OsMKK1和OsMKK6基因在水稻多重抗性中的功能研究
发布时间：2018-08-18 18:43
【摘要】：MAPK(促分裂原活化蛋白激酶)级联途径是广泛存在于真核生物中一种高度保守的信号转导途径。MAPK级联途径及其调控的基因影响植物对生物和非生物逆境的多抗性。低温、干旱和盐害等逆境严重影响了水稻生长发育、产量和品质。目前,通过酵母双杂交、体外磷酸化检测和功能验证等方法鉴定了参与水稻冷害信号转导的OsMKK6-OsMPK3途径,参与水稻盐害信号转导的OsMKK1-OsMPK4途径。但是,其下游靶蛋白及其作用机制还不明确。本研究利用Bi FC、酵母双杂交体系和MAPK磷酸化检测方法,证实了OsMKK1,OsMKK6分别与OsMPK3,OsMPK4和OsMPK6之间的互作特异性。而且,创建了OsMKK1和OsMKK6基因的超表达和RNAi抑制表达转基因植株,初步研究了它们的抗性功能。本文主要结果如下:1.OsMKK1和OsMKK6在原生质体中的亚细胞定位。将p D1301S-EGFP-MKK1、p D1301S-EGFP-MKK6融合蛋白基因表达载体转化水稻原生质体。荧光共聚焦显微镜观察发现,OsMKK1定位在内质网上,与内质网探针m Cherry-HDEL完全重合;OsMKK6定位在细胞质中;而对照GFP蛋白在细胞质、细胞膜和细胞核中都有表达。2.双分子荧光互补实验证明,OsMKK1与OsMPK3,OsMPK4在水稻原生质体中互作;OsMKK6与OsMPK3,OsMPK4在水稻原生质体中互作。酵母双杂交实验证明,OsMKK1与OsMPK4,OsMPK6互作,OsMKK6与OsMPK4互作。3.OsMKK1和OsMKK6蛋白的原核诱导和激酶活性检测。在大肠杆菌中诱导表达His-OsMKK1和His-OsMKK6-SUMO融合蛋白。体外磷酸化检测表明,OsMKK1和OsMKK6具有自磷酸化活性。利用体外点突变PCR构建了GST-MPK3-M、GST-MPK4-M和GST-MPK6-M自磷酸化失活的融合蛋白基因表达载体,在大肠杆菌BL21(DE3)中诱导表达GST-MPK3-M,GST-MPK4-M和GST-MPK6-M融合蛋白。体外磷酸化与Phos-tag-gel SDS-PAGE技术证明了GST-MPK3-M,GST-MPK4-M和GST-MPK6-M融合蛋白没有自磷酸化活性,可以用于与OsMKK1,OsMKK6激酶互作研究。4.利用农杆菌介导法获得了OsMKK1和OsMKK6基因超表达和抑制表达转基因水稻。繁殖加代,潮霉素筛选获得了T4转基因纯合株系。5.OsMKK1和OsMKK6转基因水稻萌发期的耐冷性鉴定。12℃低温处理12 d,OsMKK1超表达转基因水稻幼芽生长速度比野生型快,可溶性糖积累大于野生型;OsMKK6超表达转基因水稻幼芽生长速度大于野生型。6.OsMKK1转基因水稻苗期的耐冷性鉴定。低温处理三叶期水稻幼苗,OsMKK1超表达转基因水稻幼苗存活率高于野生型,可溶性糖积累大于野生型。超表达OsMKK1增强水稻苗期的耐冷性。7.OsMKK6转基因水稻苗期的耐盐性鉴定。150 m M Na Cl胁迫处理下,OsMKK6超表达转基因水稻幼苗存活率高于野生型水稻,OsMKK6抑制表达转基因水稻存活率低于野生型,脯氨酸积累大于野生型。OsMKK6增强水稻苗期耐盐性。
[Abstract]:Mitogen-activated protein kinase (MAPK) cascade pathway is a highly conserved signal transduction pathway in eukaryotes. Low temperature, drought and salt damage have seriously affected the growth, yield and quality of rice. At present, the OsMKK6-OsMPK3 pathway involved in rice chilling injury signal transduction and the OsMKK1-OsMPK4 pathway involved in rice salt injury signal transduction were identified by yeast two-hybrid, in vitro phosphorylation detection and functional verification. However, its downstream target protein and its mechanism of action are unclear. In this study, the interaction specificity of OsMKK1OsMKK6 with OsMPK3OsMPK4 and OsMPK6 was confirmed by using BiFC, yeast two-hybrid system and MAPK phosphorylation assay. Moreover, the overexpression of OsMKK1 and OsMKK6 genes and the inhibition of RNAi expression were established, and their anti-sexual function was studied. The main results are as follows: 1. Subcellular localization of OsMKK1 and OsMKK6 in protoplasts. The expression vector of pD1301S-EGFP-MKK1P D1301S-EGFP-MKK6 fusion protein gene was transformed into rice protoplast. Fluorescence confocal microscopy showed that OsMKK1 was located on the endoplasmic reticulum, and was located in cytoplasm with the endoplasmic reticulum probe m Cherry-HDEL, while the control GFP protein was expressed in cytoplasm, cell membrane and nucleus. The bimolecular fluorescence complementary experiment showed that OsMKK1 and OsMPK3 OsMPK4 interacted in rice protoplasts. OsMKK6 and OsMPK4 interacted with OsMPK4 in rice protoplasts. Yeast two-hybrid experiments demonstrated the interaction between OsMKK1 and OsMPK4 OsMPK6 and the interaction between OsMKK6 and OsMPK4. 3. The prokaryotic induction and kinase activity of OsMKKK1 and OsMKK6 proteins. Expression of His-OsMKK1 and His-OsMKK6-SUMO fusion protein was induced in Escherichia coli. In vitro phosphorylation assay showed that OsMKK1 and OsMKK6 had self phosphorylation activity. The fusion gene expression vector of GST-MPK3-MGST-MPK4-M and GST-MPK6-M self-phosphorylated fusion protein was constructed by using point mutation PCR in vitro. The fusion protein GST-MPK4-M and GST-MPK6-M fusion protein were induced to express GST-MPK3-MGST-MPK4-M in Escherichia coli BL21 (DE3). In vitro phosphorylation and Phos-tag-gel SDS-PAGE techniques demonstrated that GST-MPK3-MGST-MPK4-M and GST-MPK6-M fusion proteins had no autophosphorylation activity and could be used to study the interaction between GST-MPK3-MGST-MPK4-M and OsMKK6 kinase. The overexpression and inhibition of OsMKK1 and OsMKK6 genes in transgenic rice were obtained by Agrobacterium tumefaciens mediated. In addition, hygromycin screening was used to identify the cold tolerance of transgenic T4 transgenic lines. 5. OsMKKK1 and OsMKK6 transgenic rice at germination stage. The growth rate of overexpressed transgenic rice buds was faster than that of wild-type transgenic rice treated at low temperature for 12 days at 12 鈩，
本文编号：2190346
[Abstract]:Mitogen-activated protein kinase (MAPK) cascade pathway is a highly conserved signal transduction pathway in eukaryotes. Low temperature, drought and salt damage have seriously affected the growth, yield and quality of rice. At present, the OsMKK6-OsMPK3 pathway involved in rice chilling injury signal transduction and the OsMKK1-OsMPK4 pathway involved in rice salt injury signal transduction were identified by yeast two-hybrid, in vitro phosphorylation detection and functional verification. However, its downstream target protein and its mechanism of action are unclear. In this study, the interaction specificity of OsMKK1OsMKK6 with OsMPK3OsMPK4 and OsMPK6 was confirmed by using BiFC, yeast two-hybrid system and MAPK phosphorylation assay. Moreover, the overexpression of OsMKK1 and OsMKK6 genes and the inhibition of RNAi expression were established, and their anti-sexual function was studied. The main results are as follows: 1. Subcellular localization of OsMKK1 and OsMKK6 in protoplasts. The expression vector of pD1301S-EGFP-MKK1P D1301S-EGFP-MKK6 fusion protein gene was transformed into rice protoplast. Fluorescence confocal microscopy showed that OsMKK1 was located on the endoplasmic reticulum, and was located in cytoplasm with the endoplasmic reticulum probe m Cherry-HDEL, while the control GFP protein was expressed in cytoplasm, cell membrane and nucleus. The bimolecular fluorescence complementary experiment showed that OsMKK1 and OsMPK3 OsMPK4 interacted in rice protoplasts. OsMKK6 and OsMPK4 interacted with OsMPK4 in rice protoplasts. Yeast two-hybrid experiments demonstrated the interaction between OsMKK1 and OsMPK4 OsMPK6 and the interaction between OsMKK6 and OsMPK4. 3. The prokaryotic induction and kinase activity of OsMKKK1 and OsMKK6 proteins. Expression of His-OsMKK1 and His-OsMKK6-SUMO fusion protein was induced in Escherichia coli. In vitro phosphorylation assay showed that OsMKK1 and OsMKK6 had self phosphorylation activity. The fusion gene expression vector of GST-MPK3-MGST-MPK4-M and GST-MPK6-M self-phosphorylated fusion protein was constructed by using point mutation PCR in vitro. The fusion protein GST-MPK4-M and GST-MPK6-M fusion protein were induced to express GST-MPK3-MGST-MPK4-M in Escherichia coli BL21 (DE3). In vitro phosphorylation and Phos-tag-gel SDS-PAGE techniques demonstrated that GST-MPK3-MGST-MPK4-M and GST-MPK6-M fusion proteins had no autophosphorylation activity and could be used to study the interaction between GST-MPK3-MGST-MPK4-M and OsMKK6 kinase. The overexpression and inhibition of OsMKK1 and OsMKK6 genes in transgenic rice were obtained by Agrobacterium tumefaciens mediated. In addition, hygromycin screening was used to identify the cold tolerance of transgenic T4 transgenic lines. 5. OsMKKK1 and OsMKK6 transgenic rice at germination stage. The growth rate of overexpressed transgenic rice buds was faster than that of wild-type transgenic rice treated at low temperature for 12 days at 12 鈩，
本文编号：2190346
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