Cloning， Expression Analysis， and Functional Characterization of the HbTRXh2 Gene from Hevea brasiliensis

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

Abstract

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) was 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 used 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 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

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, 2026, 42(6): 250-257.

Figures/Tables 8

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

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

Fig. 2 Phylogenetic tree of HbTRXh2 and plant TRXs

Fig. 3 Expression pattern of HbTRXh2 gene in different tissues of rubber treeDifferent lowercase letters denote significant differences between tissues (P0.05). The same below

Fig. 4 Expression analysis of HbTRXh2 under different abiotic stresses

Fig. 5 Expression analysis of HbTRXh2 after different phytohormone treatments

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

Fig. 7 Growth 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

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