Screening of Populus simonii×Populus nigra WRKY70-interactive proteins by the yeast two-hybrid system
-
摘要: 小黑杨PsnWRKY70是能够响应盐胁迫的转录因子。为了进一步探究该转录因子在参与盐胁迫应答途径中与哪些蛋白质之间存在相互作用关系,本研究以140 mmol/L NaCl溶液处理过的小黑杨叶片为材料,利用热稳定核酸酶(DSN)构建了均一化的pGADT7-DEST酵母双杂交cDNA文库,将PsnWRKY70基因亚克隆至pGBKT7载体,形成BD-WRKY重组质粒,并以之为诱饵蛋白表达载体筛选小黑杨酵母双杂交cDNA文库。经过2轮酵母双杂交筛选以及回转验证试验,初步选出了5种与PsnWRKY70相互作用的蛋白质。对所筛选出的5种蛋白质进行保守结构域分析发现:有2种预测蛋白(HP1和HP2,其中HP1包含ClpP结构域),1种环化酶相关蛋白(CAP1),1种包含RNA识别基序的蛋白(RRM)以及1种泛素样修饰蛋白酶(Ulp1);对CAP1、HP1、RRM、HP2和Ulp1的毛果杨同源基因编码区上游2 000 bp范围内的顺式作用元件进行分析发现:CAP1、HP1、RRM、HP2和Ulp1的毛果杨同源基因上游启动子区均富含能够特异性结合WRKY转录因子的W-box。采用GST-pull down技术验证CAP1、HP1、RRM、HP2、Ulp1蛋白全长与PsnWRKY70转录因子之间是否存在直接的相互作用关系,将等量的His-X(X:CAP1、HP1、RRM、HP2或Ulp1)融合蛋白分别上样于正常谷胱甘肽琼脂糖树脂以及结合了GST或GST-WRKY蛋白的谷胱甘肽琼脂糖树脂中,SDS-PAGE电泳分离互作蛋白,最终Western blot结果显示:在体外状态下,HP1、RRM和Ulp1蛋白能够直接与PsnWRKY70相互作用,而CAP1和HP2则不能直接与PsnWRKY70相互作用。Abstract: Populus simonii×Populus nigra WRKY70 (PsnWRKY70) is a salt stress responsive transcription factor. In order to further identify the proteins that interact with PsnWRKY70 in salt stress response pathway, we used P. simonii×P. nigra leaves treated by 140 mmol/L NaCl solution as materials to construct a homogeneous pGADT7-DEST yeast two-hybrid cDNA library by DSN. PsnWRKY70 gene was subcloned to pGBKT7 vector to construct BD-WRKY recombinant plasmid, and then the BD-WRKY plasmid was used as a bait to screen the yeast two-hybrid cDNA library of P. simonii×P. nigra. After twice screening tests and a rotary experiment, five proteins that interact with PsnWRKY70 were detected. We analyzed the conserved domains of the five proteins and found that there were two hypothetical proteins (HP1 and HP2, HP1 contains a ClpP domain), one cyclase associated protein (CAP1), one RNA recognition motif-containing family protein (RRM) and one Ulp1 protease family protein (Ulp1) among them. The cis-elements that exist in 2 000 bp upstream of the orthologous Populus trichocarpa genes of CAP1, HP1, RRM, HP2 and Ulp1 were also analyzed, and the results suggest that the promoter regions of all the five orthologous P. trichocarpa genes contained plenty of W-box which can specifically bind to WRKY transcription factors. GST-pull down technology was utilized to validate the direct interaction between the complete CAP1/HP1/RRM/HP2/Ulp1 proteins and PsnWRKY70. An equal amount of His-X (X: CAP1/HP1/RRM/HP2/Ulp1) fusion proteins was dropped into normal glutathione agarose resin and GST or GST-WRKY protein-bound glutathione agarose resin respectively, then the interactive proteins were separated by SDS-PAGE. The final Western blot analysis indicated that HP1, RRM and Ulp1 can directly interact with PsnWRKY70 in vitro, while CAP1 or HP2 cannot.
-
Keywords:
- Populus simonii×P. nigra /
- PsnWRKY70 /
- salt stress /
- cDNA library /
- yeast two hybrid /
- interaction
-
-
[1] LIU P L, MA Y M, LIU S. Variety selection and breeding and cultivation of poplar [M]. Harbin: Northeast Forestry University Press, 2003.
[1] YAMAGUCHI S K, SHINOZAKI K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses [J]. Annu Rev Plant Biol, 2006, 57: 781-803.
[2] VINOCUR B, ALTMAN A. Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations [J]. Curr Opin Biotechnol, 2005, 16(2): 123-132.
[2] CHEN S, JIANG J, LI H Y, et al. The salt-responsive transcriptome of Populus simonii×Populus nigra via DGE [J]. Gene, 2012, 504(2): 203-212.
[3] LI J. The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense [J]. Plant Cell Online, 2004, 16(2): 319-331.
[3] SINGH K B, FOLEY R C, SANCHEZ L O. Transcription factors in plant defense and stress responses [J]. Current Opinion in Plant Biology, 2002, 5(5): 430-436.
