橡胶树HbTRXh9基因克隆及功能分析

Cloning and functional analysis of HbTRXh9 gene in Hevea brasiliensis

  • 摘要: 【目的】 克隆橡胶树h型硫氧还蛋白(TRX)基因HbTRXh9,解析其表达模式,并通过转基因酵母验证其在非生物胁迫中的抗性功能,为后续橡胶树非生物胁迫抗性及割面干涸(TPD)抗性的遗传改良提供理论参考。【方法】 PCR克隆橡胶树HbTRXh9基因,采用实时荧光定量PCR检测HbTRXh9基因的组织表达特性及在非生物胁迫下的表达模式,并构建pYES2-HbTRXh9酵母表达载体,将其转化酵母INVSc1感受态细胞,在非生物胁迫处理后对转HbTRXh9基因酵母和转pYES2空载体的酵母进行生长情况比较。【结果】 HbTRXh9基因编码区(CDS)序列长度为417 bp,编码138个氨基酸残基,蛋白相对分子质量为15.52 kD,理论等电点为4.90,亚细胞定位于细胞质,属于非分泌型亲水性蛋白,含有TRX保守结构域(pfam00085)和CGPC氧化还原活性位点。HbTRXh9与麻风树、木薯的TRXh氨基酸序列相似性较高,分别为84%和80%,三者在系统发育树中处于同一分支,均被聚在h型TRX第III组。HbTRXh9基因在不同组织中均有表达,但相对表达量存在明显差异,其中在胶乳中的相对表达量显著高于其他组织(P<0.05,下同),且在TPD橡胶树胶乳和树皮中的相对表达量显著低于健康树。干旱和低温胁迫下,HbTRXh9基因的相对表达量在处理0~12 h呈上升趋势,但在处理24 h时显著下降,在处理48 h时又升至峰值,显著高于其他处理时间。盐和氧化胁迫下,HbTRXh9基因的相对表达量整体呈先上升后下降的变化趋势,其中在盐胁迫处理12 h上升至峰值,随后显著下降;氧化胁迫处理6 h显著上升至峰值,随后逐渐下降。氧化和低温胁迫处理不同时间后,在同一稀释倍数条件下转HbTRXh9基因酵母的菌落数多于转pYES2空载体的酵母,而盐胁迫处理不同时间后,转HbTRXh9基因酵母的菌落数少于转pYES2空载体的酵母。【结论】 HbTRXh9基因参与橡胶树TPD发生及非生物胁迫应答,酵母中表达HbTRXh9基因可提高抗氧化性和抗寒性,但会降低抗盐性。

     

    Abstract: 【Objective】 This study aimed to clone the h-type thioredoxin (TRX) gene HbTRXh9 in Hevea brasiliensis,analyze its expression pattern,and verify its function in abiotic stress tolerance using transgenic yeast,thereby providing theoretical reference for abiotic stress tolerance and tapping panel dryness (TPD) tolerance in Hevea brasiliensis genetic improvement. 【Method】 The HbTRXh9 gene in Hevea brasiliensis was cloned by PCR, and real-time fluorescence quantitative PCR was used to detect the tissue-specific expression of HbTRXh9 gene and its expression patterns under abiotic stresses. The yeast expression vector pYES2-HbTRXh9 was constructed and transformed into yeast INVSc1 competent cells, and the growth of HbTRXh9 transgenic yeast and pYES2 empty vector-transformed yeast after abiotic stress treatments were compared. 【Result】 The coding sequence (CDS) of HbTRXh9 gene was 417 bp in length and encoded 138 amino acid residues, with the protein relative molecular mass of 15.52 kD and theoretical isoelectric point of 4.90. Subcellular localization was in cytoplasm, and the protein was classified as a non-secretory, hydrophilic protein containing a conserved TRX domain (pfam00085) and CGPC redox-active site. HbTRXh9 shared high amino acid sequence similarity with TRXh in Jatropha curcas and Manihot esculenta,reaching 84% and 80% respectively. These three proteins were in the same branch and clustered into the group III of h-type TRXs. The HbTRXh9 gene was expressed in different tissues, with distinct differences in relative expression, and the relative expression was significantly higher in latex than in other tissues (P<0.05,the same below). The relative expression of TPD in the latex and bark of Hevea brasiliensis was significantly lower than that in healthy trees. Under drought and low temperature stresses, the relative expression of HbTRXh9 increased from 0-12 h, decreased significantly at 24 h, and then peaked at 48 h, which was significantly higher than that at other time. Under salt and oxidative stresses, the relative expression of HbTRXh9 exhibited an overall variation trend of first increasing and then decreasing, and the relative expression peaked at 12 h under salt stress and declined significantly subsequently; under oxidative stress, it significantly climbed to peak at 6 h, followed by a gradual reduction. After treated with oxidative and low-temperature stresses for different time, the colony number of HbTRXh9-transgenic yeast was higher than that of the pYES2 empty vector-transformed yeast under the same dilution condition; under salt stress at different treatment time, the colony number of HbTRXh9-transgenic yeast was lower than that of the pYES2 empty vector-transformed yeast. 【Conclusion】 The HbTRXh9 is involved in TPD occurrence and abiotic stress responses of Hevea brasiliensis, and expression of HbTRXh9 gene in yeast can improve oxidation tolerance and cold tolerance, but reduce salt tolerance.

     

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