大豆锌指转录因子GmDi19-5对高温的响应及互作蛋白的筛选

The Response to Heat and Screening of the Interacting Proteins of Zinc Finger Protein Gm Di19-5 in Soybean

【目的】高温胁迫已经成为威胁作物生长发育的主要非生物胁迫因素之一,转录因子在植物非生物胁迫响应中起着重要作用。通过对大豆锌指转录因子基因Gm Di19-5在高温胁迫下的响应和功能鉴定,以及利用酵母双杂交技术从大豆c DNA文库筛选与其互作的候选蛋白,研究Gm Di19-5对高温胁迫的响应机制。【方法】以大豆c DNA为模板,利用实时荧光定量PCR检测Gm Di19-5在高温处理不同时间段的表达模式;通过Plant CARE和PLACE数据库预测Gm Di19-5启动子元件,并对Gm Di19-5启动子转基因拟南芥在高温处理下进行的组织化学染色,分析Gm Di19-5启动子在高温胁迫下的活性;构建p GBKT7-Gm Di19-5诱饵载体,验证自激活活性;利用酵母双杂交技术,以p GBKT7-Gm Di19-5为诱饵筛选大豆c DNA文库,筛选与其互作的候选蛋白;进一步用酵母双杂交系统验证Gm Di19-5与候选蛋白的互作;利用实时荧光定量PCR检测候选蛋白基因在对高温处理的响应情况;构建融合表达载体,用GFP-Gm Di19-5融合表达载体转化拟南芥原生质体,检测Gm Di19-5蛋白的亚细胞定位情况。【结果】实时荧光定量PCR结果显示,Gm Di19-5在高温胁迫下上调表达;启动子元件分析表明,Gm Di19-5包含多种与胁迫应答相关的顺式作用元件,包括热响应元件HSE;组织化学染色分析发现,经高温处理后,过表达拟南芥幼苗的地上部分和地下部分均有报告基因的表达;亚细胞定位结果表明,Gm Di19-5蛋白定位于拟南芥原生质体的细胞核中;通过酵母双杂交技术筛选到了与Gm Di19-5互作的候选蛋白,试验进一步验证了Gm Di19-5与Gm Dna J在酵母细胞中互作;另外,实时荧光定量PCR结果显示,互作蛋白基因Gm Dna J受高温胁迫诱导表达。【结论】Gm Di19-5受高温诱导表达,Gm Di19-5可能与Gm Dna J互作,表明Gm Di19-5功能的发挥可能需要Gm Dna J的参与。

[Objective] Heat stress has become one of main abiotic stresses that threaten plant growth and development. Transcription factors play important roles in plant abiotic stress responses. Drought-induced protein Dil9 play an important role in stress signal transduction process in plant. To further explore the functional mechanism of GmDi19-5, the function was identified in high temperature stress and its interacting proteins were screened by yeast two-hybrid system. [Method] The real-time PCR was used to analyze the expression patterns of GmDil9-5 under high temperature stress treatment in different time periods. The 1,206 bp sequence upstream of GmDil9-5 was analyzed in PlantCARE and PLACE database. The promoter activity of GmDil9-5 was also analyzed using GUS staining in transgenic Arabidopsis. GmDil9-5 was overexpressed in Arabidopsis to statistically analyze its seed germination and root length under high temperature stress conditions. Bait plasmid pGBKT7-GmDil9-5 was constructed and the self-activation was detected. To obtain the candidate proteins of GmDi 19-5, the mixture of recombinant plasmid pGBKT7-GmDi 19-5, pGADT7 with soybean cDNA library was introduced into yeast cell AH109. The yeast two-hybrid system was used to screen interactive candidate proteins. Furthermore, the interaction between GmDil9-5 and candidate proteins was analyzed using the yeast two-hybrid system. The response of candidate genes to heat stress was examined by Real-time PCR; GmDil9-Swas fused with GFP to detect its subcellular localization in protoplast cells of Arabidopsis. [Result] The expression pattern analysis showed that GmDil9-5 was involved in responses to abiotic stresses and it was induced by high temperature stress in soybean. After high temperature stress treatment, GUS activities driven by promoter significantly increased in roots, leaf primordium and young leaves in transgenic Arabidopsis. PlantCARE and PLACE database analysis showed that the GmDil9-5 promoter contained a series of cis-element, including heat shock response element （HSE）. The subcellular localization analysis revealed that green fluorescence of GFP-GmDil9-5 was mainly in the nucleus. The seed germination and root length assays revealed that GmDil9-5 transgenic Arabidopsis increased sensitivity to high temperature stress. Yeast two-hybrid results showed that candidate protein GmDnaJ interacted with GmDil9-5. Real-time PCR showed that the interactive candidate gene GmDnaJ was induced by heat stress. [ Conclusion ] The expression of GmDil 9-5 was induced by high temperature stress and its overexpression inhibited the growth and development of transgenic plants. Yeast two-hybrid analysis showed that GmDnaJ maybe interact with GmDi 19-5 in yeast cells. This result suggests that GmDil9-5 maybe function in abiotic stress responses by interacting with GmDnaJ.