[4] TRIPATHI P, RABARA R C, RUSHTON P J. A systems biology perspective on the role of WRKY transcription factors in drought responses in plants [J]. Planta, 2014, 239(2): 255-266.
[5] RUSHTON P J, SOMSSICH I E, RINGLER P, et al. WRKY transcription factors [J]. Trends Plant Sci, 2010, 15(5): 247-258.
[6] EULGEM T, RUSHTON P J, ROBATZEK S, et al. The WRKY superfamily of plant transcription factors [J]. Trends Plant Sci, 2000, 5(5): 199-206.
[7] 刘培林, 马伊民, 刘实. 杨树良种选育与栽培 [M]. 哈尔滨: 东北林业大学出版社, 2003. [9] ZHAO H, WANG S, CHEN S, et al.Phylogenetic and stress-responsive expression analysis of 20 WRKY genes in Populus simonii×Populus nigra [J]. Gene, 2015, 565(1): 130-139.
[11] EULGEM T. Dissecting the WRKY web of plant defense regulators [J]. PLoS Pathog, 2006, 2(11): e126.
[12] LI J, BRADER G, KARIOLA T, et al. WRKY70 modulates the selection of signaling pathways in plant defense [J]. Plant J, 2006, 46(3): 477-491.
[13] BESSEAU S, LI J, PALVA E T. WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana [J]. Journal of Experimental Botany, 2012, 63(7): 2667-2679.
[14] POPESCU S C, POPESCU G V, BACHAN, S, et al. MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays [J]. Genes Dev, 2009, 23(1): 80-92.
[15] LIANG W, YANG B, YU B J, et al. Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.) [J]. BMC Genomics, 2013, 14: 392.
[16] CHANG I F, CURRAN A, WOOLSEY R, et al. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana [J]. Proteomics, 2009, 9(11): 2967-2985.
[17] PARK C Y, LEE J H, YOO J H, et al. WRKY group IId transcription factors interact with calmodulin [J]. FEBS Lett, 2005, 579(6): 1545-1550.
[18] KIM K C, LAI Z, FAN B, et al. Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense [J]. Plant Cell, 2008, 20(9): 2357-2371.
[19] SHEN Q H, SAIJO Y, MAUCH S, et al. Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses [J]. Science, 2007, 315: 1098-1103.
[20] XU X, CHEN C, FAN B, et al.Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors [J]. Plant Cell, 2006, 18(5): 1310-1326.
[21] XIE Z, ZHANG Z L, ZOU X, et al. Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells [J]. Plant J, 2006, 46(2): 231-242.
[22] FIELDS S, SONG O. A novel genetic system to detect protein-protein interactions [J]. Nature, 1989, 340: 245-246.
[23] LUBAN J, STEPHEN G. The yeast two-hybrid system for studying protein-protein interactions[J]. Current Opinion in Biotech-nology, 1995, 6(1): 59-64.
[24] BUTLER N M, HANNAPEL D J. Promoter activity of polypyrimidine tract-binding protein genes of potato responds to environmental cues [J]. Planta, 2012, 236(6): 1747-1755.
[25] CONTI L, PRICE G, ODONNELL E, et al. Small ubiquitin-like modifier proteases overly tolerant to salt1 and-2 regulate salt stress responses in Arabidopsis [J]. Plant Cell Online, 2008, 20(10): 2894-2908.
[26] RAKWAL R, AGRAWAL G K, KUBO A, et al. Defense/stress responses elicited in rice seedlings exposed to the gaseous air pollutant sulfur dioxide [J]. Environmental and Experimental Botany, 2003, 49(3): 223-235.
[27] CIOLKOWSKI I, WANKE D, BIRKENBIHL R P, et al. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function [J]. Plant Mol Biol, 2008, 68(1-2): 81-92.
-
期刊类型引用(6)
1. 刘树超，邵全琴，杨帆，郭兴健，王东亮，黄海波，汪阳春，刘纪远，樊江文，李愈哲. 黄河源区放牧家畜数量及空间分布无人机遥感调查. 地球信息科学学报. 2021(07): 1286-1295 . 
百度学术
2. 郑硕，薛兴盛，白杨，吴艳兰. 徐淮地区冬季AOD时空特征及气象因子研究. 环境科学与技术. 2020(01): 78-85 . 百度学术
3. 柴曼，周滨，徐威杰，陈晨，刘文权. 基于多尺度分割GF-1影像地表覆盖信息提取研究. 内蒙古煤炭经济. 2020(06): 1-4 . 百度学术
4. 贾亮亮，汪小钦，苏华，王峰. 台湾岛高分一号卫星WFV数据气溶胶反演与验证. 环境科学学报. 2018(03): 1117-1127 . 百度学术
5. 李裕冬，罗艳，赵海涛，王程亮，杜杰. 基于MaxEnt模型的九寨沟国家级自然保护区川金丝猴适宜生境研究. 四川动物. 2018(05): 585-591 . 百度学术
6. 贾亮亮，汪小钦，王峰. 基于波段运算和纹理特征的高分一号多光谱数据云检测. 遥感信息. 2018(05): 62-68 . 百度学术
其他类型引用(6)
计量
- 文章访问数: 2042
- HTML全文浏览量: 275
- PDF下载量: 48
- 被引次数: 12
下载:
