柑橘AOS1-2的克隆及其响应溃疡病菌侵染的表达分析

doi:10.16420/j.issn.0513-353x.2022-0296

摘要/Abstract

摘要：

对柑橘中茉莉酸（Jasmonic acid，JA）生物合成途径中的关键限速酶丙二烯氧化物合酶AOS家族基因进行注释，确定其受溃疡病菌Xanthomonas citri subsp. citri（Xcc）诱导的表达模式。从甜橙数据库中共注释出3个AOS家族成员，其中CsAOS1-2对溃疡病菌响应最强烈；CsAOS1-2在‘晚锦橙’（甜橙，易感溃疡病）和‘金弹’（金柑，抗溃疡病）中均受病原菌诱导，但在‘晚锦橙’中表达量上调幅度远高于金弹；CsAOS1-2全长2 656 bp，开放阅读框1 599 bp，编码532个氨基酸，分子量为59 499.81，为非分泌性蛋白，其349 ~ 510 aa为典型的P450功能域；CsAOS1-2的表达无组织特异性；‘晚锦橙’和‘金弹’的AOS1-2启动子均含有参与植物激素和逆境应答的顺式作用元件，但在数量和类型上存在差异；烟草瞬时转化亚细胞定位分析显示CsAOS1-2定位在叶绿体中。CsAOS1-2强烈响应柑橘溃疡病菌的侵染，推测其与柑橘溃疡病敏感性具有密切的关系。

关键词: 柑橘, 溃疡病, 茉莉酸, 限速酶, AOS, 克隆, 表达

Abstract:

The AOS genes，encoding the key rate limiting enzyme in the jasmonic acid（JA）biosynthetic pathway in citrus，are annotated，and their expression patterns in response to Xanthomonas citri subsp. citri（Xcc）were determined by qRT-PCR. Based on the reference genome databases of sweet orange（Citrus sinensis），three AOS genes（CsAOS1-1，CsAOS1-2 and CsAOS1-3）were detected in citrus. Gene expression analysis showed that CsAOS1-2 is the most responsive to Xcc，compared with the other two AOS genes. The expression of CsAOS1-2 was increased by Xcc in both‘Wanjincheng’orange（Citrus sinensis Osbeck，canker-susceptible cultivar）and‘Jindan’（Fortunella crassifolia Swingle，canker-resistant cultivar）. But its expression level induced by Xcc in‘Wanjincheng’orange was significantly higher than that in‘Jindan’. A 2 656 bp long sequence of CsAOS1-2 was amplified from Wanjincheng orange. Bioinformatics analysis showed that CsAOS1-2 had a 1 599 bp open reading frame which encodes a 59 499.81 protein with 532 amino acids. CsAOS1-2 is a non-secretory protein and the 349-510 aa is a typical P450 domain. The similar expression levels of CsAOS1-2 were found in leaf，stem，rind and seed tissues. The AOS1-2 promoters of‘Wanjincheng’orange and‘Jindan’contained multiple cis-acting elements involved in plant adversity and hormone responses，but there were differences in their number and type. Subcellular localization analysis showed CsAOS1-2 located in tobacco chloroplasts. The study suggested that CsAOS1-2 strongly respond to Xcc infection and is closely related to susceptibility to citrus canker in citrus.

Key words: citrus, canker, JA, rate-limiting enzyme, AOS, cloning, expression

中图分类号:

S666

张乐欢, 邹昌玉, 王兆昊, 杨雯, 邹修平, 何永睿, 陈善春, 龙琴. 柑橘AOS1-2的克隆及其响应溃疡病菌侵染的表达分析[J]. 园艺学报, 2023, 50(6): 1355-1367.

ZHANG Lehuan, ZOU Changyu, WANG Zhaohao, YANG Wen, ZOU Xiuping, HE Yongrui, CHEN Shanchun, LONG Qin. Cloning and Expression Analysis of CsAOS1-2 in Responding to Citrus Canker Disease[J]. Acta Horticulturae Sinica, 2023, 50(6): 1355-1367.

