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

摘要/Abstract

摘要： 【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GmDi19-5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cDNA文库筛选与其互作的候选蛋白，研究GmDi19-5对高温胁迫的响应机制。【方法】以大豆cDNA为模板，利用实时荧光定量PCR检测GmDi19-5在高温处理不同时间段的表达模式；通过PlantCARE和PLACE数据库预测GmDi19-5启动子元件，并对GmDi19-5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GmDi19-5启动子在高温胁迫下的活性；构建pGBKT7-GmDi19-5诱饵载体，验证自激活活性；利用酵母双杂交技术，以pGBKT7-GmDi19-5为诱饵筛选大豆cDNA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GmDi19-5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-GmDi19-5融合表达载体转化拟南芥原生质体，检测GmDi19-5蛋白的亚细胞定位情况。【结果】实时荧光定量PCR结果显示，GmDi19-5在高温胁迫下上调表达；启动子元件分析表明，GmDi19-5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，GmDi19-5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GmDi19-5互作的候选蛋白，试验进一步验证了GmDi19-5与GmDnaJ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GmDnaJ受高温胁迫诱导表达。【结论】GmDi19-5受高温诱导表达，GmDi19-5可能与GmDnaJ互作，表明GmDi19-5功能的发挥可能需要GmDnaJ的参与。

关键词: 大豆, 锌指转录因子, 高温, 酵母双杂交, 蛋白互作

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

赵娟莹，刘佳明，冯志娟，陈明，周永斌，陈隽，徐兆师，郭长虹. 大豆锌指转录因子GmDi19-5对高温的响应及互作蛋白的筛选[J]. 中国农业科学, 2017, 50(12): 2389-2398.

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.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.12.019

https://www.chinaagrisci.com/CN/Y2017/V50/I12/2389

参考文献

[1] Paz-Ares J, Ghosal D, Wienand U, Peterson P A, Saedler H. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. The EMBO Journal, 1987, 6(12): 3553-3558.

[2] Singh K B, Foley R C, Oñate-Sánchez L. Transcription factors in plant defense and stress responses. Current Opinion in Plant Biology, 2002, 5(5): 430-436.

[3] Takatsuji H. Zinc-finger proteins: the classical zinc finger emerges in contemporary plant science. Plant Molecular Biology, 1999, 39(6): 1073-1078.

[4] Hu W, Ma H. Characterization of a novel putative zinc finger gene MIF1: involvement in multiple hormonal regulation of Arabidopsis development. The Plant Journal, 2006, 45(3): 399-422.

[5] Pabo C O, Peisach E, Grant R A. Design and selection of novel Cys2His2 zinc finger proteins. Annual Review of Biochemistry, 2001, 70(1): 313-340.

[6] Milla, Miguel A, Rodriguez, Townsend J, Chang I F, Cushman J C. The Arabidopsis AtDi19 gene family encodes a novel type of Cys2/His2 zinc-finger protein implicated in ABA- independent dehydration, high-salinity stress and light signaling pathways. Plant Molecular Biology, 2006, 61(1): 13-30.

[7] Li G, Tai F J, Zheng Y, Luo J, Gong S Y, Zhang Z T, Li X B. Two cotton Cys2/His2-type zinc-finger proteins, GhDi19-1 and GhDi19-2, are involved in plant response to salt/drought stress and abscisic acid signaling. Plant Molecular Biology, 2010, 74(4): 437-452.

[8] Li S, Xu C, Yang Y, Xia G. Functional analysis of TaDi19A, a salt-responsive gene in wheat. Plant, Cell & Environment, 2010, 33(1): 117-129.

[9] Liu W X, Zhang F C, Zhang W Z, Song L F, Wu W H, Chen Y F. Arabidopsis Di19 functions as a transcription factor and modulates PR1, PR2, and PR5 expression in response to drought stress. Molecular Plant, 2013, 6(5): 1487-1502.

[10] Qin L X, Li Y, Li D D, Xu W L, Zheng Y, Li X B. Arabidopsis drought-induced protein Di19-3 participates in plant response to drought and high salinity stresses. Plant Molecular Biology, 2014, 86(6): 609-625.

[11] Kang X, Chong J, Ni M. HYPERSENSITIVE TO RED AND BLUE 1, a ZZ-type zinc finger protein, regulates phytochrome B–mediated red and cryptochrome-mediated blue light responses. The Plant Cell, 2005, 17(3): 822-835.

[12] Wang L, Yu C, Xu S, Zhu Y, Huang W. OsDi19-4 acts downstream of OsCDPK14 to positively regulate ABA response in rice. Plant, Cell & Environment, 2016.

[13] Searles M A, Lu D, Klug A. The role of the central zinc fingers of transcription factor IIIA in binding to 5S RNA1. Journal of Molecular Biology, 2000, 301(1): 47-60.

[14] Fukamatsu Y, Mitsui S, Yasuhara M, Tokioka Y, Ihara N, Fujita S, Kiyosue T. Identification of LOV KELCH PROTEIN2 (LKP2)-interacting factors that can recruit LKP2 to nuclear bodies. Plant and Cell Physiology, 2005, 46(8): 1340-1349.

[15] Kiyosue T, Wada M. LKP1 (LOV kelch protein 1): a factor involved in the regulation of flowering time in Arabidopsis. The Plant Journal, 2000, 23(6): 807-815.

