茶树NPR基因家族成员鉴定与表达分析及冷诱导CsNPR3的基因克隆

doi:10.3969/j.issn.1004-1524.20220895

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (7): 1511-1522.DOI: 10.3969/j.issn.1004-1524.20220895

茶树NPR基因家族成员鉴定与表达分析及冷诱导CsNPR3的基因克隆

薛承进¹(), 赵兰馨¹, 赵德刚¹^,², 黄小贞¹^,³^,^*()

1.贵州大学生命科学学院/农业工程研究院, 山地植物资源保护与保护种质创新教育部重点实验室, 贵州贵阳 550025
2.贵州省农业科学研究院,贵州贵阳 550006
3.贵州大学茶学院, 贵州贵阳 550025

收稿日期:2022-06-16 出版日期:2023-07-25 发布日期:2023-08-17
作者简介:薛承进(1999—),男,海南儋州人,硕士研究生,主要从事茶树发育生物学研究。E-mail: 1327466293xue@sina.cn
通讯作者: *黄小贞, E-mail: xzhuang@gzu.edu.cn
基金资助:
国家自然科学基金(31960615)

Identification and expression analysis of NPR gene family members and cloning of cold-induced CsNPR3 gene in tea plants (Camellia sinensis)

XUE Chengjin¹(), ZHAO Lanxin¹, ZHAO Degang¹^,², HUANG Xiaozhen¹^,³^,^*()

1. The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region, Ministry of Education, College of Life Sciences/Agricultural Engineering Research Institute, Guizhou University, Guiyang 550025, China
2. Guizhou Academy of Agricultural Sciences, Guiyang 550006, China
3. College of Tea Sciences, Guizhou University, Guiyang 550025, China

Received:2022-06-16 Online:2023-07-25 Published:2023-08-17
Contact: HUANG Xiaozhen

摘要/Abstract

摘要：

冷胁迫是影响茶树(Camellia sinensis) 产量和品质的主要非生物胁迫。病程相关基因非表达子(non-expresser of pathogenesis-related genes,NPRs)是植物抗病途径中的主要免疫调节蛋白。目前,茶树中NPR基因家族及其在冷响应中的作用研究相对较少。该研究首先利用生物信息学分析,在茶树基因组中筛选鉴定出3个定位于不同的染色体的NPR基因,命名为CsNPR1、CsNPR2和CsNPR3(GenBank No.ON794747)。这些基因均包含家族保守的BTB/POZ、Ankyrin 重复结构域以及多个半胱氨酸残基。进化分析发现,茶树的NPR家族成员的数量虽然与其他物种存在差异,但进化中表现出很强的保守性,与拟南芥及其他物种的分类结果相同。基因结构分析表明,茶树NPR家族与拟南芥具有相似的内含子-外显子结构。利用实时荧光定量PCR(qRT-PCR)技术,探明CsNPR1、CsNPR2和CsNPR3的组织表达模式以及对冷刺激和水杨酸(SA)的响应情况。进一步克隆被冷信号和SA信号同时诱导表达的CsNPR3,比对发现该蛋白氨基酸序列存在着品种差异性。该研究对探明茶树NPR基因家族参与冷胁迫与水杨酸的交叉调控有一定参考意义。

关键词: 茶树, 冷胁迫, NPR, 基因家族

Abstract:

Cold stress is the main abiotic stress that affects the yield and quality of tea plants (Camellia sinensis). The non-expresser of pathogenesis-related genes, NPRs are major immune regulatory proteins which have been implicated in plant disease resistance pathway. However, the function of NPR genes has not been well investigated in tea plants. In this study, we identified three members of NPR gene family which located on different tea plant chromosomes by using bioinformatics technology. They were named as CsNPR1, CsNPR2 and CsNPR3 (GenBank No. ON794747) respectively. These genes all contain highly conserved BTB/POZ domain, ankyrin repeat domain and multiple cysteine residues. Although the phylogenetic analysis showed that the number of NPR family members in tea plants was different from that of other species, it still showed highly conservation in evolution, which was the same as that of Arabidopsis and other species. Gene structure analysis showed that tea NPR family and Arabidopsis had similar exons-intron structure. Real-time fluorescent quantitative PCR (qRT-PCR) technique was used to identify the different tissue expression patterns of CsNPR1, CsNPR2 and CsNPR3, as well as their responses to cold stimulation and hormone salicylic acid (SA). Furthermore, CsNPR3, which was induced both by cold signal and SA was further cloned. It was found that there were variety differences in the amino acid sequence of the protein. This study can provide important scientific and theoretical basis for further clarifying the involvement of NPR gene family in cold stress and salicylic acid regulation in tea plants.

