橡胶树HbTRXh2的克隆、表达及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0895

摘要/Abstract

摘要：

目的探究橡胶树硫氧还蛋白（TRX）基因HbTRXh2的表达特性及其在低温胁迫中的功能，为橡胶树抗寒性遗传改良提供可用的基因资源。方法基于RT-PCR技术从橡胶树克隆HbTRXh2，运用生物信息学手段解析其序列特征及系统进化关系。采用实时荧光定量PCR（RT-qPCR）技术系统测定HbTRXh2在橡胶树不同组织中的表达水平以及各种非生物胁迫和激素处理后该基因的表达变化。此外，基于酵母表达系统，评估了HbTRXh2在应对低温、盐、干旱和氧化胁迫中的生物学功能。结果橡胶树HbTRXh2的开放阅读框（ORF）为405 bp，编码134个氨基酸。生物信息学预测显示，该蛋白理论等电点（pI）为5.78，分子量为14.64 kD。HbTRXh2含有TRX保守结构域，系统进化树分析显示其属于h型TRX。RT-qPCR分析显示，HbTRXh2在稳定期叶片中呈现最高表达量。非生物胁迫（低温、干旱、盐和氧化胁迫）以及植物激素（脱落酸、水杨酸、乙烯和茉莉酸）处理均显著上调了HbTRXh2表达。比较pYES2-HbTRXh2转化酵母和空载体pYES2转化（对照）酵母在低温、盐、干旱和氧化胁迫处理后的存活差异发现，与对照相比，转pYES2-HbTRXh2酵母在低温、盐和氧化胁迫处理后的存活率显著提高，而在山梨醇模拟干旱胁迫处理后的存活率显著降低。转HbTRXh2提高了酵母对低温、盐和氧化胁迫的抗性，但降低了对干旱胁迫的抗性。结论 HbTRXh2参与橡胶树对非生物胁迫以及植物激素的应答，其在酵母中的异源表达显著增强了对低温、盐和氧化胁迫的抗性，但降低了抗旱性。

关键词: 硫氧还蛋白, 橡胶树, 酵母表达系统, 抗寒性, 非生物胁迫

Abstract:

Objective This study investigates the expression characteristics of the thioredoxin (TRX) gene HbTRXh2 in rubber tree (Hevea brasiliensis) and its function in responding to low-temperature stress, aiming to provide valuable gene resources for improving the tolerance to cold in rubber tree. Method RT-PCR technology was adapted to clone the HbTRXh2 gene from rubber tree. Bioinformatics methods were employed to analyze its sequence characteristics and phylogenetic relationships. Quantitative real-time PCR (RT-qPCR) were used to examine the expression patterns of HbTRXh2 in various rubber tree tissues, as well as under different abiotic stresses and hormone treatments. Furthermore, the yeast expression system was to evaluate the function of HbTRXh2 in responding to low-temperature, salt, drought and oxidative stresses. Result The open reading frame (ORF) of rubber tree HbTRXh2 was 405 bp in length, encoding 134 amino acids. The predicted molecular weight of the HbTRXh2 protein was 14.64 kD, with a theoretical isoelectric point (pI) of 5.78. HbTRXh2 contained a conserved TRX domain, and phylogenetic tree analysis indicated that it belonged to the h-type TRX. RT-qPCR analysis revealed that HbTRXh2 had the highest expression in mature leaves. Abiotic stresses (low temperature, drought, salt and oxidative stress) and plant hormones (abscisic acid, salicylic acid, ethylene and jasmonic acid) treatments significantly up-regulated the expression of HbTRXh2. Survival assays after low-temperature, salt, drought and oxidative stresses revealed significantly higher viability in HbTRXh2-expressing yeasts compared to controls carrying the empty pYES2 vector. The results showed that, compared with the control yeast, the yeast transformed with pYES2-HbTRXh2 had a significantly higher survival rate after low-temperature, salt, and oxidative stress treatments, while its survival rate was significantly lower after sorbitol-induced drought stress treatment. HbTRXh2 expression in yeast improved the tolerance to low-temperature, salt, and oxidative stress, but reduced the tolerance to drought stress. Conclusion HbTRXh2 is involved in the rubber tree's responses to abiotic stresses and phytohormones. Heterologous expression of HbTRXh2 in yeast enhances the tolerance to low temperature, salt and oxidative stresses, but reduces the tolerance to drought stress.

