柑橘CsMYB41和CsMYB63响应溃疡病菌侵染的表达

doi:10.16420/j.issn.0513-353x.2021-1282

摘要/Abstract

摘要：

MYB转录因子不仅在植物生长发育中起着重要作用，且在植物抗病反应中承担着重要的调节功能。本研究中以溃疡病感病品种纽荷尔脐橙（Citrus sinensis Osbeck）和抗病品种四季橘（C. madurensis）为材料，基于前期构建的溃疡病诱导柑橘转录组数据库，筛选获得2个受柑橘溃疡病菌Xanthomonas citri subsp. citri（Xcc）显著诱导的MYB基因：CsMYB41（Cs1g06220）和CsMYB63（Cs4g12760）。利用PCR技术克隆了纽荷尔脐橙的CsMYB41和CsMYB63，并对其结构特征及表达特性进行分析。PCR克隆测序分析发现，这2个基因的全长分别为1 304 bp、1 398 bp，开放阅读框（ORF）为1 047 bp、1 170 bp，各编码349、390个氨基酸。蛋白质同源序列比对和结构分析表明，这2个基因属于MYB类转录因子家族中的R2R3-MYB亚家族；进化树分析表明，CsMYB41和CsMYB63蛋白分别与可可、蓖麻等MYB41和MYB63蛋白高度同源。亚细胞定位结果显示，CsMYB41和CsMYB63定位在细胞核中。实时荧光定量PCR（qRT-PCR）分析表明，CsMYB41和CsMYB63均受Xcc诱导显著上调表达。激素诱导试验表明，在纽荷尔脐橙中，乙烯利（ETH）、水杨酸（SA）、脱落酸（ABA）诱导CsMYB41上调表达，而ETH、SA、茉莉酸（JA）、ABA诱导CsMYB63上调表达。在四季橘中，CsMYB41受ETH、SA诱导上调表达，CsMYB63仅受IAA诱导上调表达。以上结果表明，这2个MYB基因可能在应答柑橘溃疡病菌侵染中起重要作用。

关键词: 柑橘, 溃疡病, MYB转录因子, qRT-PCR

Abstract:

MYB transcription factors not only play a pivotal role in plant growth and development，but also have an important regulatory function in plant disease resistance. In this study，based on the previously constructed transcriptome database of highly susceptible Newhall Navel Orange（Citrus sinensis Osbeck）and highly resistant Calamondin（C. madurensis）in response to citrus canker，two MYB genes CsMYB41（Cs1g06220）and CsMYB63（Cs4g12760）were cloned from Newhall Navel Orange. The full length of the cDNA of CsMYB41 and CsMYB63 were 1 304 and 1 398 bp，which had 1 047 and 1 170 bp reading frames（ORF），respectively. Protein sequences alignment analysis showed that CsMYB41 and CsMYB63 belonged to the R2R3-MYB subfamily of the MYB transcription factor family. Phylogenetic tree analysis showed that CsMYB41 and CsMYB63 were highly homologous with cocoa MYB41 and castor MYB63，respectively. Subcellular location analysis displayed that both CsMYB41 and CsMYB63 proteins located in nucleus. qRT-PCR analysis showed that both CsMYB41 and CsMYB63 genes were significantly up-regulated by Xanthomonas citri subsp. citri. Hormone induction experiments demonstrated that CsMYB41 was significantly up-regulated by SA and ABA，and CsMYB63 was significantly up-regulated by SA，JA and ABA in Newhall Navel Orange. In Calamondin，CsMYB41 expression was increased by SA，and CsMYB63 expression was hardly affected by hormone treatment. The above results indicate that the two MYB genes may play an important role in response to citrus canker.

Key words: citrus, citrus canker, MYB transcription factor, qRT-PCR

中图分类号:

S666

刘语诺, 曹亚, 王帅, 杜美霞, 郑林, 陈善春, 邹修平. 柑橘CsMYB41和CsMYB63响应溃疡病菌侵染的表达[J]. 园艺学报, 2023, 50(3): 495-507.

LIU Yunuo, CAO Ya, WANG Shuai, DU Meixia, ZHENG Lin, CHEN Shanchun, ZOU Xiuping. Expression Analysis of CsMYB41 and CsMYB63 Genes in Response to Citrus Canker[J]. Acta Horticulturae Sinica, 2023, 50(3): 495-507.