Key words: Camellia sinensis, cold stress, NPR, gene family

中图分类号:

S571.1

薛承进, 赵兰馨, 赵德刚, 黄小贞. 茶树NPR基因家族成员鉴定与表达分析及冷诱导CsNPR3的基因克隆[J]. 浙江农业学报, 2023, 35(7): 1511-1522.

XUE Chengjin, ZHAO Lanxin, ZHAO Degang, HUANG Xiaozhen. Identification and expression analysis of NPR gene family members and cloning of cold-induced CsNPR3 gene in tea plants (Camellia sinensis)[J]. Acta Agriculturae Zhejiangensis, 2023, 35(7): 1511-1522.

图/表 10

表1 荧光定量引物设计

Table 1 Primers sequences of qRT-PCR

基因名称 Gene name	基因座 Gene locus	引物序列 Primer sequence(5'→3')	用途 Usage	产物大小 Product size/bp
CsNPR1	CSS0034352	F: TTGGTTGCTCTGTATCAGAG R: ATGATGTCAACATCGGACCT	qRT-PCR	161
CsNPR2	CSS0007599	F: ATGAGGAGTCGCTTGAGCCAT R: GCTGTTCTAGATGACAGCTAAG	qRT-PCR	160
CsNPR3	CSS0050405	F: TGTTGGCACACACATTGCA R: TCCTCAATTGATGCCTTCTC	qRT-PCR	140
CsNPR3	CSS0050405	F:TCTAGAATGACCCTTGATGATTCCT R:CCCGGGATATTGATGAGGGTGGTGGT	基因克隆 Cloning of gene	1 443
GAPDH		F: TTGGCATCGTTGAGGGTCT R: AGTGGGAACACGGAAAGC	内参基因 Reference gene	200

表2 茶树NPR家族成员编码蛋白的基本特征

Table 2 Basic characteristics of the putative proteins encoded by NPR gene family in Camellia sinensis

基本特征Basic characteristics	CsNPR1	CsNPR2	CsNPR3
基因登录号Accession number	CSS0034352	CSS0007599	CSS0050405
染色体Chromosome	Chr14	Chr8	Chr10
位置Location	73 483 419~73 488 814	162 894 169~162 902 685	154 564 692~154 567 524
编码蛋白质长度CDS/bp	1 764	1 761	1 443
蛋白质Protein/aa	587	586	480
分子量Molecular weight/ku	65 085.66	65 520.01	52 658.93
等电点pI	5.59	5.68	6.06
亚细胞定位Subcellular localization	胞浆Cytoplasmic	胞浆Cytoplasmic	胞浆Cytoplasmic

表3 茶树与拟南芥中NPR家族蛋白的氨基酸同源性比较

Table 3 Identity of putative amino acid sequences of NPR family homologues between Camellia sinensis and Arabidopsis thaliana%

NPR homologues	CsNPR2	CsNPR3	AtNPR1	AtNPR2	AtNPR3	AtNPR4	AtNPR5	AtNPR6
CsNPR1	45.69	32.43	53.06	53.47	44.88	41.98	31.47	32.61
CsNPR2		34.29	39.33	40.29	62.48	57.63	34.28	35.17
CsNPR3			29.12	30.79	29.58	29.93	75.98	73.22
AtNPR1				62.29	39.07	37.12	28.65	29.72
AtNPR2					39.77	38.48	28.22	30.14
AtNPR3						68.22	31.96	31.50
AtNPR4							30.10	31.28
AtNPR5								80.70

图1 茶树和不同物种 NPR基因家族的进化树分析

Fig.1 Phylogenetic analysis of the NPR homologue proteins from tea and other plants

图2 茶树和拟南芥NPR家族基因的多重序列比对

Fig.2 Multiple alignment of amino acid sequences of tea plants and Arabidopsis NPR homologues

图3 茶树NPR家族的基因结构和启动子元件分析 A,保守基序分析;B,保守结构域分析;C,启动子元件分析;D,基因结构分析。

Fig.3 The gene structure and promoter element analysis of NPR in tea plants A, Conserved sequence motif analysis; B, Conserved domain analysis; C, Promoter element analysis; D, Gene structure analysis.

