甜橙硫氧还蛋白基因<i>CsTRXh</i>1克隆与表达分析

王淘; 杨澄; 闫亚娜; 王雨潇; 李瑞民

doi:10.19303/j.issn.1008-0384.2022.007.008

甜橙硫氧还蛋白基因CsTRXh1克隆与表达分析

doi: 10.19303/j.issn.1008-0384.2022.007.008

赣南师范大学生命科学学院，江西　赣州　341000

基金项目: 国家自然科学基金项目（32160621）；江西省重大科技研发专项（20194ABC28007）

详细信息

作者简介:
王淘（2001-），女，研究方向：生物科学（E-mail：2879899484@qq.com）

通讯作者:
李瑞民（1990-），男，博士，讲师，研究方向：耐黄龙病基因资源发掘与利用（E-mail：1600045@gnnu.edu.cn）

中图分类号: S 664.9
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- 被引次数: 0
出版历程
- 收稿日期: 2022-04-11
- 修回日期: 2022-05-26
- 网络出版日期: 2022-08-29
- 刊出日期: 2022-07-28

Cloning and Expression of a Thioredoxin Gene CsTRXh1 from Citrus sinensis

College of Life Science, Gannan Normal University, Ganzhou, Jiangxi　341000, China

摘要

摘要: 目的通过甜橙硫氧还蛋白基因CsTRXh1克隆与表达分析，以期为解析甜橙硫氧还蛋白基因的功能及胁迫应答机制提供参考依据。方法基于甜橙基因组数据使用同源克隆法克隆甜橙硫氧还蛋白基因，利用生物信息学分析CsTRXh1蛋白与其他物种同源蛋白的相似性、系统进化和理化性质，使用qRT-PCR方法分析CsTRXh1基因在健康甜橙树和感染黄龙病甜橙树叶片中的表达模式，通过瞬时转化烟草叶片分析CsTRXh1蛋白在植物细胞中的亚细胞定位。结果获得1个甜橙硫氧还蛋白基因CsTRXh1，GenBank登录号为ON125405。蛋白质序列比对分析表明，CsTRXh1与其他植物来源的硫氧还蛋白具有较高的序列相似度，包含保守结构域Thioredoxin。聚类分析表明，CsTRXh1为h型硫氧还蛋白。表达分析结果表明，CsTRXh1基因在感染黄龙病甜橙叶片中上调表达。亚细胞定位分析表明，CsTRXh1定位于细胞质和细胞膜。结论 CsTRXh1基因受黄龙病菌侵染诱导表达，推测CsTRXh1在黄龙病菌侵染柑橘过程中参与生理生化调控功能。
- 甜橙 /
- 硫氧还蛋白 /
- 基因克隆 /
- 表达分析 /
- 亚细胞定位
Abstract: Objective A thioredoxin gene, CsTRXh1, was cloned from Citrus sinensis to determine the expression and subcellular localization for studying its function and role played in stress response. Method CsTRXh1 from the homology sequence in the genome of C. sinensis was cloned. Homology, phylogenetic relationship, and physicochemical properties of the gene were analyzed, and expressions in healthy and Huanglongbing-infected trees detected. The transient expression of the gene in tobacco leaves was used to find its subcellular localization. Result The successfully cloned CsTRXh1 was assigned with the GenBank accession ON125405. A multi-protein sequences alignment showed that it contained a conserved thioredoxin domain with a high similarity with other plant thioredoxin proteins. The phylogenetic analysis indicated it to be an h-type thioredoxin protein. CsTRXh1 was upregulated in the leaves of Huanglongbing-infected trees and localized in the cytoplasm and cytomembrane. Conclusion The expression induced in the Huanglongbing-infected trees revealed a possible association of CsTRXh1 with the physiological and biochemical regulations of insect-disseminated Candidatus Liberibacter asiaticus that causes the destructive, incurable citrus greening disease.
- Citrus sinensis /
- thioredoxin /
- gene cloning /
- expression analysis /
- subcellular localization

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图 1 CsTRXh1基因序列分析

Figure 1. Sequence analysis on CsTRXh1

下载: 全尺寸图片幻灯片

图 2 CsTRXh1及其同源蛋白序列比对

图中蓝线标注序列区域为Thioredoxin保守结构域。

Figure 2. Multiple sequence alignment between CsTRXh1 and homology proteins

Dark blue line indicates conserved thioredoxin domain.

