柑橘响应黄龙病菌侵染的NAC基因的克隆及表达分析

doi:10.16420/j.issn.0513-353x.2021-0553

摘要/Abstract

摘要：

挖掘柑橘抗黄龙病（Huanglongbing,HLB）基因是抗病育种的基础和关键。以感染黄龙病菌亚洲种Candidatus Liberibacter asiaticus（CLas）早期（2个月）锦橙根和叶片中脉比较转录组数据为基础,筛选到9个响应柑橘黄龙病侵染诱导的NAC基因,从中选3个差异表达水平较高的基因克隆,分别命名为CsNAC21/22、CsNAC68和CsNAC78。生物信息分析表明3个基因均符合NAC基因家族的特征;烟草亚细胞定位结果表明,CsNAC68定位在细胞核,CsNAC21/22和CsNAC78定位在细胞核和细胞质。实时荧光定量PCR（qRT-PCR）分析表明,3个候选基因在易感HLB的锦橙、耐病的马蜂柑和高耐病的九里香的组织和病原菌诱导表达特征呈现明显差异。以健康植株为对照,CsNAC68和CsNAC78主要在锦橙的根中响应CLas感染,显著上调表达,CsNAC68在九里香叶肉和马蜂柑根中响应CLas感染,显著上调表达;CsNAC21/22主要在锦橙根和马蜂柑叶肉中显著下调表达。以‘锦橙’叶片为试材通过qRT-PCR分析候选基因响应SA、JA、ABA、ETH诱导的表达特征,结果表明,3个基因可能参与ABA的信号转导途径,CsNAC68可能参与SA和JA的信号转导途径,而CsNAC78可能参与ETH的信号转导途径。

关键词: 柑橘, 黄龙病, NAC转录因子, 基因克隆, 表达分析

Abstract:

Citrus Huanglongbing（HLB）has become a major disease and limiting factor of production in citrus areas that have become infected. It can cause production extinction when the damage to citrus industry is serious,and there is no effective prevention and treatment method for this disease. The key to solve this problem is to excavate citrus resistance genes for disease resistance breeding. Therefor,in this study,based on the transcriptome data of Candidatus Liberibacter asiaticus（CLas）in the root and midrib in the early stage of infection（2 months）,nine NAC（NAM,ATAF1/2,CUC2）genes were screened in response to HLB infection,and three genes with high differential expression were cloned,named as CsNAC21/22,CsNAC68 and CsNAC78,respectively. Bioinformatics analysis showed that CsNAC21/22,CsNAC68 and CsNAC78 encoded 306,503 and 152 amino acids respectively,and the isoelectric point（pI） was 5.43,6.59 and 8.39,respectively. The conserved domain analysis showed that CsNAC21/22,CsNAC68 and CsNAC78 contained five subdomains of the conserved domain A-E,and the A,C and D parts were relatively conserved,which was consistent with the characteristics of the NAC gene family. Phylogenetic tree analysis showed that CsNAC21/22 was closely related to Glycine max and Populus trichocarpa,and CsNAC68 and CsNAC78 were closely related to Arabidopsis thaliana and Populus trichocarpa. Subcellular localization analysis showed that CsNAC68 located in the nucleus,and CsNAC21/22 and CsNAC78 located in the nucleus and cytoplasm. Real-time fluorescent quantitative PCR（qRT-PCR）analysis showed that the three candidate genes showed significantly different tissue and pathogen induced expression characteristics in HLB susceptible Jincheng（Citrus sinensis）and HLB resistant Mafenggan（C. hystrix）and Jiulixiang（Murraya exotica）. With healthy plants as the control,the expression levels of CsNAC68 and CsNAC78 were significantly up-regulated in the Jincheng root in response to CLas infection,while CsNAC68 was in response to CLas infection in the mesophyll of Jiulixiang and the root of Mafenggan significantly up-regulated expression. The expression of CsNAC21/22 was significantly down-regulated in the root of Jincheng and the mesophyll of Mafenggan. Using Jincheng leaves as the test material,the expression characteristics of candidate genes in response to plant hormone induction were analyzed by qRT-PCR. The results showed that the three genes may be involved in the signal transduction pathway of abscisic acid（ABA）,and CsNAC68 may be involved in the signal transduction pathway of salicylic acid（SA）and jasmonic acid（JA）,and CsNAC78 may be involved in the signal transduction pathway of ethylene（ETH）.