图/表 8

表1 本研究中所用引物

Table 1 Primers used in this study

用途 Use	引物 Primer	引物序列 Primer sequence
cDNA扩增cDNA amplification	CsAOS1-2	F：GGACTAGTATGGCATCCACTTCTCTA；R：CGGAATTCTCAAAAGCTTGCTCTCTT
启动子引物Promoter	CsAOS1-2-P	F：GCAGATCGAACCCGAGGAGT；R：CTGAATGAGAAACGCGACGC
	FjAOS1-2-P	F：ATAGAGTCGTGGAAGGTGTGCG；R：GCGGAATCAAACAAGGGTCC
基因表达	Actin-qRT	F：CATCCCTCAGCACCTTCC；R：CCAACCTTAGCACTTCTCC
Gene expression	CsAOS1-1-qRT	F：AGCTTTTGGGTCAATTTCGGAT；R：TCACGAAATGTTCCAAATAGAAACT
	CsAOS1-2-qRT	F：TTGGTTCGGTAAGAACCCCG；R：GGTAGGCCCAGGGAGAGTAA
	CsAOS1-3-qRT	F：CGGCTCTCTCCATCTTTCCC；R：ATCAATTGCGACCAGCGGTA

表2 柑橘AOS家族基因的序列分析

Table 2 Sequence analysis of AOS gene family in Citrus

基因 Gene	CAP序号 CAP ID	氨基酸数 Number of amino acid	分子量 Molecular weigh	等电点 pI	亚细胞定位 Subcellular localization	外显子数	内含子数	开放阅读框/bp
基因 Gene	CAP序号 CAP ID	氨基酸数 Number of amino acid	分子量 Molecular weigh	等电点 pI	亚细胞定位 Subcellular localization	Exon number	Intron number	Open reading frame
CsAOS1-1	Cs_ont_2g018770	499	55 760.06	5.99	未知 Unknown	2	1	1 500
CsAOS1-2	Cs_ont_3g004330.1	532	59 499.81	9.10	叶绿体 Chloroplast	1	0	1 599
CsAOS1-3	Cs_ont_8g022950.1	481	54 195.31	8.53	内质网 Endoplasmic reticulum	1	0	1 446

图1 柑橘与其他物种AOS进化树（A）、结构域（B）及功能域（C）桃：Prunus persica；苹果：Malus × domestica；草莓：Fragaria vesca；葡萄：Vitis vinifera；马铃薯：Solanum buberosum番茄：Solanum lycopersicum；白木香：Aquilaria sinensis；甜橙：Citrus sinensis；雷蒙棉：Gossypium raimondii；大豆：Glycine max；红皮柳：Salix purpurea；毛果杨：Populus trichocarpa；拟南芥：Arabidopsis thaliana；大麦：Hordeum vulgare；水稻：Oryza sativa.

Fig. 1 AOS evolutionary tree（A, structural domain（B）and functional domain（C）of citrus and other species

图2 AOS蛋白的多重序列比对 AGP25595.1：白木香AOS；Prupe.1G386300.1：桃AOS；SapurV1A.0017s0540.1：红皮柳；Gorai.009G274700.1：雷蒙棉；AT5G42650：拟南芥；LOC Os03g55800.1：水稻AOS；KAE8799605.1：大麦AOS。红色方框表示AOS 基因家族特有保守结构域序列I-Helix region、ETLR-motif和Heme-Binding domain；▼表示细胞色素P450结构域。

Fig. 2 Multiple sequence alignment of AOS protein AGP25595.1：Aquilaria sinensis AOS；Prupe.1G386300.1：Prunus persica AOS；SapurV1A.0017s0540.1：Salix purpurea AOS；Gorai.009G274700.1：Gossypium raimondii AOS；AT5G42650：Arabidopsis thaliana AOS；LOC Os03g55800.1：Oryza sativa AOS；KAE8799605.1：Hordeum vulgare AOS. The AOS gene motifs of I-Helix region, ETLR-motif and Heme-Binding domain are red boxed. Represents the cytochrome P450 binding domain by ▼.

