蛋白激发子Hrip1互作蛋白的筛选及其原核表达

doi:10.13560/j.cnki.biotech.bull.1985.20170001

摘要/Abstract

摘要： 蛋白激发子Hrip1是从极细链格孢菌（Alternaria tenuissima）中分离纯化出的一种新型超敏反应诱导蛋白,能激发烟草、拟南芥和水稻等多种植物的免疫防御反应,提高广谱抗病性。为了探究Hrip1诱导植物抗性反应的分子机制,以Hrip1为诱饵蛋白,通过酵母双杂交,在水稻cDNA文库中筛选Hrip1的互作蛋白。经回转验证和x-a-gal染色得到了一个Hrip1互作蛋白-CSN5。构建了互作蛋白CSN5的原核表达载体pGEX-6P-2-CSN5,并转化大肠杆菌表达菌株BL21。经IPTG诱导后获得了可溶性的融合表达蛋白,用GST亲和层析柱纯化获得单一条带的融合表达蛋白,并用Western blot鉴定了融合表达蛋白的正确性。研究结果为进一步鉴定Hrip1的互作蛋白及其功能奠定了基础。

关键词: 蛋白激发子, 互作蛋白, 酵母双杂交, 原核表达

Abstract: The protein elicitor Hrip1 is a novel hypersensitive response-inducing protein secreted by necrotrophic fungus,Alternaria tenuissima,and it induces defense response and enhances broad-spectrum disease resistance in tobacco,Arabidopsis and rice plants. In order to investigate the molecular mechanism of Hrip1 inducing plant disease-resistance,we screened its interaction partners from a rice cDNA library using Hrip1 as bait protein and via yeast two hybrid system. After retransformation assay and x-a-gal staining,Hirp1-interacting protein CSN5 was obtained. Then,the recombinant pGEX-6P-2-CSN5 plasmid was constructed and transformed into the chemical competent cells of Escherichia coli BL21. The recombinant fusion protein in a single band was purified with a GST affinity chromatography column,and identified by Western blot. The results provided a base for further identification of the protein interacted with Hrip1 and its functions.

Key words: protein elicitor, interacting protein, yeast two hybrid system, prokaryotic expression

李书鹏, 杨秀芬, 袁京京, 邱德文. 蛋白激发子Hrip1互作蛋白的筛选及其原核表达[J]. 生物技术通报, 2017, 33(6): 182-189.

LI Shu-peng, YANG Xiu-fen, YUAN Jing-jing, QIU De-wen. Screening of Interacting Proteins with Protein Elicitor Hrip1 and Its Prokaryotic Expression[J]. Biotechnology Bulletin, 2017, 33(6): 182-189.

参考文献

[1] Zhang Y, Yang X, Liu Q, et al. Purification of novel protein elicitor from Botrytis cinerea that induces disease resistance and drought tolerance in plants[J]. Microbiological Research, 2010, 165（2）：142-151.
[2] Gómez-Gómez L, Felix G, Boller T. A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana[J]. The Plant Journal, 1999, 18（3）：277-284.
[3] Mao J, Liu Q, Yang X, et al. Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco[J]. Annals of Applied Biology, 2010, 156（3）：411-420.
[4] Bu B, Qiu D, Zeng H, et al. A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton[J]. Plant Cell Reports, 2014, 33（3）：461-470.
[5] Okinaka Y, Yang CH, Perna NT, et al. Microarray profiling of Erwinia chrysanthei 3937 genes that are regulated during plant infection[J]. Molecular Plant-Microbe Interactions, 2002, 15（7）：619-629.
[6] Garcia-Brugger A, Lamotte O, Vandelle E, et al. Early signaling events induced by elicitors of plant defenses[J]. Molecular Plant-Microbe Interactions, 2006, 19（7）：711-724.
[7] Schwessinger B, Ronald PC. Plant innate immunity：perception of conserved microbial signatures[J]. Plant Biology, 2012, 63.
[8] Boller T, Felix G. A renaissance of elicitors：perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors[J]. Annual Review of Plant Biology, 2009, 60：379-406.
[9] Zipfel C, Robatzek S, Navarro L, et al. Bacterial disease resistance in Arabidopsis through flagellin perception[J]. Nature, 2004, 428（6984）：764-767.
[10] Kunze G, Zipfel C, Robatzek S, et al. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants[J]. The Plant Cell, 2004, 16（12）：3496-3507.
[11] Zhang XC, Wu X, Findley S, et al. Molecular evolution of lysin motif-type receptor-like kinases in plants[J]. Plant Physiology, 2007, 144（2）：623-636.
[12] Kulye M, Liu H, Zhang Y, et al. Hrip1, a novel protein elicitor from necrotrophic fungus, Alternaria tenuissima, elicits cell death, expression of defence-related genes and systemic acquired resistance in tobacco[J]. Plant, Cell & Environment, 2012, 35（12）：2104-2120.
[13] Peng XC, Qiu DW, Zeng HM, et al. Inducible and constitutive expression of an elicitor gene Hrip1 from Alternaria tenuissima enhances stress tolerance in Arabidopsis[J]. Transgenic Research, 2015, 24（1）：135-145.
[14] Boller T, He SY. Innate immunity in plants：an arms race between pattern recognition receptors in plants and effectors in microbial pathogens[J]. Science, 2009, 324（5928）：742-744.
[15] Dodds PN, Rathjen JP. Plant immunity：towards an integrated view of plant-pathogen interactions[J]. Nature Reviews Genetics, 2010, 11（8）：539-548.
[16] Kwok S F, Solano R, Tsuge T, et al. Arabidopsis homologs of a c-Jun coactivator are present both in monomeric form and in the COP9 complex, and their abundance is differentially affected by the pleiotropic cop/det/fus mutations[J]. The Plant Cell, 1998, 10（11）：1779-1790.
[17] Wei N, Deng XW. COP9：a new genetic locus involved in light-regulated development and gene expression in Arabidopsis[J]. The Plant Cell, 1992, 4（12）：1507-1518.
[18] Wei N, Serino G, Deng XW. The COP9 signalosome：more than a protease[J]. Trends in Biochemical Sciences, 2008, 33（12）：592-600.
[19] Schwechheimer C, Serino G, Callis J, et al. Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response[J]. Science, 2001, 292（5520）：1379-1382.
[20] Lyapina S, Cope G, Shevchenko A, et al. Promotion of NEDD8-CUL1 conjugate cleavage by COP9 signalosome[J]. Science, 2001, 292（5520）：1382-1385.
[21] Franciosini A, Serino G, Deng XW. COP9 Signalosome Network[J]. Molecular Biology, 2014：313-332.
[22] Cope GA, Suh GSB, Aravind L, et al. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1[J]. Science, 2002, 298（5593）：608-611.
[23] Craig A, Ewan R, Mesmar J, et al. E3 ubiquitin ligases and plant innate immunity[J]. Journal of Experimental Botany, 2009, 60 （4）：1123-1132.
[24] Mukhtar MS, Carvunis AR, Dreze M, et al. Independently evolved virulence effectors converge onto hubs in a plant immune system network[J]. Science, 2011, 333（6042）：596-601.