下载: 全尺寸图片幻灯片

图 3 CsTRXh1及其同源蛋白系统发育分析

Figure 3. Phylogenetic analysis of CsTRXh1 and its homology proteins

下载: 全尺寸图片幻灯片

图 4 CsTRXh1基因在感染黄龙病与健康纽荷尔叶片中的表达分析

**表示单因素ANOVA方差分析P值小于0.01。

Figure 4. Relative expressions of CsTRXh1 in leaves of healthy and Huanglongbing-infected Newhall trees

** indicates P＜0.01 based on univariate ANOVA analysis.

下载: 全尺寸图片幻灯片

图 5 CsTRXh1蛋白的亚细胞定位分析

绿色荧光场和叶绿体场激发波长为488 nm；白光场为可见光，叠加为绿色荧光场、白光场和叶绿体场图片重叠；Free GFP代表对照蛋白绿色荧光定位情况，CsTRXh1代表目的蛋白绿色荧光定位情况。

Figure 5. Subcellular location of CsTRXh1

The excitation wavelength of GFP field and Chloroplast field was 488 nm; the bright field was visible light, and the GFP field, bright field and chloroplast field were merged to generate an overlapped image. Free GFP stands for the location of control green fluorescent protein, CsTRXh1 stands for the location of CsTRXh1.

下载: 全尺寸图片幻灯片

表 1 基因CsTRXh1克隆、定量分析和载体构建所用引物序列

Table 1. Primer sequence used for cloning, expression analysis, and vector construction of CsTRXh1

引物名称　　　　　　 Primer names	引物序列（5'-3'）　　　　　　　　　　 Prime sequences	退火温度 Tm/℃	用途 Usage
CsTRXh1-F	ATGGCAGCAGCAGAAGAGGG	61.2	基因克隆 Gene cloning
CsTRXh1-R	TTAGGCAGAGGCAGTTGCCAG	61.2	基因克隆 Gene cloning
CsTRXh1-2300-F	AGAACACGGGGGACGAGCTCATGGCAGCAGCAGAAGAGGG	60.9	植物双元表达载体构建 Construction of plant binary expression vector
CsTRXh1-2300-R	ACCATGGTGTCGACTCTAGAGGCAGAGGCAGTTGCCAG	60.9	植物双元表达载体构建 Construction of plant binary expression vector
CsTRXh1-q-F	GCCGTTTCATTGCTCCTTTC	56.6	实时荧光定量 Real time fluorescence quantification
CsTRXh1-q-F	AGTCAGTGGCAACACTCTTC	56.6	实时荧光定量 Real time fluorescence quantification
GAPDH-F	GAAAGGTCTTGCCTGCTTTG	56.8	内参 Internal reference
GAPDH-R	TCCTTCTCCAGCCTCACTGT	56.8	内参 Internal reference
表中下划线标注分别为Sac I、Xba I酶切位点。 Recognition site of Sac I, Xba I enzyme are underlined.

下载: 导出CSV

表 2 CsTRXh1蛋白的二级结构组成

Table 2. Secondary structure of CsTRXh1

类型 Type	位点数量 Number	比例 Ratio /%
α-螺旋 Alpha helix	57	47.90
延伸链 Extended strand	23	19.33
β-转角 Beta turn	12	10.08
无规则卷曲 Random coil	27	22.69

下载: 导出CSV

参考文献(21)