Key words: thioredoxin, rubber tree, yeast expression system, tolerance to cold, abiotic stress

杨洁, 王玉婷, 冯成天, 何其光, 张明亮, 袁坤, 王真辉, 刘辉. 橡胶树HbTRXh2的克隆、表达及功能分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0895.

YANG Jie, WANG Yu-ting, FENG Cheng-tian, HE Qi-guang, ZHANG Ming-liang, YUAN Kun, WANG Zhen-hui, LIU Hui. Cloning， Expression Analysis， and Functional Characterization of the HbTRXh2 Gene from Hevea brasiliensis[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0895.

图/表 8

表1 引物序列和用途

Table 1 Primer sequences and their applications

引物名称 Primer name	引物序列 Primer sequence （5'‒3'）	用途 Purpose
HbTRXh2-GF	GGTACCATGGGAGGTTTATTCTCTGCT	酵母表达载体构建
HbTRXh2-GR	TCTAGATCAAGTGGCCCTGTGTTTC
HbTRXh2-QF	ATTTTGCTGCATCGTGGTGT	RT-qPCR
HbTRXh2-QR	ACGTTGGCATAGCCTGAACT
HbUBC4-QF	TTTCTTTGGTGACGCTGCAA	橡胶树内参基因
HbUBC4-QR	TCACCCTGAACCTGATAGCC
pYES2-YF	TAATACGACTCACTATAGGG	pYES2载体检测
pYES2-YR	ATTAAAGCCTTCGAGCGTCC
Actin-F	AGTTGCCCCAGAAGAACACC	酵母内参基因
Actin-R	TACCGGCAGATTCCAAACCC

图1 HbTRXh2编码区核苷酸序列及其编码的氨基酸序列黄色表示TRX结构域，下划线表示CGPC氧化还原活性中心

Fig. 1 Nucleotide sequences of the coding region of HbTRXh2 gene and its encoded amino acid sequenceThe TRX domain is highlighted in yellow background, and the CGPC redox-active center is underlined

图2 HbTRXh2与植物TRXs的系统进化树

Fig. 2 Phylogenetic tree of HbTRXh2 and plant TRXs

图3 HbTRXh2在橡胶树不同组织中的表达模式不同小写字母表示组织之间存在显著差异（P<0.05）。下同

Fig. 3 Expression pattern of HbTRXh2 gene in different tissues of rubber treeDifferent lowercase letters denote significant differences between tissues (P<0.05). The same below

图4 不同非生物胁迫下HbTRXh2的表达分析

Fig. 4 Expression analysis of HbTRXh2 under different abiotic stresses

图5 不同植物激素处理后HbTRXh2的表达分析

Fig. 5 Expression analysis of HbTRXh2 after different phytohormone treatments

图6 HbTRXh2重组酵母的分子检测A：HbTRXh2重组酵母PCR验证；B：转化酵母中HbTRXh2的半定量PCR分析。M：DL2000 DNA marker（TaKaRa）；N1、N2：未加模板的阴性对照；P1：pYES2质粒阳性对照；P2：pYES2-HbTRXh2质粒阳性对照；1‒3：pYES2转化酵母单克隆；4‒6：pYES2-HbTRXh2转化酵母单克隆

Fig. 6 Molecular verification of HbTRXh2 recombinant yeastA: PCR verification of HbTRXh2 recombinant yeast. B: Semi-quantitative PCR analysis of HbTRXh2 expression in transformed yeast. M: DL2000 DNA marker (TaKaRa); N1 and N2: Negative control without template; P1: pYES2 plasmid positive control; P2: pYES2-HbTRXh2 plasmid positive control; 1-3: pYES2 transformed yeast monoclonal; 4-6: pYES2-HbTRXh2 transformed yeast monoclonal

图7 转HbTRXh2酵母与转pYES2空载体对照酵母在氧化（A）、低温（B）、盐（C）和干旱（D）胁迫处理后的存活差异

Fig. 7 Survival differences between HbTRXh2 transgenic yeast and pYES2 empty vector-transformed yeast (Control) after oxidative (A), low temperature (B), salt (C), and drought (D) stress treatments

