Cloning and Expression Analysis of CsAOS1-2 in Responding to Citrus Canker Disease

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (6): 1355-1367.doi: 10.16420/j.issn.0513-353x.2022-0296

• Plant Protection • Previous Articles Next Articles

Cloning and Expression Analysis of CsAOS1-2 in Responding to Citrus Canker Disease

ZHANG Lehuan, ZOU Changyu, WANG Zhaohao, YANG Wen, ZOU Xiuping, HE Yongrui, CHEN Shanchun(), LONG Qin()

National Citrus Engineering Research Center，Citrus Research Institute，Southwest University/Chinese Academy of Agricultural Sciences，Chongqing 400712，China

Received:2022-11-10 Revised:2023-01-26 Online:2023-06-25 Published:2023-06-27
Contact: * （E-mail：chenshanchun@cric.cn，longqin@cric.cn）

Abstract

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

CLC Number:

S666

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.

Figures/Tables 8

References 45

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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

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

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

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

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		+

Cloning and Expression Analysis of CsAOS1-2 in Responding to Citrus Canker Disease

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