Gene Cloning and Expression Analysis of NAC Gene in Citrus in Response to Huanglongbing

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

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (7): 1441-1457.doi: 10.16420/j.issn.0513-353x.2021-0553

• Research Papers • Previous Articles Next Articles

Gene Cloning and Expression Analysis of NAC Gene in Citrus in Response to Huanglongbing

ZHENG Lin, WANG Shuai, LIU Yunuo, DU Meixia, PENG Aihong, HE Yongrui, CHEN Shanchun(), ZOU Xiuping()

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

Received:2022-02-08 Revised:2022-04-25 Online:2022-07-25 Published:2022-07-29
Contact: CHEN Shanchun,ZOU Xiuping E-mail:chenshanchun@cric.cn;zouxiuping@cric.cn

Abstract

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

CLC Number:

S666

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.

Figures/Tables 14

References 56

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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

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

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

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

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

Gene Cloning and Expression Analysis of NAC Gene in Citrus in Response to Huanglongbing

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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

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

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