参考文献 32

[1]	Fukushi Y, Yokochi Y, Hisabori T, et al. Plastidial thioredoxin-like proteins are essential for normal embryogenesis and seed development in Arabidopsis thaliana [J]. J Plant Res, 2025, 138(2): 337-345.
[2]	Zhou JF, Song TQ, Zhou HW, et al. Genome-wide identification, characterization, evolution, and expression pattern analyses of the typical thioredoxin gene family in wheat (Triticum aestivum L.) [J]. Front Plant Sci, 2022, 13: 1020584.
[3]	Chibani K, Pucker B, Dietz KJ, et al. Genome-wide analysis and transcriptional regulation of the typical and atypical thioredoxins in Arabidopsis thaliana [J]. FEBS Lett, 2021, 595(21): 2715-2730.
[4]	Cui XY, Gu JM, Liu PK, et al. Genome-wide identification and characterization of the thioredoxin (TRX) gene family in tomato (Solanum lycopersicum) and a functional analysis of SlTRX2 under salt stress [J]. Plant Physiol Biochem, 2025, 220: 109478.
[5]	Geigenberger P, Thormählen I, Daloso DM, et al. The unprecedented versatility of the plant‎ thioredoxin system [J]. Trends Plant Sci, 2017, 22(3): 249-262.
[6]	Ji MG, Park HJ, Cha JY, et al. Expression of Arabidopsis thaliana Thioredoxin-h2 in Brassica napus enhances antioxidant defenses and improves salt tolerance [J]. Plant Physiol Biochem, 2020, 147: 313-321.
[7]	Ji H, Liu D, Zhang Z, et al. A bacterial F-box effector suppresses SAR immunity through mediating the proteasomal degradation of OsTrxh2 in rice [J]. Plant J, 2020, 104(4): 1054-1072.
[8]	Meng L, Wong JH, Feldman LJ, et al. A membrane-associated thioredoxin required for plant growth moves from cell to cell, suggestive of a role in intercellular communication [J]. Proc Natl Acad Sci, 2010, 107(8): 3900-3905.
[9]	Chae HB, Jung YJ, Paeng SK, et al. Functional changes of OsTrxm from reductase to molecular chaperone under heat shock stress [J]. Plant Physiol Biochem, 2023, 203: 108005.
[10]	Digiuni S, Schellmann S, Geier F, et al. A competitive complex formation mechanism underlies trichome patterning on Arabidopsis leaves [J]. Mol Syst Biol, 2008, 4(1): 217.
[11]	Park SK, Jung YJ, Lee JR, et al. Heat-shock and redox-dependent functional switching of an h-type Arabidopsis thioredoxin from a disulfide reductase to a molecular chaperone [J]. Plant Physiol, 2009, 150(2): 552-561.
[12]	Tian Y, Fan M, Qin Z, et al. Hydrogen peroxide positively regulates brassinosteroid signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription factor [J]. Nat Commun, 2018, 9(1): 1063.
[13]	Kamoun H, Feki K, Tounsi S, et al. The thioredoxin h-type TdTrxh2 protein of durum wheat confers abiotic stress tolerance of the transformant Arabidopsis plants through its protective role and the regulation of redox homoeostasis [J]. Protoplasma, 2024, 261(2): 317-331.
[14]	Cejudo FJ, González MC, Pérez-Ruiz JM. Redox regulation of chloroplast metabolism [J]. Plant Physiol, 2021, 186(1): 9-21.
[15]	De Brasi-Velasco S, Sánchez-Guerrero A, Castillo MC, et al. Thioredoxin TRXo1 is involved in ABA perception via PYR1 redox regulation [J]. Redox Biol, 2023, 63: 102750.
[16]	Matsuzawa A. Thioredoxin and redox signaling: Roles of the thioredoxin system in control of cell fate [J]. Arch Biochem Biophys, 2017, 617: 101-105.
[17]	卫晋瑶, 郑红裕, 石钰欣. 2024年我国天然橡胶产业形势分析及未来展望 [J]. 中国热带农业, 2025(2): 17-22, 7.