图/表 9

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引物名称 Primer	序列（5′-3′） Sequences	引物名称 Primer	序列（5′-3′） Sequences
qCs1g06220-F	AAGCTTGTCGAGTAATTGAAATGGG	qCs1g06220-R	TTATTATACAAGTCCAATCGCATCC
qCs4g12760-F	ATTATCTCAAATGGGAAAAGGGAGA	qCs4g12760-R	AATTATTTGATCACTCGCAAACGGA
Cs1g06220-F	CGGGGTACC ATGGGAAGGCCACCAAGCTC	Cs1g06220-R	CGCGGATCC TACAAGTCCAATCGCATCCA
Cs4g12760-F	CGGGGTACC ATGGGAAAAGGGAGAGCTCC	Cs4g12760-R	CGCGGATCC CTCGCAAACGGAACTTTGTA
Actin-F	CATCCCTCAGCACCTTCC	Actin-R	CCAACCTTAGCACTTCTCC

引物名称 Primer	序列（5′-3′） Sequences	引物名称 Primer	序列（5′-3′） Sequences
qCs1g06220-F	AAGCTTGTCGAGTAATTGAAATGGG	qCs1g06220-R	TTATTATACAAGTCCAATCGCATCC
qCs4g12760-F	ATTATCTCAAATGGGAAAAGGGAGA	qCs4g12760-R	AATTATTTGATCACTCGCAAACGGA
Cs1g06220-F	CGGGGTACC ATGGGAAGGCCACCAAGCTC	Cs1g06220-R	CGCGGATCC TACAAGTCCAATCGCATCCA
Cs4g12760-F	CGGGGTACC ATGGGAAAAGGGAGAGCTCC	Cs4g12760-R	CGCGGATCC CTCGCAAACGGAACTTTGTA
Actin-F	CATCCCTCAGCACCTTCC	Actin-R	CCAACCTTAGCACTTCTCC

基因ID Gene ID	基因描述 Gene description	表达差异倍数 log₂ fold change
		纽荷尔脐橙 Newhall Navel Orange			四季橘 Calamondin
		1 d	3 d	5 d	1 d	3 d	5 d
Cs1g01670	MYB93一种新的侧根发育负调控因子 MYB93 a novel negative regulator of lateral root development	0.0	0.0	0.0	0.0	3.6	4.7
Cs1g06220	MYB41参与创伤和渗透应激反应 MYB41 involved in wounding and osmotic stress response	0.0	4.5	4.7	0.0	0.0	0.0
Cs1g19400	MYB7参与原花青素的合成 MYB7 involved in proanthocyanidins synthesis	2.5	4.0	6.1	1.1	0.4	3.3
Cs1g19970	MYB78响应脱落酸和盐胁迫 MYB78 responses to abscisic acid and salt stress	4.3	4.4	5.5	3.0	3.9	4.7
Cs2g06950	MYB样蛋白102参与创伤和渗透应激 MYB-like 102 involved in wounding and osmotic stress	3.4	5.1	7.1	3.1	2.3	2.8
Cs3g02020	MYB同源结构域样超家族蛋白 MYB Homeodomain-like superfamily protein	2.2	3.4	5.2	1.0	1.0	1.8
Cs3g22220	MYB6参与调节花青素生物合成 MYB6 involved in regulating anthocyanin biosynthesis	3.2	4.0	5.0	2.5	3.0	4.3
Cs3g23070	MYBR1参与多种非生物刺激反应 MYBR1 involved in a variety of abiotic stimiuli response	1.9	3.3	3.8	1.3	1.9	2.5
Cs4g07780	MYB103是花药发育所必需的 MYB103 is required for anther development	4.0	3.4	5.4	2.6	3.7	4.9
Cs4g12760	MYB63参与细胞壁发育 MYB63 involved in cell wall development	8.5	10.3	11.2	6.2	5.9	7.5
Cs4g18910	MYB36参与侧根边界的发育 MYB36 involved in develompent of lateral root boundaries	1.0	-0.5	-0.8	1.2	2.0	2.4
Cs5g10670	MYB96在干旱胁迫期间介导脱落酸信号传导 MYB96 mediates abscisic acid signaling during drought stress	-1.5	-1.4	-8.1	-0.9	-1.2	-3.4
Cs5g26420	同源结构域样超家族蛋白 Homeodomain-like superfamily protein	-1.1	-1.9	-3.8	-0.5	-0.8	-2.1
Cs5g27440	MYB117在器官模式中发挥功能 MYB117 functions in organ patterning	2.0	1.1	-0.8	1.7	1.1	0.7
Cs5g29830	MYB14	2.1	0.6	1.1	3.6	2.1	1.5
Cs6g17340	MYB20	4.2	2.4	0.6	3.8	2.8	1.4
Cs7g26930	MYB62参与赤霉素生物合成的调节 MYB62 involved in regulation of gibberellic acid biosynthesis	3.0	3.7	4.0	1.9	1.6	2.0
Cs7g32400	TBP1具有单个MYB端粒DNA结合域 TBP1 with a single MYB telomeric DNA-binding domain	0.2	0.8	3.2	0.0	0.0	1.0
Cs8g12680	MYB4相关蛋白 MYB-related protein MYB4	1.5	2.1	2.6	2.5	2.5	2.2
Cs9g01750	推测转录因子MYB108 putative transcription factor MYB108	2.1	5.0	6.1	1.9	2.5	4.7
Orange1.1t00897	MYB38调节腋生分生组织的形成 MYB38 regulates axillary meristem formation	2.9	4.2	5.6	2.4	3.9	3.7