图4 茶树NPR家族成员在不同组织器官中的相对表达量 R、S、B、ML、OL、F分别表示根、茎、芽、成叶、老叶和花。相同字母代表没有显著性差异(n=4,P<0.05)。

Fig.4 Relative expression levels of NPR family members in different tissues and organs of tea plants R, S, B, ML, OL, F represent roots, stems, buds, mature leaves, old leaves and flowers. Means do not differ if they are labeled with the same letter (n=4, P<0.05).

图5 4 ℃处理后NPR基因在茶树中的表达情况 *,与0 h相比,差异显著(n=4,P<0.05)。下同。

Fig.5 Expression of NPR homologues in response to 4 ℃ in tea plants *, Extremely significant difference compared with 0 h (n=4, P<0.05). The same as below.

图6 SA处理后NPR基因在茶树中的表达情况 **,与0 h相比,差异极显著(P<0.01)。

Fig.6 Expression of NPR homologues in response to SA in tea plants **, Extremely significant difference compared with 0 h (P<0.01).

图7 CsNPR3全长核苷酸序列及编码的氨基酸序列黑框表示氨基酸变异位点。

Fig.7 Full length nucleotide sequence and putative amino acid sequence of CsNPR3 Black box indicates amino acid variation site.

参考文献 27

[1]	GUY C L. Cold acclimation and freezing stress tolerance: role of protein metabolism[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 1990, 41: 187-223.
[2]	DING Y L, SHI Y T, YANG S H. Advances and challenges in uncovering cold tolerance regulatory mechanisms in plants[J]. New Phytologist, 2019, 222(4): 1690-1704.
[3]	DING Y L, YANG S H. Surviving and thriving: how plants perceive and respond to temperature stress[J]. Developmental Cell, 2022, 57(8): 947-958.
[4]	DONG C H, AGARWAL M, ZHANG Y Y, et al. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(21): 8281-8286.
[5]	KUNKEL B N, BROOKS D M. Cross talk between signaling pathways in pathogen defense[J]. Current Opinion in Plant Biology, 2002, 5(4): 325-331.
[6]	MIURA K, OHTA M. SIZ1, a small ubiquitin-related modifier ligase, controls cold signaling through regulation of salicylic acid accumulation[J]. Journal of Plant Physiology, 2010, 167(7): 555-560.
[7]	MIURA K, TADA Y. Regulation of water, salinity, and cold stress responses by salicylic acid[J]. Frontiers in Plant Science, 2014, 5: 4.
[8]	SCOTT I M, CLARKE S M, WOOD J E, et al. Salicylate accumulation inhibits growth at chilling temperature in Arabidopsis[J]. Plant Physiology, 2004, 135(2): 1040-1049.
[9]	OLATE E, JIMÉNEZ-GÓMEZ J M, HOLUIGUE L, et al. NPR1 mediates a novel regulatory pathway in cold acclimation by interacting with HSFA1 factors[J]. Nature Plants, 2018, 4(10): 811-823.
[10]	韩永光, 马利刚, 赵乐, 等. 植物抗性基因NPR1研究进展[J]. 安徽农业科学, 2018, 46(26): 18-20.
	HAN Y G, MA L G, ZHAO L, et al. Research progress on resistance gene NPR1 in plants[J]. Journal of Anhui Agricultural Sciences, 2018, 46(26): 18-20. (in Chinese with English abstract)
[11]	WANG P, ZHAO Z, ZHANG Z, et al. Genome-wide identification and analysis of NPR family genes in Brassica juncea var. tumida[J]. Gene, 2021, 769(4): 145210.
[12]	BAN Q Y, WANG X W, PAN C, et al. Comparative analysis of the response and gene regulation in cold resistant and susceptible tea plants[J]. PLoS One, 2017, 12(12): e0188514.