Key words: Citrus, Huanglongbing, NAC, gene cloning, expression analysis

中图分类号:

S666

郑林, 王帅, 刘语诺, 杜美霞, 彭爱红, 何永睿, 陈善春, 邹修平. 柑橘响应黄龙病菌侵染的NAC基因的克隆及表达分析[J]. 园艺学报, 2022, 49(7): 1441-1457.

ZHENG Lin, WANG Shuai, LIU Yunuo, DU Meixia, PENG Aihong, HE Yongrui, CHEN Shanchun, ZOU Xiuping. Gene Cloning and Expression Analysis of NAC Gene in Citrus in Response to Huanglongbing[J]. Acta Horticulturae Sinica, 2022, 49(7): 1441-1457.

图/表 14

表1 普通PCR引物

Table 1 The sequences of common PCR primer pairs

用途Amplification	名称Name	序列（5′-3′）Sequence
黄龙病常规 PCR 检测 Huanglongbing routine PCR test	OI1 OI2C	F:GCGCGTATGCAATACGAGCGGCA
黄龙病常规 PCR 检测 Huanglongbing routine PCR test	OI1 OI2C	R:GCCTCGCGACTTCGCAACCCAT
基因克隆Gene cloning	CsNAC21/22	F:CGCGGATCCATGGGGCTAAGAGATATTGGAGCTAC
基因克隆Gene cloning	CsNAC21/22	R:CCGGAATTCTCATAGGAAAACCATGCTGTTATCCAAT
	CsNAC68	F:TCCCCCGGGATGCCCAAAGAGGACCGAG
	CsNAC68	R:GCGTCGACTCAATGAAGATACGCCATT
	CsNAC78	F:CGCGGATCCATGGACTATAATTGCCTGGGATACAG
	CsNAC78	R:CCGGAATTCCTATATGAAGAAAAACAAGTTGTATAGAACAGAAG
亚细胞定位Subcellular localization	CsNAC21/22	F:GGGGTACCATGGGGCTAAGAGATATTGG
亚细胞定位Subcellular localization	CsNAC21/22	R:ACGCGTCGACTAGGAAAACCATGCTGTTAT
	CsNAC68	F:GGGGTACCATGCCCAAAGAGGACCGAGT
	CsNAC68	R:ACGCGTCGACATGAAGATACGCCATTTCCC
	CsNAC78	F:GGGGTACCATGGACTATAATTGCCTGGG
	CsNAC78	R:ACGCGTCGACTATGAAGAAAAACAAGTTGT

表2 qRT-PCR引物

Table 2 The sequences of qRT-PCR primer pairs

目的基因 Target gene	引物序列（5′-3′） Primer sequence
Cs1g09640.1	F:AGCCATGGGACTTACCAACG;R:ACCAGTAAGGCCGTTTCCAG
Cs4g12500.1	F:GATCATCAGCTTCCGGGGTT;R:TTCGATGCTGAGCGGTTTCT
Cs5g29650.1(CsNAC21/22)	F:GGTGGCAAAGCTCAATGCAA;R:GCGTCTTCCTCATCCCTACG
Cs6g14200.1	F:AAGGGAAGTGCCTCAACGAG;R:TCGTCTCCGGAGGATGAACT
Cs8g14700.1	F:GTCAAGGAGACACCGAGCAA;R:AGCCTGTGGCCTTCCAATAC
orange1.1t00587.1	F:AGGAATTTGTCCTCCTTCGTT;R:AGCCGAAGGTTGAGGAGTTG
orange1.1t00588.1	F:TACAAAGGTCGTGGCTCCAG;R:TGCAGCTCGGGTATAGTCCT
orange1.1t00589.1(CsNAC68)	F:CGAAGGAATTTGTCCTCTTCCG;R:AGACCGCGGTTTATCTGTGG
orange1.1t00590.1(CsNAC78)	F:ACTGGTGGCGATCGTCAAAT;R:AGAACAGAAGAGATTGCCTGATGA

图1 柑橘NAC转录因子的无根系统发育树

Fig. 1 An unrooted phylogenetic tree of the NAC transcription factors of citrus

表3 CsNAC家族在锦橙中应答CLas侵染的转录组比较

Table 3 Comparative analysis of CsNAC family transcriptome in response to CLas infection in Jincheng

