柑橘Rboh家族鉴定及其对激素和柑橘溃疡病的响应

doi:10.3864/j.issn.0578-1752.2020.20.008

摘要/Abstract

摘要：

【目的】活性氧（reactive oxygen species,ROS）是植物抗病反应中重要的信号分子,而Rboh是参与活性氧产生的关键酶之一。本研究通过鉴定和分析柑橘全基因组中Rboh基因家族生物信息学特性、生物胁迫相关植物激素和溃疡病菌（Xanthomonas citri subsp. citri,Xcc）侵染诱导下表达模式,为研究Rboh基因家族与柑橘抗溃疡病过程的相关性,进一步研究和利用柑橘Rboh基因打下基础。【方法】利用过氧化物酶数据库RedOxiBase和柑橘（甜橙）CAP数据库获得柑橘Rboh家族序列信息;利用生物信息学软件对CsRboh进行理化性质、系统进化关系、染色体定位、基因结构、蛋白功能结构域、保守基序和启动子元件系统分析。通过实时荧光定量PCR（qRT-PCR）分析植物激素水杨酸（salicylic acid,SA）、茉莉酸（jasmonic acid,JA）、脱落酸（abscisic acid,ABA）和溃疡病菌处理下CsRboh表达模式。【结果】鉴定得到7个柑橘Rboh家族成员（CsRboh01—CsRboh07）,其编码蛋白包含784—946个不等的氨基酸残基,等电点（PI）分布在8.67—9.40,主要定位于细胞膜结构和细胞质。根据系统进化树可将柑橘Rboh家族划分为I—IV 4个亚家族。CsRboh家族基因不均匀分布在甜橙的5条染色体,均含有典型的呼吸爆发NADPH氧化酶结构域（NADPH_Ox）、铁还原酶跨膜组件（Ferric_reduct）、FAD结合结构域（FAD_binding）和铁还原酶NAD结合结构域（NAD_binding）4个功能结构域。CsRboh家族各基因的启动子区域含不同激素响应元件,数目和类型各有差异。qRT-PCR结果显示CsRboh基因家族各成员可响应激素和溃疡病菌诱导,在不同激素和溃疡病菌处理下感病品种晚锦橙和抗病品种四季橘中表达模式具有差异。结合系统进化树聚类分析、同源基因功能研究及其启动子区域顺式作用元件分析发现,CsRboh02、CsRboh04和CsRboh06在抗、感两个品种中受柑橘溃疡病菌诱导表现出明显不同的表达趋势和表达量。【结论】CsRboh02、CsRboh04和CsRboh06可能与柑橘品种抗、感性密切相关,是3个有潜力的抗溃疡病分子育种候选基因,初步确定CsRboh家族基因在柑橘响应溃疡病过程中发挥关键作用。

关键词: 柑橘溃疡病, Rboh家族, 柑橘溃疡病菌, 水杨酸, 茉莉酸

Abstract:

【Objective】Rboh is the main producer of reactive oxygen species (ROS), which is the key molecule involved in plant disease resistance signaling. To clarify the relationships between CsRboh family and citrus bacterial canker (CBC), the Rboh family of Citrus sinensis was identified and the bioinformatics characteristics as well as the expression patterns induced by biotic stress-related plant hormones and Xanthomonas citri subsp. citri (Xcc) were analyzed. It is also aimed to lay the foundation for further research of citrus Rboh genes in molecular breeding.【Method】Firstly, the RedOxiBase database and the citrus (sweet orange) CAP database were used to identify the citrus Rboh family sequences from the whole genome of C. sinensis. Then, series of bioinformatics tools were used to perform analysis of physical and chemical properties, phylogenetic relationships, chromosomal localizations, gene structures, protein functional domains, the conserved motifs and promoter elements systematically. Finally, qRT-PCR was used to analyze the expression patterns of CsRbohs induced by biotic stress-related plant hormones (salicylic acid, SA; jasmonic acid, JA; abscisic acid, ABA) and Xcc.【Result】A total of 7 members of the Rboh family in C. sinensis genome were identified (CsRboh01-CsRboh07). These genes encoded proteins contain 784-946 amino acid (AA) residues with isoelectric points (PI) ranging from 8.67 to 9.40. CsRbohs mainly distribute on the cell membrane system and cytoplasmic. The CsRbohs family can be divided into I to IV sub-groups based on the phylogenetic tree. These 7 genes are unevenly distributed on 5 sweet orange chromosomes. Each of the CsRbohs contains 4 typical functional domains, namely the respiratory burst NADPH oxidase domain (NADPH_Ox), the ferric reductase like transmembrane component (Ferric_reduct), the FAD binding domain (FAD_binding) and the ferric reductase NAD binding domain (NAD_binding). The promoter regions of CsRboh family genes contain different hormone response elements with different numbers and types. qRT-PCR showed that the expression of CsRboh gene family members in susceptible variety Wanjincheng and resistant variety Calamondin had some differences induced by different hormones and Xcc. According to the findings from phylogeny, homologous analysis and cis-acting elements analysis, CsRboh02, CsRboh04 and CsRboh06 had different expression patterns and levels induced by Xcc in resistant variety Calamondin and susceptible variety Wanjincheng.【Conclusion】CsRboh02, CsRboh04 and CsRboh06 may be closely related to the resistance and sensibility of citrus varieties, and they are three potential candidate genes for molecular breeding of resistance to canker disease, and the CsRboh family genes are preliminarily determined to play a key role in the process of citrus canker response.