[13]	HAO X Y, TANG H, WANG B, et al. Integrative transcriptional and metabolic analyses provide insights into cold spell response mechanisms in young shoots of the tea plant[J]. Tree Physiology, 2018, 38(11): 1655-1671.
[14]	PENG J, LI N N, DI T M, et al. The interaction of CsWRKY4 and CsOCP3 with CsICE1 regulates CsCBF1/3 and mediates stress response in tea plant (Camellia sinensis)[J]. Environmental and Experimental Botany, 2022, 199: 104892.
[15]	周旋, 申璐, 金媛, 等. 外源水杨酸对盐胁迫下茶树生长及主要生理特性的影响[J]. 西北农林科技大学学报(自然科学版), 2015, 43(7): 161-167.
	ZHOU X, SHEN L, JIN Y, et al. Effects of exogenous salicylic acid on growth and physiological characteristics of tea plant(Camellia sinensis)under salt stress[J]. Journal of Northwest A & F University (Natural Science Edition), 2015, 43(7): 161-167. (in Chinese with English abstract)
[16]	XIA E H, TONG W, HOU Y, et al. The reference genome of tea plant and resequencing of 81 diverse accessions provide insights into its genome evolution and adaptation[J]. Molecular Plant, 2020, 13(7): 1013-1026.
[17]	WEI C L, YANG H, WANG S B, et al. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(18): E4151-E4158.
[18]	SUNG D Y, KAPLAN F, LEE K J, et al. Acquired tolerance to temperature extremes[J]. Trends in Plant Science, 2003, 8(4): 179-187.
[19]	WU Y, ZHANG D, CHU J Y, et al. The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid[J]. Cell Reports, 2012, 1(6): 639-647.
[20]	BACKER R, MAHOMED W, REEKSTING B J, et al. Phylogenetic and expression analysis of the NPR1-like gene family from Persea americana(Mill.)[J]. Frontiers in Plant Science, 2015, 6: 300.
[21]	CAO H, LI X, DONG X. Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(11): 6531-6536.
[22]	ZHANG J K, JIAO P, ZHANG C, et al. Apple NPR1 homologs and their alternative splicing forms may contribute to SA and disease responses[J]. Tree Genetics & Genomes, 2016, 12(5): 1-14.
[23]	BOYLE P, LE SU E, ROCHON A, et al. The BTB/POZ domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function[J]. The Plant Cell, 2009, 21(11): 3700-3713.
[24]	江慎秀, 尚海, 李涛, 等. 油桐NPR1家族全基因组鉴定及表达模式分析[J]. 植物遗传资源学报, 2021, 22(2): 521-531.
	JIANG S X, SHANG H, LI T, et al. Genome-wide identification and expression pattern analysis of NPR1 family in Vernicia fordii[J]. Journal of Plant Genetic Resources, 2021, 22(2): 521-531. (in Chinese with English abstract)
[25]	YOCGO R E, GEZA E, CHIMUSA E R, et al. A post-gene silencing bioinformatics protocol for plant-defence gene validation and underlying process identification: case study of the Arabidopsis thaliana NPR1[J]. BMC Plant Biology, 2017, 17(1): 218.
[26]	MA Y, DAI X Y, XU Y Y, et al. COLD1 confers chilling tolerance in rice[J]. Cell, 2015, 160(6): 1209-1221.
[27]	DUAN D D, ZHANG H M. A single SNP in NRT1.1B has a major impact on nitrogen use efficiency in rice[J]. Science China Life Sciences, 2015, 58(8): 827-828.

