大豆锌指转录因子GmDi19-5对高温的响应及互作蛋白的筛选

doi:10.3864/j.issn.0578-1752.2017.12.019

Abstract

Abstract: 【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 Di19 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 GmDi19-5 under high temperature stress treatment in different time periods. The 1,206 bp sequence upstream of GmDi19-5 was analyzed in PlantCARE and PLACE database. The promoter activity of GmDi19-5 was also analyzed using GUS staining in transgenic Arabidopsis. GmDi19-5 was overexpressed in Arabidopsis to statistically analyze its seed germination and root length under high temperature stress conditions. Bait plasmid pGBKT7-GmDi19-5 was constructed and the self-activation was detected. To obtain the candidate proteins of GmDi19-5, the mixture of recombinant plasmid pGBKT7-GmDi19-5, 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 GmDi19-5 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; GmDi19-5was fused with GFP to detect its subcellular localization in protoplast cells of Arabidopsis. 【Result】The expression pattern analysis showed that GmDi19-5 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 GmDi19-5 promoter contained a series of cis-element, including heat shock response element (HSE). The subcellular localization analysis revealed that green fluorescence of GFP-GmDi19-5 was mainly in the nucleus. The seed germination and root length assays revealed that GmDi19-5 transgenic Arabidopsis increased sensitivity to high temperature stress. Yeast two-hybrid results showed that candidate protein GmDnaJ interacted with GmDi19-5. Real-time PCR showed that the interactive candidate gene GmDnaJ was induced by heat stress. 【Conclusion】The expression of GmDi19-5 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 GmDi19-5 in yeast cells. This result suggests that GmDi19-5 maybe function in abiotic stress responses by interacting with GmDnaJ.

Key words: Glycine max, zinc finger protein, high temperature, yeast two-hybrid, protein interaction

ZHAO JuanYing, LIU JiaMing, FENG ZhiJuan, CHEN Ming, ZHOU YongBin, CHEN Jun, XU ZhaoShi, GUO ChangHong. The Response to Heat and Screening of the Interacting Proteins of Zinc Finger Protein GmDi19-5 in Soybean[J].Scientia Agricultura Sinica, 2017, 50(12): 2389-2398.

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The Response to Heat and Screening of the Interacting Proteins of Zinc Finger Protein GmDi19-5 in Soybean

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