Key words: citrus bacterial canker (CBC), Rboh family, Xanthomonas citri subsp. citri (Xcc), salicylic acid (SA), jasmonic acid (JA)

秦秀娟,祁静静,窦万福,陈善春,何永睿,李强. 柑橘Rboh家族鉴定及其对激素和柑橘溃疡病的响应[J]. 中国农业科学, 2020, 53(20): 4189-4203.

QIN XiuJuan,QI JingJing,DOU WanFu,CHEN ShanChun,HE YongRui,LI Qiang. Identification of Rboh Family and the Response to Hormone and Citrus Bacterial Canker in Citrus[J]. Scientia Agricultura Sinica, 2020, 53(20): 4189-4203.

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基因Gene	上游引物Forward primer	下游引物Reverse primer
CsRboh01	GCTCTCGGCTTCAACTTCCT	TCAAAGCTCGAGCCGCTAAA
CsRboh02	ATGGAGCACCAGCTCAAGAC	AGTAGCCCCAATTCCAAGCC
CsRboh03	TTGGCCAGCAGATTGGAGAG	CTGCGAAACTTGCCTCAACC
CsRboh04	ACGCCCGTCTTCAGATTTTCT	GCCATAAGTGACTGACACGCT
CsRboh05	CCGGCGCAATCTGTTAAAGG	TGTAGGCTTCAGCTTCTGGC
CsRboh06	CGCATGAAAGGGACACCGAAT	CAAATGAGCCTTGTTCCCTTGTT
CsRboh07	AACCGGTGATCAAGTGGTCG	GCATGGCTACAAAATTTGAGGGAA
CsActin	CATCCCTCAGCACCTTCC	CCAACCTTAGCACTTCTCC

名称 Name	CAP号 CAP ID	大小 Size (aa)	分子量 Molecular weight (Da)	等电点 Isoelectric point (PI)	亲水性平均值 Grand average of hydropathicity	脂肪指数 Aliphatic index	不稳定系数 Instability index	亚细胞定位 Subcellular localization
CsRboh01	Cs5g02940.1	946	106370.8	9.40	-0.247	87.68	50.16	细胞外膜Outer membrane
CsRboh02	Cs8g12000.1	912	103139.3	9.05	-0.299	83.84	42.57	细胞质Cytoplasmic
CsRboh03	Cs3g14240.1	889	101017.9	9.10	-0.265	85.43	40.96	细胞外膜Outer membrane
CsRboh04	Cs4g06920.1	875	99488.8	9.31	-0.252	82.61	48.38	细胞内膜Inner membrane
CsRboh05	Cs7g19320.1	811	91648.2	8.67	-0.269	86.94	40.91	细胞质Cytoplasmic
CsRboh06	Cs8g17640.1	844	96907.0	8.99	-0.173	84.69	40.42	细胞内膜Inner membrane
CsRboh07	Cs5g11890.1	784	89837.0	9.09	-0.015	89.06	44.93	细胞内膜Inner membrane

名称 Name	α-螺旋 Alpha helix (%)	β-转角 Beta turn (%)	不规则卷曲 Random coil (%)	伸展链 Extended strand (%)
CsRboh01	44.61	4.76	36.79	13.85
CsRboh02	43.75	5.26	34.87	16.12
CsRboh03	44.43	5.29	35.32	14.96
CsRboh04	41.83	4.57	38.29	15.31
CsRboh05	42.17	4.19	37.36	16.28
CsRboh06	43.60	5.21	36.85	14.34
CsRboh07	43.24	5.48	36.48	14.80

基因 Gene	茉莉酸响应元件 CGTCA-motif	水杨酸响应元件 TCA-element	脱落酸响应元件 ABRE
Csrboh01	3	1	7
Csrboh02	1	1	1
Csrboh03	2	-	5
Csrboh04	3	-	5
Csrboh05	-	-	6
Csrboh06 Csrboh07	2 1	1 -	1 -