橡胶树HbTRXh5基因在酵母中的表达及抗逆性分析

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

摘要/Abstract

摘要：

【目的】克隆橡胶树硫氧还蛋白基因HbTRXh5，分析其表达特性，探究其在非生物胁迫中的功能，为橡胶树抗逆性遗传改良提供基因资源。【方法】采用RT-PCR方法克隆橡胶树HbTRXh5基因，利用生物信息学方法分析其序列特性和系统进化关系。采用实时荧光定量PCR分析HbTRXh5基因在橡胶树各组织以及非生物胁迫下的表达。构建HbTRXh5基因酵母表达载体并转入酵母，比较转基因酵母和对照酵母在低温、盐和氧化胁迫处理后的存活差异。【结果】HbTRXh5基因编码区长354 bp，编码117个氨基酸。HbTRXh5含有硫氧还蛋白保守结构域和CGPC活性位点，属于h型硫氧还蛋白第I亚组。HbTRXh5在橡胶树各组织中均有表达，以胶乳中的表达量最高。低温、盐以及H₂O₂和甲基紫精诱导的氧化胁迫处理均能诱导HbTRXh5的表达。成功将HbTRXh5基因转入酿酒酵母INVSc1菌株中并诱导表达。同转pYES2空载体对照酵母相比，转HbTRXh5基因酵母在H₂O₂处理后具有更高的存活率。相反，在低温和盐胁迫处理后，转HbTRXh5基因酵母的存活率较对照酵母明显降低。【结论】橡胶树HbTRXh5的表达受低温、盐和氧化胁迫调控，酵母中表达HbTRXh5提高了重组酵母对氧化胁迫的抗性，但降低了对低温和盐胁迫的抗性。

关键词: 橡胶树, 硫氧还蛋白, 酵母表达系统, 非生物胁迫, 氧化胁迫, 抗寒性

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

吴双, 逯锐琳, 冯成天, 袁坤, 王真辉, 刘进平, 刘辉. 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 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.

图/表 6

图1 HbTRXh5基因编码区核苷酸序列及其编码的氨基酸序列蓝色背景部分为硫氧还蛋白（TRX）结构域，下划线部分为CGPC活性中心

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

图2 HbTRXh5和其他植物硫氧还蛋白的系统进化树

Fig. 2 Phylogenetic tree of HbTRXh5 and other plant thioredoxins

图3 HbTRXh5基因在橡胶树各组织中的表达不同小写字母表示差异显著（P<0.05）。下同

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

图4 低温、盐以及H2O2或MV诱导的氧化胁迫下HbTRXh5的表达变化

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

图5 pYES2或pYES2-HbTRXh5转化酵母的分子检测 A：pYES2或pYES2-HbTRXh5转化酵母的PCR检测；B：半定量RT-PCR检测HbTRXh5基因在转化酵母中的表达；M：DL2000 DNA marker（TaKaRa）；N1和N2：未加模板的阴性对照；P1：pYES2质粒阳性对照；P2：pYES2-HbTRXh5质粒阳性对照；1-3和7-9：pYES2转化酵母单克隆；4-6和10-12：pYES2-HbTRXh5转化酵母单克隆

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

图6 非生物胁迫处理下转HbTRXh5基因酵母和转pYES2空载体对照酵母的存活差异 A：H2O2诱导的氧化胁迫处理（1 d）；B：低温（-20℃）胁迫处理；C：盐胁迫（2.5 mol/L NaCl）处理

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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