	Wei JY, Zheng HY, Shi YX. Analysis of China’s natural rubber industry in 2024 and future outlook [J]. China Trop Agric, 2025(2): 17-22, 7.
[18]	刘琰琰, 韩冬, 杨菲, 等. 气象灾害对橡胶树的影响及风险评估综述 [J]. 福建林业科技, 2016, 43(3): 244-252.
	Liu YY, Han D, Yang F, et al. Studies for impact of meteorological disasters on Hevea brasiliensis and risk assessment [J]. J Fujian For Sci Technol, 2016, 43(3): 244-252.
[19]	孙永帅, 田维敏, 翟德利, 等. 我国橡胶树育种的技术瓶颈与创新发展建议 [J]. 中国科学院院刊, 2024, 39(1): 191-197.
	Sun YS, Tian WM, Zhai DL, et al. Technological bottlenecks and innovative developments for rubber tree breeding in China [J]. Bull Chin Acad Sci, 2024, 39(1): 191-197.
[20]	吴双, 逯锐琳, 冯成天, 等. 橡胶树HbTRXh5基因在酵母中的表达及抗逆性分析 [J]. 生物技术通报, 2024, 40(12): 136-144.
	Wu S, Lu RL, Feng CT, et al. Expression of HbTRXh5 gene of Hevea brasiliensis in yeast and analysis on its resistance to stress [J]. Biotechnol Bull, 2024, 40(12): 136-144.
[21]	王帅, 袁坤, 何其光, 等. 橡胶树硫氧还蛋白基因HbCXXS1的克隆及表达分析 [J]. 生物技术通报, 2022, 38(12): 214-222.
	Wang S, Yuan K, He QG, et al. Cloning and expression analysis of HbCXXS1, a thioredoxin gene in Hevea brasiliensis [J]. Biotechnol Bull, 2022, 38(12): 214-222.
[22]	李双江, 冯成天, 胡义钰, 等. 橡胶树HbDHAR2基因克隆及表达分析 [J]. 华北农学报, 2021, 36(3): 25-32.
	Li SJ, Feng CT, Hu YY, et al. Cloning and expression analysis of HbDHAR2 gene from Hevea brasiliensis [J]. Acta Agric Boreali Sin, 2021, 36(3): 25-32.
[23]	Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative Ct method [J]. Nat Protoc, 2008, 3(6): 1101-1108.
[24]	Nascimento CP, da Fonseca-Pereira P, Ferreira-Silva M, et al. Functional analysis of the extraplastidial TRX system in germination and early stages of development of Arabidopsis thaliana [J]. Plant Sci, 2025, 350: 112310.
[25]	Reichheld JP, Mestres-Ortega D, Laloi C, et al. The multigenic family of thioredoxin h in Arabidopsis thaliana: specific expression and stress response [J]. Plant Physiol Biochem, 2002, 40(6-8): 685-690.
[26]	Timm S, Klaas N, Niemann J, et al. Thioredoxins o1 and h2 jointly adjust mitochondrial dihydrolipoamide dehydrogenase-dependent pathways towards changing environments [J]. Plant Cell Environ, 2024, 47(7): 2540-2558.
[27]	Jiang CL, Dai JX, Han HL, et al. Determination of thirteen acidic phytohormones and their analogues in tea (Camellia sinensis) leaves using ultra high performance liquid chromatography tandem mass spectrometry [J]. J Chromatogr B, 2020, 1149: 122144.
[28]	Kavi Kishor PB, Tiozon RN Jr, Fernie AR, et al. Abscisic acid and its role in the modulation of plant growth, development, and yield stability [J]. Trends Plant Sci, 2022, 27(12): 1283-1295.
[29]	Verma V, Ravindran P, Kumar PP. Plant hormone-mediated regulation of stress responses [J]. BMC Plant Biol, 2016, 16(1): 86.
[30]	Ben Saad R, Ben Romdhane W, Bouteraa MT, et al. Lobularia maritima thioredoxin-h2 gene mitigates salt and osmotic stress damage in tobacco by modeling plant antioxidant system [J]. Plant Growth Regul, 2022, 97(1): 101-115.
[31]	Lee ES, Park JH, Wi SD, et al. Redox-dependent structural switch and CBF activation confer freezing tolerance in plants [J]. Nat Plants, 2021, 7(7): 914-922.
[32]	Xu A, Wei N, Hu H, et al. Thioredoxin h2 inhibits the MPKK5-MPK3 cascade to regulate the CBF-COR signaling pathway in Citrullus lanatus suffering chilling stress [J]. Hortic Res, 2023, 10(2): uhac256.