Karimzadegan Vahid, Jalali Javaran Vahid, Shams Bakhsh Masoud, Jalali Javaran Mokhtar
Department of Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
Genetic Recourse Management Plan, Department of Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
Mol Biotechnol. 2019 Feb;61(2):84-92. doi: 10.1007/s12033-018-0138-8.
The aim of this study is to assess the effect of methyl jasmonate (MeJA) and temperature on the valuable pharmaceuticals expression in a virus-mediated transient expression system, and so the Zuchini Yellow Mosaic Virus (ZYMV) based vector was used for transferring the GFP reporter gene and recombinant tissue plasminogen activator (rtPA) gene (K2S) to cucurbit (Cucurbita pepo L.). MeJA, temperature and time (days after inoculation), were evaluated as a factorial experiment in a completely randomized design (CRD). At first, the effect of all treatment combinations on GFP expression was assessed. At this step, the ELISA test was used to select the optimum treatment combination. ELISA method revealed the significant difference between applied treatments. The optimized treatment significantly increased the expression of rtPA compared to the control. The Real-Time PCR reaction for both GFP and rtPA genes showed no significant differences between optimum and control treatments, however, transcripts of the small subunit of RuBisCO were extremely down-regulated in optimum treatment condition. Reduction in RuBisCO expression at protein level was tangible under treatment condition based on the ELISA test. Therefore, it can be inferred that suppressing the expression of RuBisCO, probably resulted in higher access of expression system to free amino acids inside the cell. In this study, MeJA has been shown to be a positive factor, but the low temperature (17 °C), unlike previous studies, suppressed the expression of recombinant protein unexpectedly, probably due to the incompatibility of the viral construct with low temperature. In conclusion, the use of a suitable gene construct, which is not sensitive to temperature, is likely to result in a more favorable outcome.
本研究的目的是评估茉莉酸甲酯（MeJA）和温度对病毒介导的瞬时表达系统中珍贵药物表达的影响，因此使用基于西葫芦黄花叶病毒（ZYMV）的载体将绿色荧光蛋白（GFP）报告基因和重组组织型纤溶酶原激活剂（rtPA）基因（K2S）转移到葫芦科植物（西葫芦）中。将MeJA、温度和时间（接种后天数）作为析因试验，采用完全随机设计（CRD）进行评估。首先，评估所有处理组合对GFP表达的影响。在这一步骤中，使用酶联免疫吸附测定（ELISA）试验来选择最佳处理组合。ELISA方法显示所应用的处理之间存在显著差异。与对照相比，优化后的处理显著提高了rtPA的表达。针对GFP和rtPA基因的实时聚合酶链反应（Real-Time PCR）显示，最佳处理和对照处理之间没有显著差异，然而，在最佳处理条件下，核酮糖-1,5-二磷酸羧化酶/加氧酶（RuBisCO）小亚基的转录本被极度下调。基于ELISA试验，在处理条件下，RuBisCO在蛋白质水平的表达降低是明显的。因此，可以推断，RuBisCO表达的抑制可能导致表达系统更容易获取细胞内的游离氨基酸。在本研究中，MeJA已被证明是一个积极因素，但与先前的研究不同，低温（17℃）意外地抑制了重组蛋白的表达，这可能是由于病毒构建体与低温不相容所致。总之，使用对温度不敏感的合适基因构建体可能会产生更有利的结果。
