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Expression analysis of anthocyanin transcription factor PyWRKY26 in pear fruits

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梨果实花青苷调控因子 PyWRKY26 蛋白表达分析

Journal Information

J. Hefei Univ. Tech. (Nat. Sci.), Feb.2025, Vol.48 No.2: 227-231

DOI: 10.3969/j.issn.1003-5060.2025.02.012

Authors

YAN Longfei, ZHANG Hua, XIAO Yuwei, YAO Gaifang, HU Kangdi

(School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China)

Abstract and Keywords

Abstract: Anthocyanins are the main pigment of Pyrus pyrifolia, and the synthesis of anthocyanins is regulated by a series of transcription factors. PyWRKY26 is the main transcription factor involved in the anthocyanin synthesis. However, the structure of the protein is not clear. In this study, the coding sequence (CDS) full length of PyWRKY26 gene was cloned by polymerase chain reaction (PCR) from the pericarp of Starkrimson pear. PyWRKY26 was ligated with the prokaryotic expression vector pCOLD with His tag using homologous recombination technology, and the recombinant expression vector was transformed into Rosetta(DE3). Then, the recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result showed that the CDS full length of PyWRKY26 gene was successfully amplified from the pericarp of Starkrimson pear and the recombinant expression vector of PyWRKY26-pCOLD was constructed. The PyWRKY26-pCOLD fusion protein was successfully expressed by 0.5 mmol/L IPTG for 16 h at 22 °C and purified by His-Ni column combined with SDS-PAGE. This study lays an important foundation for further studying the structure and function of PyWRKY26.

Keywords: Starkrimson pear; PyWRKY26 gene; gene cloning; prokaryotic expression; protein purification

作者信息

颜龙飞，张华，肖钰薇，姚改芳，胡康棣

（合肥工业大学食品与生物工程学院，安徽合肥230601）

摘要和关键词

摘要: 花青苷是形成红皮梨的主要色素类物质，其合成受一系列转录因子的调控。PyWRKY26是参与红皮梨花青苷合成的主要转录因子，但是其蛋白结构特征并不清楚。文章以“红茄梨”果皮为实验材料，利用聚合酶链式反应（polymerase chain reaction，PCR）克隆PyWRKY26基因的编码序列（coding sequence，CDS）全长；采用同源重组克隆技术将PyWRKY26与带His标签的pCOLD原核表达载体进行连接，将重组表达载体转化到大肠杆菌Rosetta（DE3），通过异丙基 $ \beta $-D-硫代半乳糖苷（IPTG）法诱导重组蛋白表达，利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表达产物。结果表明：在“红茄梨”果皮中能成功扩增出PyWRKY26基因的CDS全长，并构建了PyWRKY26-pCOLD重组表达载体；将重组质粒的大肠杆菌置于22℃条件下，用0.5 mmol/L IPTG诱导16 h，与对照相比，该诱导条件下能成功表达蛋白；通过His镍柱纯化法并结合SDS-PAGE检测技术得到了纯化后含量较高的PyWRKY26-pCOLD融合蛋白，为进一步研究转录因子PyWRKY26的蛋白结构及功能奠定了重要基础。

关键词: 红茄梨；PyWRKY26基因；基因克隆；原核表达；蛋白纯化

Funding

国家自然科学基金资助项目（31901993；32170315）；安徽省自然科学基金资助项目（1908085MC72）

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