过氧化氢酶基因CsKat01与柑橘溃疡病相关性分析

doi:10.16420/j.issn.0513-353x.2020-0132

摘要/Abstract

摘要：

为了探究过氧化氢酶基因CsKat01在柑橘抵御溃疡病过程中的作用,对感病品种‘晚锦橙’和抗病品种‘四季橘’中CsKat01进行了生物信息和表达分析,并探究了溃疡病菌接种处理后两品种中CAT酶活性和H₂O₂含量的关系。克隆CsKat01的编码序列并对该基因及其编码蛋白进行结构和功能分析,发现CsKat01编码框全长为1 482 bp,共编码493个氨基酸残基,蛋白序列中含有典型的CAT结构域;通过启动子序列克隆和分析发现‘晚锦橙’和‘四季橘’核心启动子区域均含有水杨酸、茉莉酸和脱落酸的响应元件,但数量不同;利用qRT-PCR分析水杨酸、茉莉酸、脱落酸和柑橘溃疡病菌（Xcc）对CsKat01的诱导表达特性,发现CsKat01的高表达可能使柑橘相对更感病;结合Xcc诱导后植物组织中CsKat01酶活性和H₂O₂含量,综合分析CsKat01、CAT酶活、H₂O₂含量和柑橘对溃疡病抗性之间的关系,推测CsKat01基因可能通过调控CAT的酶活性,改变H₂O₂含量进而影响柑橘对溃疡病的抗性。

关键词: 柑橘, 溃疡病, 过氧化氢酶, CsKat01, 过氧化氢

Abstract:

In order to investigate the correlation between CsKat01 and citrus bacterial canker disease （CBCD）response,bioinformatics and expression analysis of CsKat01 as well as the relationship between CAT activity and Hydrogen peroxide（H₂O₂）contents in CBCD susceptible variety Wanjincheng and resistant variety Calamondin were performed. Firstly,the coding sequence（CDS）of the CsKat01 gene was cloned and the structure and function of the coding protein were analyzed. The full-length CDS of CsKat01 is 1 482 bp and it encodes 493 amino acid residues with a typical catalase domain. Secondly,by cloning and analyzing the promoter sequence,we studied the promoter regulatory elements and found that the core promoters of both Wanjincheng and Calamondin contained salicylic acid（SA）,jasmonic acid（JA）and abscisic acid（ABA）response elements whose numbers are different. Thirdly,qRT-PCR was used to analyze the induced expression profiles of plant hormones and Xanthomonas citri subsp. citri（Xcc）on CsKat01,and then the correlation between the enzyme and CBCD was determined. Finally,the relations between CsKat01,CAT activity,H₂O₂ content and CBCD resistance were analyzed. We speculate that CsKat01 may regulate CAT enzyme activity,which further regulates H₂O₂ accumulation,and then regulates CBCD resistance.

Key words: citrus, bacterial canker disease, catalase, CsKat01, hydrogen peroxide

中图分类号:

S666

祁静静, 秦秀娟, 谢宇, 陈善春, 何永睿, 李强. 过氧化氢酶基因CsKat01与柑橘溃疡病相关性分析[J]. 园艺学报, 2021, 48(1): 26-36.

QI Jingjing, QIN Xiujuan, XIE Yu, CHEN Shanchun, HE Yongrui, LI Qiang. Correlation Analysis of Citrus Catalase Gene CsKat01 and Citrus Canker Disease[J]. Acta Horticulturae Sinica, 2021, 48(1): 26-36.

图/表 7

图1 CsKat01信息学分析

Fig. 1 Bioinformatics analysis of CsKat01

图2 多个物种过氧化氢酶蛋白序列比对 Cs:甜橙;Egl:桉树;Vv:葡萄;Nt:烟草;Pt:杨树;At:拟南芥。

Fig. 2 Multiple alignment of catalases of several organisms Cs:Citrus sinensis;Egl:Eucalyptus globulus;Vv:Vitis vinifera;Nt:Nicotiana tabacum;Pt:Populus trichocarpa;At:Arabidopsis thaliana.

图3 不同物种CAT的系统进化树分支上的数字代表分支的可靠程度,长度代表进化距离。

Fig. 3 The phylogenetic tree of CAT from different species The number on the branch represents the reliability of the branch,and the length represents the evolutionary distance.

表1 Plant CARE预测CsKat01启动子区域顺式作用元件

Table 1 cis-Acting regulatory elements in promoter of CsKat01 predicted by Plant CARE

元件名称 Cis elements	功能 Funtion	序列 Sequence	位置 Position		方向 Strand
元件名称 Cis elements	功能 Funtion	序列 Sequence	晚锦橙 Wanjincheng	四季橘 Calamondin	晚锦橙 Wanjincheng	四季橘 Calamondin
ABRE	脱落酸响应 ABA-responsiveness	ACGTG	733	733	+	+
				1 536		-
CCAAT-box	MYBHv1结合位点 MYBHv1 binding site	CAACGG	1 307	1 320	-	-
CGTCA-motif	茉莉酸响应 JA-responsiveness	CGTCA	943	1 470	+	-
			1 455		-
TCA-element	水杨酸响应元件 SA-responsiveness	CCATCTTTTT	1 298	1 128	-	+

图4 水杨酸、茉莉酸和脱落酸诱导下CsKat01的相对表达 a、a’分别表示四季橘和晚锦橙的数据显著性差异,不同字母表示差异显著,P < 0.05。下同。

Fig. 4 The relative expression of CsKat01 induced by SA,JA and ABA The data significant difference of Calamondin and Wanjincheng is indicated by a and a’ respectively,and different letters indicate significant difference,P < 0.05. The same below.

图5 柑橘溃疡病菌诱导下CsKat01的相对表达量

Fig. 5 The relative expression of CsKat01 induced by Xcc infection

图6 溃疡病诱导下CAT酶活和H2O2含量变化

Fig. 6 CAT enzyme activitie and H2O2 content induced by Xcc infection* P < 0.05;** P < 0.01.

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