutated, may not have MeSA lipase activity
Cs7g24810	CsSABP2-6	—	—	3.34	8.30E-04	缺失突变,可能无MeSA脂酶活性 Deletion mutation, may not have MeSA lipase activity