StFBL25基因的克隆及转基因马铃薯获得

Cloning of StFBL25 and Generation of Transgenic Potato

摘要

马铃薯（Solanum tuberosum L.）F-Box蛋白功能研究尚不完善，尤其是低温响应机制缺乏系统性分析。目前研究主要聚焦于F-Box蛋白的互作网络，其具体功能尚缺乏系统研究，尤其在马铃薯中研究较少。实验室前期通过甲基化扩增敏感多态性技术获得了马铃薯低温响应基因StFBL25，鉴定为一类F-Box蛋白编码基因。通过生物信息学分析StFBL25蛋白结构与功能，构建StFBL25基因亚细胞定位载体，观察StFBL25蛋白的亚细胞定位情况。构建StFBL25基因的过表达和干扰表达载体，用于马铃薯的遗传转化。StFBL25基因全长1 455 bp，编码484个氨基酸，预测含61个潜在磷酸化位点，蛋白结构预测显示，其主要由α-螺旋、无规则卷曲和延伸链组成。亚细胞定位显示StFBL25定位于细胞核。成功获得StFBL25基因过表达与干扰表达的转基因马铃薯植株，qRT-PCR结果显示，过表达株系3-10中StFBL25表达量较野生型上调213倍，干扰表达株系5-6中StFBL25基因的表达量为野生型的0.097倍。研究结果可为马铃薯抗低温基因的筛选和功能研究提供理论基础。

Abstract

Research on the function of potato (Solanum tuberosum L.) F-Box proteins remains incomplete, particularly regarding the lack of systematic analysis of their low-temperature response mechanisms. Current studies primarily focus on the interaction networks of F-Box proteins, however comprehensive research on their specific functions is still limited, especially in potato. Previous work in the laboratory identified the low-temperature responsive gene StFBL25 using Methylation-Sensitive Amplification Polymorphism technology, classifying it as an F-Box protein-coding gene. The structure and function of the StFBL25 protein were analyzed through bioinformatic approaches. A subcellular localization vector for the StFBL25 gene was constructed to determine the subcellular localization of the StFBL25 protein. Additionally, overexpression and gene silencing vectors of StFBL25 were developed for potato genetic transformation. The StFBL25 gene has a full length of 1 455 bp, encoding 484 amino acids. Bioinformatics prediction revealed 61 potential phosphorylation sites. Protein structure prediction shows that alpha-helices, random coils, and extended strands primarily constitute StFBL25. Subcellular localization experiments demonstrate that StFBL25 localizes to the nucleus. Transgenic potato plants overexpressing and with knocked down expression of StFBL25 gene were successfully obtained. qRT-PCR results show that compared to wild-type plants, StFBL25 gene expression increased by 213-fold in the overexpression line 3-10, while expression decreased to 0.097-fold in the knockdown line 5-6. Collectively, these findings provide a foundation for screening and functionally studying low-temperature resistance genes in potato.

关键词

StFBL25蛋白 / 生物信息学预测 / 亚细胞定位 / 马铃薯

Key words

StFBL25 protein / bioinformatics prediction / subcellular localization / potato

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基金资助

国家自然科学基金项目(32060498)