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本研究以富含γ-氨基丁酸的巨胚稻“Tge B”基因组DNA为模板,利用PCR技术扩增得到GABA合成关键酶基因Os GAD3的上游959 bp启动子序列。序列分析表明:Os GAD3启动子不仅含有转录必备的TATA box、CAAT box元件,还含有分生组织特异性表达调控元件CAT-box、CCGTCC-box以及一些非生物胁迫和激素响应元件等。将该启动子序列与GUS基因融合构建表达载体,转化水稻后进行GUS组织化学染色分析,结果显示,Os GAD3启动子驱动的GUS基因在转基因植株的根、茎中具有较高的表达活性,且在根尖和茎初生韧皮部区域的表达更为明显,在种胚及叶片切口处也能检测到GUS表达。浸水后种胚中的GUS活性显著提高,且随着浸水时间的延长呈逐渐增加趋势。研究结果为了解巨胚稻Os GAD3的表达调控机制提供了依据。
In this study, the GbA synthetic key enzyme gene Os GAD3 upstream 959 bp promoter sequence was amplified by PCR using γ-aminobutyric acid-rich giant embryo rice “Tge B” genomic DNA as a template. Sequence analysis showed that the Os GAD3 promoter contained not only the transcriptional TATA box and CAAT box elements but also CAT-box, CCGTCC-box and some abiotic stresses and hormone response elements. The promoter sequence was fused with GUS gene to construct the expression vector. After transformation of rice, GUS histochemical staining analysis showed that GUS gene driven by Os GAD3 promoter had higher expression activity in the roots and stems of the transgenic plants, and The expression of GUS in the apical phloem and the primary phloem region was more obvious, and the GUS expression was also detected at the embryo and leaf incision. The GUS activity in seed embryos increased significantly after immersion, and gradually increased with the extension of immersion time. The results provide a basis for understanding the regulation mechanism of Os GAD3 in giant embryo rice.