Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India.
Gene. 2013 Sep 15;527(1):332-8. doi: 10.1016/j.gene.2013.06.016. Epub 2013 Jun 22.
The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.
甜菊叶 [甜菊（Stevia rebaudiana (Bertoni)] 是甜菊糖苷的丰富来源，这些糖苷在世界上许多国家被用作无热量的甜味剂。甜菊糖苷的甜菊醇部分是通过质体 2C-甲基-D-赤藓醇 4-磷酸途径合成的，其中（E）-4-羟基-3-甲基-2-丁烯基二磷酸还原酶（HDR）是关键酶。HDR 催化（E）-4-羟基-3-甲基-2-丁烯基二磷酸同时转化为五个碳异戊烯基单位、异戊烯基二磷酸和二甲基烯丙基二磷酸。甜菊 HDR（SrHDR）通过基因互补成功挽救了 HDR 致死突变株 MG1655 ara<>ispH，表明 SrHDR 编码一种功能性蛋白质。该基因表现出昼夜变化的表达。为了鉴定可能的调控元件，克隆了该基因的上游区域，并通过计算机分析检测到了假定的顺式作用元件。电泳迁移率变动分析（EMSA）使用假定的光响应元件 GATA 显示，白天从叶片中分离出的核蛋白（NP）与叶片中的 NP 结合，但在黑暗期间的 NP 不与 GATA 结合。数据表明，GATA 盒参与了甜菊中 SrHDR 的光介导基因调控。
