马铃薯StSN2信号肽缺失对其定位及蛋白互作的影响  

The Impact of Signal Peptide Deletion on the Localization and Protein-protein Interaction of Potato StSN2 Protein

作　　者：温和 蔡诚诚 李罗品[1,2] 刘石锋 梁晓 王强[1,2] 王西瑶[1,2] WEN He;CAI Chengcheng;LI Luopin;LIU Shifeng;LIANG Xiao;WANG Qiang;WANG Xiyao(State Key Laboratory of Southwest Crop Gene Resources Exploration and Utilization,Sichuan Agricultural University,Chengdu,611130;Potato Research and Development Center,College of Agriculture,Sichuan Agricultural University,Chengdu,611130;Crop Research Institute of Sichuan Academy of Agricultural Sciences,Chengdu,611130)

机构地区：[1]四川农业大学西南作物基因资源发掘与利用国家重点实验室,成都611130 [2]四川农业大学农学院马铃薯研究与开发中心,成都611130 [3]四川省农业科学院作物研究所,成都611130

出　　处：《基因组学与应用生物学》2024年第5期755-766,共12页Genomics and Applied Biology

基　　金：国家现代农业技术体系四川薯类创新团队项目(SKL-ZY202203);西南作物基因资源与利用国家重点实验室“生物育种”揭榜挂帅项目(sccxtd-2023-09)共同资助。

摘　　要：StSN2属于马铃薯(Solanum tuberosum L.)Snakin/GASA(gibberellic acid-stimulated arabidopsis)蛋白家族,其N端具有信号肽,C端具有12个保守的半胱氨酸残基,主要在植物生长发育和逆境胁迫响应方面发挥重要调控作用。为了明确StSN2蛋白N端信号肽区域对其蛋白亚细胞定位及蛋白互作的影响,本研究首先通过信号肽预测及PCR扩增,获得含信号肽的StSN2基因片段和N端信号肽缺失的StSN2△SP片段,长度分别为315 bp和243 bp。其次,通过酶切连接方法将StSN2△SP基因片段连接在亚细胞定位载体上,获得融合蛋白YFP-StSN2△SP。瞬时表达实验结果发现,N端信号肽缺失后的StSN2△SP蛋白的亚细胞定位发生了变化,只定位在细胞核。最后通过同源重组方式将StSN2以及StSN2△SP分别构建到酵母诱饵载体上,并与pGADT7-SnRK1α进行酵母双杂交实验。结果显示,StSnRK1α与StSN2共转化酵母细胞后,其细胞不能在四缺培养基中生长,而StSnRK1α与N端信号肽缺失的StSN2△SP共转化酵母细胞后,其细胞能在四缺培养基中生长并显色,说明StSN2信号肽缺失后对其亚细胞定位及蛋白互作均有影响。本研究为StSN2蛋白的生物学功能研究提供了理论依据,为进一步完善该基因在马铃薯块茎休眠的功能研究奠定了实验基础。StSN2 belongs to the Snakin/GASA(gibberellic acid-stimulated arabidopsis)family of potato(Solanum tuberosum L.).Its N-terminal has a signal peptide,and its C-terminal has 12 conserved cysteine residues,mainly playing an important regulatory role in plant growth and development and stress response to stress.In order to understand how the N-terminal signal peptide region of StSN2 affects its subcellular localization and interactions,we conducted several experiments.First,we predicted the location of the signal peptide using computational methods and then amplified the corresponding DNA fragments.We obtained fragments of 315 bp(contai-ning the signal peptide)and 243 bp(deleted signal peptide).Next,we cloned the deleted signal peptide fragment into a subcellular lo-calization vector and expressed it as a fusion protein(YFP-StSN2△SP).Transient expression analysis revealed that the localization of the StSN2△SP protein without the signal peptide was altered and restricted to the nucleus.Finally,we created yeast bait carriers for both the full-length and deleted signal peptide versions of StSN2 and performed yeast two-hybrid experiments with StSnRK1α.The re-sults demonstrated that full-length StSN2 could not grow in a four-deficiency medium when transformed with StSnRK1α.However,the StSN2△SP variant without the signal peptide and StSnRK1αgrew normally under these conditions.These findings suggest that deleting the signal peptide from StSN2 impacts its subcellular localization and protein-protein interactions.This study provides a theoretical ba-sis for studying the biological functions of StSN2 proteins.It also lays an experimental foundation for further improving the functional studies of this gene in potato tuber dormancy.

关 键 词：马铃薯 StSN2 信号肽 亚细胞定位 酵母双杂交 

分 类 号：S532[农业科学—作物学]