Expression of HbTRXh5 Gene of Hevea brasiliensis in Yeast and Analysis on Its Resistance to Stress

doi:10.13560/j.cnki.biotech.bull.1985.2024-0488

Abstract

Abstract:

【Objective】Cloning and analysis of expression characteristics of the thioredoxin gene HbTRXh5 from rubber tree（Hevea bsinensis）, exploring its function in abiotic stress, which aims to provide genetic resources for genetic improvement of stress tolerance in rubber tree.【Method】RT-PCR method was used to clone HbTRXh5 gene from rubber tree, and bioinformatics methods were employed to analyze its sequence characteristics and phylogenetic relationship. Quantitative real-time PCR was applied to analyze the expressions of HbTRXh5 in various tissues of rubber trees and under abiotic stress. The yeast expression vector of HbTRXh5 was constructed and transformed into yeast, and then the survival differences between transgenic yeast and control yeast after cold, salt, and oxidative stress treatments were compared. 【Result】The coding region of HbTRXh5 gene was 354 bp in length and encoded 117 amino acids. HbTRXh5 contained a conserved thioredoxin domain and a CGPC active site, belonging to the subgroup I of h-type thioredoxin. HbTRXh5 was expressed in various tissues of rubber tree, with the highest expression in latex. Low temperature, salt, and oxidative stress-induced by H₂O₂ and methyl violet up-regulated the expression of HbTRXh5. HbTRXh5 gene was successfully transformed into the yeast strain INVSc1 and induced to have expression. Compared with the control yeast transformed with the pYES2 empty vector, the HbTRXh5 gene transgenic yeast had a higher survival rate after H₂O₂ treatment, but showed a lower survival rate after cold and salt stress treatments. 【Conclusion】The expression of HbTRXh5 is regulated by low temperature, salt, and oxidative stress in rubber tree. The overexpression of HbTRXh5 in yeast enhances the tolerance to oxidative stress, but reduces the tolerance to low temperature and salt stresses.

Key words: Hevea brasiliensis, thioredoxin, yeast expression system, abiotic stress, oxidative stress, cold tolerance

WU Shuang, LU Rui-lin, FENG Cheng-tian, YUAN Kun, WANG Zhen-hui, LIU Jin-ping, LIU Hui. Expression of HbTRXh5 Gene of Hevea brasiliensis in Yeast and Analysis on Its Resistance to Stress[J]. Biotechnology Bulletin, 2024, 40(12): 136-144.

Figures/Tables 6

Fig. 1 Nucleotide sequence of HbTRXh5 gene coding region and its encoded amino acid sequence The blue background indicates the thioredoxin（TRX）domain, and the underlined one shows the highly conserved CGPC active site

Fig. 2 Phylogenetic tree of HbTRXh5 and other plant thioredoxins

Fig. 3 Expression of HbTRXh5 gene in various tissues of rubber tree（Hevea brasiliensis） Different lowercase letters indicate a significant difference（P<0.05）. The same below

Fig. 4 Changes in HbTRXh5 expression under low temperature, salt, and H2O2- or MV-induced oxidative stresses

Fig. 5 Molecular detection of yeast transformed with pYES2 or pYES2-HbTRXh5 A: PCR detection of yeast transformed with pYES2 or pYES2-HbTRXh5. B: Semi-quantitative RT-PCR detection of HbTRXh5 gene expression in transformed yeast.M: DL2000 DNA marker（TaKaRa）; N1 and N2: negative control without template; P1: pYES2 plasmid positive control; P2: pYES2-HbTRXh5 plasmid positive control; 1-3 and 7-9: pYES2 transformed yeast monoclonal; 4-6 and 10-12: pYES2-HbTRXh5 transformed yeast monoclonal

Fig. 6 Survival differences between HbTRXh5 transgenic yeast and control yeast transformed with pYES2 empty vector after abiotic stress treatment A: H2O2-induced oxidative stress treatment（1 d）. B: Low temperature stress（-20℃）treatment. C: Salt stress（2.5 mol/L NaCl）treatment

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