基因ID Gene ID	基因描述 Gene description	表达差异倍数 log₂ fold change
		纽荷尔脐橙 Newhall Navel Orange			四季橘 Calamondin
		1 d	3 d	5 d	1 d	3 d	5 d
Cs1g01670	MYB93一种新的侧根发育负调控因子 MYB93 a novel negative regulator of lateral root development	0.0	0.0	0.0	0.0	3.6	4.7
Cs1g06220	MYB41参与创伤和渗透应激反应 MYB41 involved in wounding and osmotic stress response	0.0	4.5	4.7	0.0	0.0	0.0
Cs1g19400	MYB7参与原花青素的合成 MYB7 involved in proanthocyanidins synthesis	2.5	4.0	6.1	1.1	0.4	3.3
Cs1g19970	MYB78响应脱落酸和盐胁迫 MYB78 responses to abscisic acid and salt stress	4.3	4.4	5.5	3.0	3.9	4.7
Cs2g06950	MYB样蛋白102参与创伤和渗透应激 MYB-like 102 involved in wounding and osmotic stress	3.4	5.1	7.1	3.1	2.3	2.8
Cs3g02020	MYB同源结构域样超家族蛋白 MYB Homeodomain-like superfamily protein	2.2	3.4	5.2	1.0	1.0	1.8
Cs3g22220	MYB6参与调节花青素生物合成 MYB6 involved in regulating anthocyanin biosynthesis	3.2	4.0	5.0	2.5	3.0	4.3
Cs3g23070	MYBR1参与多种非生物刺激反应 MYBR1 involved in a variety of abiotic stimiuli response	1.9	3.3	3.8	1.3	1.9	2.5
Cs4g07780	MYB103是花药发育所必需的 MYB103 is required for anther development	4.0	3.4	5.4	2.6	3.7	4.9
Cs4g12760	MYB63参与细胞壁发育 MYB63 involved in cell wall development	8.5	10.3	11.2	6.2	5.9	7.5
Cs4g18910	MYB36参与侧根边界的发育 MYB36 involved in develompent of lateral root boundaries	1.0	-0.5	-0.8	1.2	2.0	2.4
Cs5g10670	MYB96在干旱胁迫期间介导脱落酸信号传导 MYB96 mediates abscisic acid signaling during drought stress	-1.5	-1.4	-8.1	-0.9	-1.2	-3.4
Cs5g26420	同源结构域样超家族蛋白 Homeodomain-like superfamily protein	-1.1	-1.9	-3.8	-0.5	-0.8	-2.1
Cs5g27440	MYB117在器官模式中发挥功能 MYB117 functions in organ patterning	2.0	1.1	-0.8	1.7	1.1	0.7
Cs5g29830	MYB14	2.1	0.6	1.1	3.6	2.1	1.5
Cs6g17340	MYB20	4.2	2.4	0.6	3.8	2.8	1.4
Cs7g26930	MYB62参与赤霉素生物合成的调节 MYB62 involved in regulation of gibberellic acid biosynthesis	3.0	3.7	4.0	1.9	1.6	2.0
Cs7g32400	TBP1具有单个MYB端粒DNA结合域 TBP1 with a single MYB telomeric DNA-binding domain	0.2	0.8	3.2	0.0	0.0	1.0
Cs8g12680	MYB4相关蛋白 MYB-related protein MYB4	1.5	2.1	2.6	2.5	2.5	2.2
Cs9g01750	推测转录因子MYB108 putative transcription factor MYB108	2.1	5.0	6.1	1.9	2.5	4.7
Orange1.1t00897	MYB38调节腋生分生组织的形成 MYB38 regulates axillary meristem formation	2.9	4.2	5.6	2.4	3.9	3.7