Rahamkulov Ilhom, Bakhsh Allah
Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, 51240 Nigde, Turkey.
3 Biotech. 2020 Oct;10(10):426. doi: 10.1007/s13205-020-02350-x. Epub 2020 Sep 11.
The present study was conducted to determine efficiency of green tissue-specific (pRCA) and stress-inducible promoters (pRD29A) to express beta-glucuronidase () gene in transgenic potatoes compared with constitutive promoter (35S CaMV). The promoter fragments were isolated from their original source and cloned upstream to in pCAMBIA-1301 binary vector to develop plant expression constructs, i.e., pRCA-pCAMBIA and pRD29A-pCAMBIA. strain GV2260 harboring recombinant plasmids were used to infect leaf discs and internodal explant of Lady Olympia cultivar. GUS histochemical analysis was performed at different stages to determine GUS activity in transgenic plants. To determine activity of stress-inducible promoter (pRD29A), transgenic plants were exposed to heat, drought and combination of both heat and drought stress. The real time (RT-qPCR) and GUS florimetric assays revealed that pRD29A promoter gets more activated under drought, heat and combination of both stresses. GUS expression levels were more than 10 folds high with pRD29A promoter compared to control. Likewise, the reduced transcripts levels of gene under control of pRCA promoter were found in tuber/roots of transgenic plants compared to 35S promoter. GUS florimetric assays also showed decreased or no GUS expression in tubers. In conclusion, the results encourage the appropriate use of promoters to drive the expression of foreign gene(s) for the development of potato lines tolerant to biotic and abiotic stress while minimizing the risks of transgenic technology in potatoes.
本研究旨在确定绿色组织特异性启动子（pRCA）和胁迫诱导型启动子（pRD29A）相较于组成型启动子（35S CaMV）在转基因马铃薯中表达β-葡萄糖醛酸酶（GUS）基因的效率。从其原始来源分离启动子片段，并克隆到pCAMBIA-1301二元载体中GUS的上游，以构建植物表达载体，即pRCA-pCAMBIA和pRD29A-pCAMBIA。携带重组质粒的根癌农杆菌菌株GV2260用于感染“奥林匹亚夫人”品种的叶片圆盘和节间外植体。在不同阶段进行GUS组织化学分析，以确定转基因植物中的GUS活性。为了确定胁迫诱导型启动子（pRD29A）的活性，将转基因植物暴露于高温、干旱以及高温和干旱的组合胁迫下。实时定量聚合酶链反应（RT-qPCR）和GUS荧光测定表明，pRD29A启动子在干旱、高温以及两种胁迫组合下的激活程度更高。与对照相比，pRD29A启动子驱动的GUS表达水平高出10倍以上。同样，与35S启动子相比，在转基因植物块茎/根中，pRCA启动子控制下的GUS基因转录水平降低。GUS荧光测定还显示块茎中GUS表达降低或无表达。总之，这些结果鼓励适当使用启动子来驱动外源基因的表达，以培育对生物和非生物胁迫具有耐受性的马铃薯品系，同时将马铃薯转基因技术的风险降至最低。
