| 马铃薯StCYP83B1基因过表达载体构建及遗传转化 |
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| 引用本文：孔乐辉，宗德乾，史青尧，殷盼盼，巫文玉，单卫星， 强晓玉.马铃薯StCYP83B1基因过表达载体构建及遗传转化[J].西北农业学报,2024,(8):1462~1479 |
| DOI:10.7606/j.issn.1004-1389.2024.08.007 |
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| 基金项目:国家自然科学基金（31872657）；国家重点研发计划（2017YFD0200602 2）；国家现代农业产业技术体系（CARS 09）；高校科研业务科技创新专项（2452020146）。 |
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| 中文摘要:旨在为解析马铃薯StCYP83B1基因的免疫功能提供重要材料基础，从四倍体马铃薯栽培种‘大西洋’的cDNA中克隆到StCYP83B1基因，将其与融合mCherry标签的35S::pART27重组载体连接，构建p35S::StCYP83B1 mCherry植物过表达载体，并通过农杆菌介导的本氏烟瞬时表达体系确认该蛋白的表达；利用农杆菌介导的马铃薯遗传转化方法，将p35S::StCYP83B1 mCherry转入马铃薯中，随机挑选3株转基因植株进行鉴定：PCR检测证明StCYP83B1已整合到马铃薯基因组中，反转录实时定量PCR检测表明StCYP83B1在转化植株中的转录水平比未转化的对照植株上调40~49倍，免疫印迹检测表明StCYP83B1蛋白可以在转化植株中正常表达。同时，对转化马铃薯植株及微型薯的表型鉴定表明，StCYP83B1过量表达并不影响马铃薯的正常生长发育。该稳定转化马铃薯植株的获得为后续深入解析StCYP83B1基因的免疫功能及作用机制奠定了重要材料基础。 |
| 中文关键词:马铃薯 StCYP83B1 表达载体 遗传转化 |
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| Construction of an Overexpression Vector for StCYP83B1 in Potato (Solanum tuberosum) and Development of StCYP83B1 Overexpressing Transgenic Potato Lines |
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| Abstract:To obtain the important transgenic potato plants for further investigation of the immune function of StCYP83B1 gene in potatoes,this study cloned StCYP83B1 gene from the cDNA of tetraploid potato cultivar ‘Atlantic’ and inserted it into the 35S::pART27 recombinant vector labeled with mCherry to create a plant overexpression vector named p35S::StCYP83B1 mCherry. The expression of StCYP83B1 protein was confirmed using an Agrobacterium mediated transient expression system in Nicotiana benthamiana. The p35S::StCYP83B1 mCherry vector was transformed into potato cultivar ‘Atlantic’ via Agrobacterium mediated genetic transformation. Three individual transgenic lines were randomly selected for identification. PCR detection confirmed the integration of StCYP83B1 into potato genome,while qRT PCR detection revealed a 40 49 fold increase in the transcription level of StCYP83B1 in transgenic lines compared to untransformed control plants. Western blotting detection confirmed the protein expression of StCYP83B1 in transgenic lines. Additionally,the phenotypic analyses conducted on the transgenic potato plants and the minitubers indicated that overexpression of StCYP83B1 did not affect the potato growth and development. These results confirmed the successful generation of StCYP83B1 OE transgenic potato plants,providing an essential materials for further functional and mechanistic studies on the StCYP83B1 gene. |
| keywords:Potato StCYP83B1 Expression vector Genetic transformation |
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