Saleem Muhammad Zafar, Arshad Fatima, Ahmad Nauman, Iftikhar Muhammad, Saleem Ammara, Jahangir Ghulam Zahra, Zulfiqar Asma, Arshed Muhammad Zeeshan, Shah Anis Ali, Shaffique Shifa, Gatasheh Mansour K
Centre for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan.
Division of Science and Technology (DSnT), Department of Botany, University of Education Lahore, Main Campus, Lahore, Pakistan.
Mol Biotechnol. 2025 Jun 5. doi: 10.1007/s12033-025-01448-y.
Plants face various biotic and abiotic stresses, necessitating the activation of defense mechanisms, including pathogenesis-related (PR) proteins. Osmotin-like proteins (OLPs), belonging to the PR-5 family, play a crucial role in plant defense by enhancing resistance to pathogens and environmental stresses. However, the functional characterization of OLPs remains limited. This study aimed to clone and express the OLP gene from the medicinal plant Solanum nigrum in Escherichia coli to facilitate further functional and structural analyses. The genomic DNA of S. nigrum was isolated from in vitro-cultured plants, and the OLP gene was amplified using primers designed via Primer3 software based on NCBI sequences. Gradient PCR optimization determined the optimal annealing temperature between 58.3 °C and 60 °C. The amplified gene was cloned into the pTZ57R/T vector and transformed into E. coli. Sequencing confirmed a 98% homology with reported OLP sequences. For expression analysis, the gene was subcloned into the expression vector pET15b and transformed into E. coli BL21 (DE3). Induction with 1 mM IPTG at 37 °C for 3 h resulted in the production of a 26 kDa protein, confirmed by SDS-PAGE and Protein Dot Blot analysis using anti-histidine antibodies. The successful cloning and expression of OLP provide a foundation for investigating its role in plant-pathogen interactions and its potential applications in agriculture and medicine. This study contributes to understanding PR proteins and offers insights into their potential for enhancing stress tolerance in crops.
植物面临各种生物和非生物胁迫，因此需要激活防御机制，包括病程相关（PR）蛋白。类渗透素蛋白（OLPs）属于PR-5家族，通过增强对病原体和环境胁迫的抗性，在植物防御中发挥关键作用。然而，OLPs的功能特性仍很有限。本研究旨在从药用植物龙葵中克隆OLP基因并在大肠杆菌中表达，以促进进一步的功能和结构分析。从体外培养的植物中分离出龙葵的基因组DNA，并使用基于NCBI序列通过Primer3软件设计的引物扩增OLP基因。梯度PCR优化确定了58.3℃至60℃之间的最佳退火温度。将扩增的基因克隆到pTZ57R/T载体中并转化到大肠杆菌中。测序证实与已报道的OLP序列有98%的同源性。为了进行表达分析，将该基因亚克隆到表达载体pET15b中并转化到大肠杆菌BL21（DE3）中。在37℃用1 mM IPTG诱导3小时，产生了一种26 kDa的蛋白质，通过SDS-PAGE和使用抗组氨酸抗体的蛋白质斑点印迹分析得到证实。OLP的成功克隆和表达为研究其在植物-病原体相互作用中的作用及其在农业和医学中的潜在应用提供了基础。本研究有助于理解PR蛋白，并为其增强作物胁迫耐受性的潜力提供了见解。