[16] Feng Z J, Cui X Y, Cui X Y, Chen M, Yang G X, Ma Y Z, He G Y, Xu Z S. The soybean GmDi19-5 interacts with GmLEA3.1 and increases sensitivity of transgenic plants to abiotic stresses. Frontiers in plant science, 2015, 6: 179.

[17] Das A, Eldakak M, Paudel B, Kim D W, Hemmati H, Basu C, Rohila J S. Leaf proteome analysis reveals prospective drought and heat stress response mechanisms in soybean. Biomed Research International, 2016: 6021047.

[18] Yamasaki Y, Randall S K. Functionality of soybean CBF/DREB1 transcription factors. Plant Science, 2016, 246: 80-90.

[19] 李林蔚, 张双喜, 周永斌, 裴丽丽, 陈明, 李连城, 马有志, 刘生祥, 徐兆师. 大豆类钙调磷酸酶B亚基GmCBL1互作候选蛋白的筛选. 植物遗传资源学报, 2015, 16(2): 359-363.

Li L W, Zhang S X, Zhou Y B, Pei L L, Chen M, Li L C, Ma Y Z, Liu S X, Xu Z S. Screening of candidate interacting proteins of the calcineurin B-like protein GmCBL1 in soybean. Journal of Plant Genetic Resources, 2015, 16(2): 359-363. (in Chinese)

[20] 于太飞, 徐兆师, 李盼松, 陈明, 李连城, 张华, 马有志. 小麦蛋白激酶TaMAPK2互作蛋白的筛选与验证. 中国农业科学, 2014, 47(13): 2494-2503.

Yu T F, Xu Z S, Li P S, Chen M, Li L C, Zhang H, Ma Y Z. Screening and identification of proteins interacting with TaMAPK2 in wheat. Scientia Agricultura Sinica, 2014, 47(13): 2494-2503. (in Chinese)

[21] Zhao W, Liu Y W, Zhou J M, Zhao S P, Zhang X H, Min D H. Genome-wide analysis of the lectin receptor-like kinase family in foxtail millet (Setaria italica L.). Plant Cell, Tissue and Organ Culture, 2016, 127(2): 335-346.

[22] He G H, Xu J Y, Wang Y X, Liu J M, Li P S, Chen M, Ma Y Z, Xu Z S. Drought-responsive WRKY transcription factor genes TaWRKY1 and TaWRKY33 from wheat confer drought and/or heat resistance in Arabidopsis. BMC Plant Biology, 2016, 16(1): 116.

[23] 杨致荣, 王兴春, 李西明, 杨长登. 高等植物转录因子的研究进展. 遗传, 2004, 26(3): 403-408.

Yang Z Y, Wang X C, Li X M, Yang C D. Advance on the study of transcription factors in higher plants. Hereditas (Beijing), 2004, 26(3): 403-408. (in Chinese)

[24] Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal, 2002, 31(3): 279-292.

[25] 张燕利, 高捍东, 吴锦华. 4种景天科植物耐热性测定. 西南林学院学报, 2010, 30(6): 52-54.

Zhang Y L, Gao H D, Wu J H. Study on the heat tolerance of four species in family crassu laceae. Journal of Southwest Forestry University, 2010, 30(6): 52-54. (in Chinese)

[26] Larkindale J, Hall J D, Knight M R, Vierling E. Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiology, 2005, 138(2): 882-897.

[27] 张海娜, 李小娟, 李存东, 肖凯. 过量表达小麦超氧化物歧化酶(SOD)基因对烟草耐盐能力的影响. 作物学报, 2008, 34(8): 1403-1408.

Zhang H N, Li X J, Li C D, Xiao K. Effects of overexpression of wheat superoxide dismutase (SOD) genes on salt tolerant capability in tobacco. Acta Agronomica Sinica, 2008, 34(8): 1403-1408. (in Chinese)

[28] Kanno K, Mae T, Makino A. High night temperature stimulates photosynthesis, biomass production and growth during the vegetative stage of rice plants. Soil Science and Plant Nutrition, 2009, 55(1): 124-131.

[29] 谢晓金, 李秉柏, 程高峰, 刘春蕾, 周千. 高温对不同水稻品种剑叶生理特性的影响. 农业现代化研究, 2009, 30(4): 483-486.

Xie X J, Li B B, Cheng G H, Liu C L, Zhou Q. Effects of high temperature on physiological characteristics in flag leaves of different rice. Research of Agricultural Modernization, 2009, 30(4): 483-486. (in Chinese)

[30] 周人纲. 拟南芥AtJ3基因的克隆和分析. 植物学报, 1999, 41(6): 597-602.

Zhou R G. Cloning and analysis of AtJ3 gene in Arabidopsis thaliana. Acta Botanica Sinica, 1999, 41(6): 597-602. (in Chinese)

[31] Cyr D M, Langer T, Douglas M G. DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends in Biochemical Sciences, 1994, 19(4): 176-181.

[32] Diefenbach J, Kindl H. The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species. European Journal of Biochemistry, 2000, 267(3): 746-754.

[33] Zhu J K, Shi J, Bressan R A, Hasegawa P M. Expression of an Atriplex nummularia gene encoding a protein homologous to the bacterial molecular chaperone DnaJ. The Plant Cell, 1993, 5(3): 341-349.