基因ID Gene ID	基因 Gene	CLas vs. 空白对照 CLas vs. Mock
基因ID Gene ID	基因 Gene	叶片中脉Midrib	根Root
Cs1g09640.1	CsNTL9-1	1.252	4.283
Cs4g12500.1	CsNAC	—	—
Cs5g29650.1	CsNAC21/22	—	—
Cs6g14240.1	CsNAC090-1	5.469	—
Cs8g14700.1	CsNAC090-2	-1.172	—
orange1.1t00587.1	CsNAC050	2.315	5.735
orange1.1t00588.1	CsNTL9-2	—	20.474
orange1.1t00589.1	CsNAC68	—	4.163
orange1.1t00590.1	CsNAC78	1.633	11.316

表4 柑橘黄龙病菌诱导锦橙9个NAC基因的相对表达量

Table 4 Relative expression levels of 9 NAC genes in Jincheng induced by HLB

基因 Gene	根 Root	叶脉 Midrib	基因 Gene	根 Root	叶脉 Midrib
CsNTL9-1	2.42 ± 0.31	1.16 ± 0.24	CsNAC050	4.24 ± 0.37	2.63 ± 0.44
CsNAC	0.93 ± 0.09	2.01 ± 0.32	CsNTL9-2	1.49 ± 0.15	1.68 ± 0.18
CsNAC21/22	0.49 ± 0.13	0.68 ± 0.10	CsNAC68	8.62 ± 0.39	2.32 ± 0.18
CsNAC090-1	0.75 ± 0.12	1.38 ± 0.22	CsNAC78	6.62 ± 0.29	1.86 ± 0.04
CsNAC090-2	0.97 ± 0.04	0.63 ± 0.10

表5 柑橘3个NAC基因基因编码的氨基酸序列分析

Table 5 Amino acid sequence analysis of three NAC genes in citrus

名称 Name	CAP号 CAP ID	ORF/bp	氨基酸数 Number of amino acid	分子量 Molecular weight	等电点 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
CsNAC21/22	Cs5g29650.1	921	306	35 107.14	5.43	-0.712	66.84	43.83	细胞核 Nuclear
CsNAC68	orange1.1t00589.1	1 512	503	56 809.43	6.59	-0.730	58.99	41.30	细胞核 Nuclear
CsNAC78	orange1.1t00590.1	459	152	17 691.99	8.39	-0.543	71.84	32.27	细胞质 Cytoplasm

图2 柑橘CsNAC与拟南芥AtNAC、水稻OsNAC氨基酸的多序列比对

Fig. 2 Multi-sequence alignment of citrus CsNAC amino acid sequences with Arabidopsis thaliana AtNAC and Oryza sativa OsNAC amino acid sequences

图3 CsNAC系统进化树分析采用蛋白的全长序列进行邻接法构建,并在每个节点指示1 000次复制的bootstrap值,分支上的数字为自展值（%）。

Fig. 3 CsNAC phylogenetic tree analysisConstructed by Neighbor-Joining method with the full-length sequence of the protein,and the Bootstrap value of 1 000 copies is indicated on each node,and the number on the branch is the self-spreading value（%）.

图4 柑橘3个NAC基因在烟草表皮细胞中的瞬时表达

Fig. 4 Transient expression of three NAC genes in tobacco epidermal cells

图5 不同抗性柑橘锦橙、马蜂柑和九里香接种Clas后3个NAC基因在不同组织中的相对表达量“*”表示与同一品种健康植株对照的差异显著性（P < 0.05）。

Fig. 5 Analysis of the relative expression levels of three NAC genes in different tissues of Jincheng,Mafenggan and Jiulixiang“*”Represents the significance of the difference with healthy plants of the same cultivar（P < 0.05）.

图6 SA诱导下CsNAC的表达量“*”表示与同一时期水处理对照的差异显著性（P < 0.05）。

Fig. 6 CsNAC expression induced by SA“*”indicates the significant difference between water treatment control in the same period（P < 0.05）.

图7 JA诱导下CsNAC的表达量“*”表示与同一时期水处理对照的差异显著性（P < 0.05）。

Fig. 7 CsNAC expression induced by JA“*”indicates the significant difference between water treatment control in the same period（P < 0.05）.

图8 ABA诱导下CsNAC的表达量“*”表示与同一时期水处理对照的差异显著性（P < 0.05）。

Fig. 8 CsNAC expression induced by ABA“*”indicates the significant difference between water treatment control in the same period（P < 0.05）.

图9 ETH诱导下CsNAC的表达量“*”表示与同一时期水处理对照的差异显著性（P < 0.05）。

Fig. 9 CsNAC expression induced by ETH“*”indicates the significant difference between water treatment control in the same period（P < 0.05）.

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