水稻BWMK1互作基因BWIP4和BWIP2功能分析
摘要
BWMK1 (Blast and Wound Induced MAPK gene)是水稻中第1个被克隆鉴定的MAPK基因,受稻瘟菌侵染和创伤强烈诱导表达。为深入研究BWMK1基因参与MAPK级联反应介导的植物抗病信号传导途径,我们采用酵母双杂交技术筛选水稻稻瘟病诱导表达后的cDNA文库,获得了7个BWMK1的互作蛋白,并命名为BWIP1-BWIP7 (BWMK1 Interacting Proteins)本研究选择其中的2个互作蛋白编码基因BWIP2和BWIP4进行功能分析,BWIP2编码一个未知假想蛋白；BWIP4,编码一个水稻磷酸盐转运蛋白,即OsPT8。
本研究中构建了BWIP4 RNAi干涉载体pAND A-BWIP4和BWIP4超表达载体NCGR-1300-BWIP4以及BWIP2 RNAi干涉载体pANDA-BWIP2和BWIP2超表达载体BWIP2通过农杆菌介导法,以粳稻品种日本晴为转基因受体,获得35个BWIP4干涉转基因株系,136个BWIP4超表达转基因株系,108个BWIP2超表达转基因株系和32个BWIP2干涉转基因株系,通过GUS、PCR和RT-PCR检测,选择其中4个BWIP4-RNAi、5个BWIP4超表达、5个BWIP2-RNAi和8个BWIP2超表达阳性转基因株系做进一步的功能分析。水稻稻瘟病菌接种实验表明,BWIP4和BWIP2干涉植株对稻瘟病菌抗性无明显变化；水稻白叶枯病菌接种实验表明,BWIP4和BWIP2干涉植株均能提高对白叶枯病菌R6-D的抗性,BWIP4和BWIP2超表达植株对白叶枯病菌R6-D的感病性增强,表明水稻BWMK1互作基因BWIP4和BWIP2可能负调控水稻对白叶枯病的抗性。水稻低磷处理实验结果表明,低磷环境能诱导水稻植株中BWIP4基因的表达,BWIP4超表达植株新根数目增多,叶片中的磷含量明显高于对照日本晴。
BWMK1 was the first reported rice MAPK gene,which was transcriptionally activated by blast fungus infection and wounding. In order to further understand the BWMK1-mediated defense signaling pathway, we identified 7 BWMK1 interacting genes by yeast two-hybrid system, designated BWIP1-BWIP7. BWIP4 and BWIP2 were selected for further functional characterization. BWIP4 encodes a putative phosphate transporter (OsPT8), but BWIP2 shows no hits to known functional groups using the BLAST search.
Through Agrobacterium mediated transformation method, RNAi constracts pANDA-BWIP4 and pANDA-BWIP2, overexpression constracts NCGR-1300-BWIP4 and pCAMBIA1301-BWIP2 were transformed into the Japonica cultivar Nipponbare,and the 35 lines,32 lines,136 lines and 108 lines had been abtained respectively.The GUS assay and PCR results showed that the target genes has been intergrated into the receptor. Further expression analysis confirmed that the target genes has been overexpressed and down-regulated in the overexpression and RNAi transgenic plants. The disease resistance of these transgenic lines to bacterial blight pathogen Xanthomonas oryzae pv.oryzae (Xoo) were evaluated. The results showed that both the RNAi lines of BWIP4 and BWIP2 enhanced resistance to Xoo, in contrary, the over-expression lines of BWIP4 and BWIP2 were more susceptible to Xoo than the wide type. These results showed that BWIP4 and BWIP2 were negative regulators for the resitance to the rice bacterial blight pathogen.The Pi content and the number of new roots were assayed in BWIP4 overexpression plants,grown in nutrient solutions with Pi-deficiency and Pi-sufficiency.The resultes showed that the expression level of BWIP4 in rice was significantly enhanced by Pi-deficiency. The number of new roots in BWIP4-overexpression plants were much more than the control at Pi-deficiency treatment. The Pi content of BWIP4-overexpression plant leaves were much higher than the Japonica cultivar Nipponbare.
本研究中构建了BWIP4 RNAi干涉载体pAND A-BWIP4和BWIP4超表达载体NCGR-1300-BWIP4以及BWIP2 RNAi干涉载体pANDA-BWIP2和BWIP2超表达载体BWIP2通过农杆菌介导法,以粳稻品种日本晴为转基因受体,获得35个BWIP4干涉转基因株系,136个BWIP4超表达转基因株系,108个BWIP2超表达转基因株系和32个BWIP2干涉转基因株系,通过GUS、PCR和RT-PCR检测,选择其中4个BWIP4-RNAi、5个BWIP4超表达、5个BWIP2-RNAi和8个BWIP2超表达阳性转基因株系做进一步的功能分析。水稻稻瘟病菌接种实验表明,BWIP4和BWIP2干涉植株对稻瘟病菌抗性无明显变化；水稻白叶枯病菌接种实验表明,BWIP4和BWIP2干涉植株均能提高对白叶枯病菌R6-D的抗性,BWIP4和BWIP2超表达植株对白叶枯病菌R6-D的感病性增强,表明水稻BWMK1互作基因BWIP4和BWIP2可能负调控水稻对白叶枯病的抗性。水稻低磷处理实验结果表明,低磷环境能诱导水稻植株中BWIP4基因的表达,BWIP4超表达植株新根数目增多,叶片中的磷含量明显高于对照日本晴。
BWMK1 was the first reported rice MAPK gene,which was transcriptionally activated by blast fungus infection and wounding. In order to further understand the BWMK1-mediated defense signaling pathway, we identified 7 BWMK1 interacting genes by yeast two-hybrid system, designated BWIP1-BWIP7. BWIP4 and BWIP2 were selected for further functional characterization. BWIP4 encodes a putative phosphate transporter (OsPT8), but BWIP2 shows no hits to known functional groups using the BLAST search.
Through Agrobacterium mediated transformation method, RNAi constracts pANDA-BWIP4 and pANDA-BWIP2, overexpression constracts NCGR-1300-BWIP4 and pCAMBIA1301-BWIP2 were transformed into the Japonica cultivar Nipponbare,and the 35 lines,32 lines,136 lines and 108 lines had been abtained respectively.The GUS assay and PCR results showed that the target genes has been intergrated into the receptor. Further expression analysis confirmed that the target genes has been overexpressed and down-regulated in the overexpression and RNAi transgenic plants. The disease resistance of these transgenic lines to bacterial blight pathogen Xanthomonas oryzae pv.oryzae (Xoo) were evaluated. The results showed that both the RNAi lines of BWIP4 and BWIP2 enhanced resistance to Xoo, in contrary, the over-expression lines of BWIP4 and BWIP2 were more susceptible to Xoo than the wide type. These results showed that BWIP4 and BWIP2 were negative regulators for the resitance to the rice bacterial blight pathogen.The Pi content and the number of new roots were assayed in BWIP4 overexpression plants,grown in nutrient solutions with Pi-deficiency and Pi-sufficiency.The resultes showed that the expression level of BWIP4 in rice was significantly enhanced by Pi-deficiency. The number of new roots in BWIP4-overexpression plants were much more than the control at Pi-deficiency treatment. The Pi content of BWIP4-overexpression plant leaves were much higher than the Japonica cultivar Nipponbare.
引文
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[3]Ligterink W, Hirt H. Mitogen-activated protein (MAP) kinase pathways in plants: versatile signaling tools[J]. Int Rev Cytol,2001,201:209-275
[4]Jonak C, Okresz L, Bogre L, et al. Complexity, cross talk and integration of plant MAP kinase signalling[J]. Curr Opin in Plant Biol,2002,5(5):415-424
[5]任琰,谌江华,黄俊浩等.水稻MAPK基因OsMPK4的克隆鉴定、蛋白表达和转基因载体构建[J].浙江大学学报,2006,32(6)：613-620.
[6]Tena G, Asai T,Chiu W L,et al. Plant mitogen-activated protein kinase signaling cascades [J]. Curr.Opin. Plant Biol.,2001,4:392-400.
[7]Pedley K F,Martin G B. Role of mitogen-activated protein kinases in plant immunity [J]. Curr. Opin. Plant Biol.,2005,8:541-547.
[8]刘强,张贵友,SHINOZAKI Ka zuo植物促分裂原活化蛋白(MAP)激酶[J].植物学报,2000,42(7)：661-667.
[9]Romeis T,Piedras P,Zhang S,et al. Rapid Avr29 and Cf292dependent activation of MAP kinases in tobacco cell cultures and leaves:convergence of resistance gene,elicitor,wound and salicylate responses [J]. Plant Cell,1999,11:273-287.
[10]Yang K Y, Liu Y, Zhang S. Activation of a mitogen-activated protein kinase pat hway is involved in disease resistance in tobacco [J]. Proc Natl Acad Sci USA,2001, 98:741-746.
[11]Ren D,Yang H,Zhang S. Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis [J]. J Biol Chem.,2002,277:559-565.
[12]Asai T,Tena G, Plotnikova J,et al. MAP kinase signaling cascade in Arabidopsis innate immunity [J]. Nature,2002,415:977-983.
[13]Jin H L,Axtell M J,Dahlbeck D,et al. NPK1,an MEKK121ike mitogen activated protein kinase,regulates innate immunity and development in plants [J] Developmental Cell,2002,3:291-297.
[14]Jin H,Liu Y, Yang K Y, et al. Function of a mitogen activated protein kinase pathway in N gene-mediated resistance in tobacco [J]. Plant J.,2003,33:719-731.
[15]Del Pozo O,Pedley K F,Martin G B. MAPKKK alpha is a positive regulator of cell death associated with both plant immunity and disease [J]. The EMBO Journal,2004, 23:3072-3082.
[16]Menke F L,van Pelt J A,Pieterse C M,et al. Silencing of the mitogen activated protein kinase MAPK6 compromises disease resistance in Arabidopsis [J]. Plant Cell, 2004,16:897-907.
[17]Jiang Y,Li Z,Schwarz E M, et al. Structure function studies of p38 mitogen activated protein kinase. Loop 1 2 influences substrate specificity and autophosphorylation,but not upstream kinase selection[J]. J Biol Chem.1997,272:11096-11102.
[18]Lodish H,Berk A,Zipursky S L,et al. Molecular Cell Biology[M]. New York:W H Freeman and Company,2000,880-881.
[19]吴涛,宗晓娟,谷令坤等.植物中MAPK及其在信号传导中的作用[J].生物技术通报,2006(5)：261-270.
[20]Ichimura et al. Mitogen activated protein kinase cascades in plants:a new nomenclature[J]. Trends Plant Sci,2002,7:301-308
[21]He C,Fong SHT,Yang D,et al. BWMKl,a novel MAP kinase induced by fungal infection and mechanical wounding in rice[J]. Mol Plant Microbe Interact,1999, 12:1064-1073.
[22]Cheong YH,Moon BC,Kim JK, et al. BWMK1,a rice mitogen activated protein kinase,locates in the nucleus and mediates pathogenesis related gene expression by activation of a transcription factor[J]. Plant Physiol,2003,132:1961-1972.
[23]Song F,Goodman RM. OsBIMK1,a rice MAP kinase gene involved in disease resistance responses[J]. Planta,2002,215:997-1005.
[24]Agrawal GK,Rakwal R,lwahashi H. Isolation of novel rice(Oryza sativa L.)multiple stress responsive MAP kinase gene,OsMSRMK2,whose mRNA accumulates rapidly in response to environmental cues[J]. Biochem Biophys Res Comm,2002,294:1009-1016.
[25]Wen JQ,Oono K,Imai R. Two novel mitogen activated protein signaling components, OsMEK1 and OsMAP1,are involved in a moderate low-temperature signaling pathway in rice[J]. Plant Physiol,2002,129:1880-1891.
[26]Huang HJ,Fu SF,Tai YH,et al. Expression of Oryza sativa MAP kinase gene is developmentally regulated and stress-responsive[J]. Physiol Plant,2002,114:572-580.
[27]Xiong LZ,Yang YN. Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid inducible mitogen activated protein kinase[J]. Plant Cell,2003,15:745-759.
[28]Song DH,Chen JH,Song FM,et al. A novel rice MAPK gene,OsBIMK2,is involved in disease resistance response[J]. Plant Biol,2006,8(5):587-596.
[29]Yeh CM,Hsiao LJ,Huang HJ. Cadmium activates a mitogen activated protein kinase gene and MAP kinases in rice[J]. Plant Cell Physiol,2004,45:1306-1312.
[30]Agrawal GK,Agrawal SK,Shibato J,et al. Novel rice MAP kinases OsMSRMK3 and OsW JUMKl involved in encountering diverse environmental stresses and developmental regulation[J]. Biocbem Biopbys Res Comm,2003,300:775-783
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