编辑推荐

Metrics

阅读次数

全文

512

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	16	0	0	496

来源	本网站	其他网站

次数	344	168
比例	67%	33%

摘要

337

最新录用	在线预览	正式出版

0	0	337

	来源	本网站

	次数	337
	比例	100%

茶树NPR基因家族成员鉴定与表达分析及冷诱导CsNPR3的基因克隆

Identification and expression analysis of NPR gene family members and cloning of cold-induced CsNPR3 gene in tea plants (Camellia sinensis)

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 10

参考文献 27

相关文章 15

编辑推荐

Metrics

本文评价

[1]	白鼎臣, 赵支飞, 龚雪, 刘源, 牛素贞, 陈正武. 贵州栽培型地方茶树叶片气孔性状全基因组关联分析[J]. 浙江农业学报, 2023, 35(7): 1550-1563.
[2]	李必聪, 李慧英, 肖遥, 罗莎, 周庆红, 黄英金, 朱强龙. 芋扩展蛋白基因家族的全基因组鉴定及其在球茎膨大中的表达分析[J]. 浙江农业学报, 2023, 35(7): 1604-1616.
[3]	张新业, 李文静, 朱姝, 孙艳香, 王聪艳, 闫训友, 周志国. 三种伞形科蔬菜作物棕榈酰基转移酶基因家族的鉴定与分析[J]. 浙江农业学报, 2023, 35(6): 1315-1327.
[4]	张淑红, 张运峰, 武秋颖, 高凤菊, 李亚子, 纪景欣, 许可, 范永山. 玉米大斑病菌醇脱氢酶基因家族的鉴定和生物信息学分析[J]. 浙江农业学报, 2023, 35(5): 1108-1115.
[5]	李春雷, 徐红梅, 刘杰, 张汝君, 马兴云, 张华. 铝在茶树叶片亚细胞中的分布及其与细胞壁的结合研究[J]. 浙江农业学报, 2023, 35(3): 509-514.
[6]	季美君, 曹孜怡, 王翌婷, 陆静茹, 汪保华. 陆地棉NCS1基因家族的全基因组成员鉴定及分析[J]. 浙江农业学报, 2023, 35(2): 249-258.
[7]	丁一, 郑旭霞, 黄海涛, 毛宇骁, 赵芸. 浙江4个主要茶树群体种资源表型性状及遗传多样性分析[J]. 浙江农业学报, 2023, 35(2): 364-372.
[8]	杨春, 孟泽洪, 李帅, 梁思慧, 乔大河, 陈正武. 十二个茶树品种对茶棍蓟马、茶小绿叶蝉抗性表现及抗性成分初步鉴定[J]. 浙江农业学报, 2022, 34(8): 1713-1724.
[9]	杨春, 乔大河, 郭燕, 梁思慧, 林开勤, 陈正武. 115份贵州茶树资源氨基酸和茶氨酸分析与特异资源筛选[J]. 浙江农业学报, 2022, 34(7): 1351-1360.
[10]	邹振浩, 孙业良, 赵玉宝, 李鑫, 张丽平, 张兰, 董春旺, 付建玉, 韩文炎, 颜鹏. 采摘花芽对春茶产量与品质的影响[J]. 浙江农业学报, 2022, 34(7): 1369-1376.
[11]	余艳玲, 罗洪林, 罗辉, 冯鹏霏, 潘传燕, 宋漫玲, 肖蕊, 张永德. 卵形鲳鲹生肌调节因子基因家族的鉴定及在胚胎中的表达[J]. 浙江农业学报, 2022, 34(4): 695-705.
[12]	刘同金, 徐铭婕, 汪精磊, 刘良峰, 崔群香, 包崇来, 王长义. 萝卜ALMT基因家族的鉴定与表达[J]. 浙江农业学报, 2022, 34(4): 746-755.
[13]	吴小清, 周菲菲, 叶影, 黄艳梅, 杨蕾玉, 黄海涛, 吴媛媛. 不同茶树品种制龙井茶的香气特征研究[J]. 浙江农业学报, 2022, 34(3): 437-446.
[14]	疏再发, 刘瑜, 邵静娜, 郑生宏, 周慧娟, 吉庆勇, 何卫中. 浙南早生茶树种质资源主要品质成分分析及优异资源鉴选[J]. 浙江农业学报, 2022, 34(11): 2438-2450.
[15]	葛诗蓓, 金迪迪, 杨明来, 王辉, 张兰, 韩文炎, 李鑫. 不同比例红蓝光对茶叶品质成分的影响与相关调控机理研究[J]. 浙江农业学报, 2022, 34(10): 2105-2111.