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利用同源克隆技术从六倍体普通小麦中获得了两个不同的双脱氢抗坏血酸还原酶(TaDHAR)基因的cDNA克隆。器官表达模式分析表明,这两个TaDHAR基因(暂时命名为TaDHAR1和TaDHAR2)在小麦根、茎、叶、幼穗以及开花后10d、20d和30d的种子中均有表达,为组成型表达基因。原生质体表达实验表明,两个基因的产物均可能定位在细胞质中。在细菌中表达并提纯了两个基因的重组蛋白。体外生化测定表明两个重组蛋白均具有将双脱氢抗坏血酸还原成抗坏血酸的能力,其最适pH为7.5,在37oC时的活性比25oC高,但25oC条件下pH6.0和7.0时,两个DHAR蛋白的活性显著不同。本研究的结果为进一步揭示TaDHAR基因在小麦抗坏血酸代谢中的生理作用奠定了基础。
Two different cDNA clones of didehydroascorbate reductase (TaDHAR) gene were obtained from hexaploid wheat using homologous cloning technique. The analysis of organ expression pattern showed that the two TaDHAR genes (temporarily named as TaDHAR1 and TaDHAR2) were expressed in wheat roots, stems, leaves, young spikes and seeds at 10d, 20d and 30d after flowering, which were constitutively expressed genes. Protoplast expression experiments show that the products of both genes may be located in the cytoplasm. The recombinant protein of both genes is expressed and purified in bacteria. In vitro biochemical assays showed that both recombinant proteins have the ability to reduce didehydroascorbate to ascorbic acid with an optimum pH of 7.5, higher activity at 25 ° C than 25 ° C at 25 ° C, two at pH 6.0 and 7.0 at 25 ° C DHAR protein activity significantly different. The results of this study laid the foundation for further revealing the physiological role of TaDHAR gene in wheat ascorbate metabolism.