图3 柑橘感病品种‘晚锦橙’和抗病品种‘金弹’中CsAOS受溃疡病菌诱导的表达量双尾t检验（* P < 0.05；** P < 0.01），与各自的水处理对照相比较。

Fig. 3 The expression of CsAOS induced by canker pathogen in citrus susceptible variety‘Wanjincheng’and resistant variety‘Jindan’ Two-tailed student’s t-test（* P < 0.05；** P < 0.01），compared with mock control.

图4 CsAOS1-2在柑橘‘晚锦橙’和‘金弹’不同组织的表达量不同字母分别表示‘晚锦橙’和‘金弹’不同组织间在P < 0.05水平下差异显著（Duncan’s test）。

Fig. 4 Expression of CsAOS1-2 in different tissues of‘Wangjincheng’and‘Jindan’ Different letters indicate‘Wanjincheng’and‘Jindan’significant different at P < 0.05 in different tissues of the same varieties（Duncan’s test）.

表3 PlantCARE预测AOS1-2启动子区顺式作用元件

Table 3 cis-Acting regulatory elements in promoter of AOS1-2 predicted by PlantCARE

顺式元件 cis-Element	特性 Characteristic	序列 Sequence	起始位/bp Strat position		方向Strand
顺式元件 cis-Element	特性 Characteristic	序列 Sequence	晚锦橙 Wanjincheng	金弹 Jindan	晚锦橙 Wanjincheng	金弹 Jindan
ABRE	脱落酸响应 Abscisic acid responsiveness	CACGTG ACGTG ACGTG ACGTG ACGTG CACGTG CACGTG	311 913 694 1 093 312 1 092 912		+ + + + + - -
AE-box	光响应元件的一部分 Part of a light responsive element	AGAAACTT	622	788	+	+
ARE	厌氧诱导 Anaerobic induction	AAACCA AAACCA	663	830 1 137	+	+ +
Box4	光响应元件的一部分 Part of a light responsive element	ATTAAT ATTAAT	1 416	451 1 420	-	+ -
CGTCA-motif	茉莉酸甲酯响应 Methyl jasmonate responsiveness	CGTCA CGTCA CGTCA	105 798	222 958 255	- +	- + -
G-box	光响应元件 Light-responsiveness element	CACGTG CACGAC CACGTG CACGAC CACGTG	311 1 208 912 1 092	1 198 1 290	+ + - -	+ -
GA-motif	光响应元件的一部分 Part of a light responsive element	ATAGATAA	872 1 052	1 032	+ +	+
MBS	干旱响应Drought response	CAACTG		1 534		+
MRE	光响应元件 Light-responsiveness element	AACCTAA AACCTAA	1 223	98 1 213	-	- -
P-box	赤霉素响应 Gibberellin-responsive	CCTTTTG	496		+
TATC-box	赤霉素响应元件 Gibberellin-responsive element	TATCCCA		841		-
TCA-element	水杨酸响应 Salicylic acid responsiveness	CCATCTTTTT		1 515		+
TCT-motif	光响应元件的一部分 Part of a light responsive element	TCTTAC	127	277	+	+
TGACG-motif	茉莉酸甲酯响应元件 Methyl jasmonate-responsive element	TGACG TGACG TGACG	105 798	222 958 255	+ -	+ - +
circadian	昼夜规律元件 Involved in circadian control	CAAAGATATC		73		+
W-box	WRKY转录因子结合位点 WRKY transcription factor binding site	TTGACC	112		+

图5 CsAOS1-2亚细胞定位

Fig. 5 Subcellular localization analysis of CsAOS1-2

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