[1]	WANG N. The Citrus huanglongbing crisis and potential solutions [J]. Molecular Plant, 2019, 12(5): 607−609. doi: 10.1016/j.molp.2019.03.008
[2]	MA W X, PANG Z Q, HUANG X E, et al. Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin [J]. Nature Communications, 2022, 13: 529. doi: 10.1038/s41467-022-28189-9
[3]	GELHAYE E, ROUHIER N, NAVROT N, et al. The plant thioredoxin system [J]. Cellular and Molecular Life Sciences CMLS, 2005, 62(1): 24−35. doi: 10.1007/s00018-004-4296-4
[4]	LEE M Y, SHIN K H, KIM Y K, et al. Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots [J]. Plant Physiology, 2005, 139(4): 1881−1889. doi: 10.1104/pp.105.067884
[5]	SWEAT T A, WOLPERT T J. Thioredoxin h5 is required for victorin sensitivity mediated by a CC-NBS-LRR gene in Arabidopsis [J]. The Plant Cell, 2007, 19(2): 673−687. doi: 10.1105/tpc.106.047563
[6]	MENG L, WONG J H, FELDMAN L J, et al. A membrane-associated thioredoxin required for plant growth moves from cell to cell, suggestive of a role in intercellular communication [J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(8): 3900−3905. doi: 10.1073/pnas.0913759107
[7]	ZHANG C J, ZHAO B C, GE W N, et al. An apoplastic h-type thioredoxin is involved in the stress response through regulation of the apoplastic reactive oxygen species in rice [J]. Plant Physiology, 2011, 157(4): 1884−1899. doi: 10.1104/pp.111.182808
[8]	JI M G, PARK H J, CHA J Y, et al. Expression of Arabidopsis thaliana Thioredoxin-h2 in Brassica napus enhances antioxidant defenses and improves salt tolerance [J]. Plant Physiology and Biochemistry, 2020, 147: 313−321. doi: 10.1016/j.plaphy.2019.12.032
[9]	WILKINS M R, GASTEIGER E, BAIROCH A, et al. Protein identification and analysis tools in the ExPASy server [J]. Methods in Molecular Biology (Clifton, N J), 1999, 112: 531−552.
[10]	GEOURJON C, DELÉAGE G. SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments [J]. Computer Applications in the Biosciences:CABIOS, 1995, 11(6): 681−684.
[11]	TAMURA K, STECHER G, PETERSON D, et al. MEGA6: Molecular evolutionary genetics analysis version 6.0 [J]. Molecular Biology and Evolution, 2013, 30(12): 2725−2729. doi: 10.1093/molbev/mst197
[12]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔC_T method [J]. Methods, 2001, 25(4): 402−408. doi: 10.1006/meth.2001.1262
[13]	WASZCZAK C, CARMODY M, KANGASJÄRVI J. Reactive oxygen species in plant signaling [J]. Annual Review of Plant Biology, 2018, 69: 209−236. doi: 10.1146/annurev-arplant-042817-040322
[14]	邱金龙, 金巧玲, 王钧. 活性氧与植物抗病反应 [J]. 植物生理学通讯, 1998, 34(1):56−63. QIU J L, JIN Q L, WANG J. Activity of oxygen and plant disease resistance [J]. Plant Physiology Communications, 1998, 34(1): 56−63.(in Chinese)
[15]	PITINO M, ARMSTRONG C M, DUAN Y P. Molecular mechanisms behind the accumulation of ATP and H₂O₂ in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection [J]. Horticulture Research, 2017, 4: 17040. doi: 10.1038/hortres.2017.40
[16]	MHAMDI A, NOCTOR G, BAKER A. Plant catalases: Peroxisomal redox guardians [J]. Archives of Biochemistry and Biophysics, 2012, 525(2): 181−194. doi: 10.1016/j.abb.2012.04.015
[17]	MATA-PÉREZ C, SPOEL S H. Thioredoxin-mediated redox signalling in plant immunity [J]. Plant Science, 2019, 279: 27−33. doi: 10.1016/j.plantsci.2018.05.001
[18]	CLARK K J, PANG Z Q, TRINH J, et al. Sec-delivered effector 1 (SDE1) of 'Candidatus Liberibacter asiaticus' promotes Citrus huanglongbing [J]. Molecular Plant-Microbe Interactions:MPMI, 2020, 33(12): 1394−1404. doi: 10.1094/MPMI-05-20-0123-R
[19]	FAN J, CHEN C X, YU Q B, et al. Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to ‘Candidatus Liberibacter asiaticus’ infection [J]. Molecular Plant-Microbe Interactions:MPMI, 2012, 25(11): 1396−1407. doi: 10.1094/MPMI-06-12-0150-R
[20]	LIAO H L, BURNS J K. Gene expression in Citrus sinensis fruit tissues harvested from huanglongbing-infected trees: Comparison with girdled fruit [J]. Journal of Experimental Botany, 2012, 63(8): 3307−3319. doi: 10.1093/jxb/ers070
[21]	HU Y, ZHONG X, LIU X L, et al. Comparative transcriptome analysis unveils the tolerance mechanisms of Citrus hystrix in response to ‘Candidatus Liberibacter asiaticus’ infection [J]. PLoS One, 2017, 12(12): e0189229. doi: 10.1371/journal.pone.0189229