OsMKK1和OsMKK6基因在水稻多重抗性中的功能研究
發(fā)布時(shí)間：2018-08-18 18:43
【摘要】：MAPK(促分裂原活化蛋白激酶)級(jí)聯(lián)途徑是廣泛存在于真核生物中一種高度保守的信號(hào)轉(zhuǎn)導(dǎo)途徑。MAPK級(jí)聯(lián)途徑及其調(diào)控的基因影響植物對(duì)生物和非生物逆境的多抗性。低溫、干旱和鹽害等逆境嚴(yán)重影響了水稻生長發(fā)育、產(chǎn)量和品質(zhì)。目前,通過酵母雙雜交、體外磷酸化檢測和功能驗(yàn)證等方法鑒定了參與水稻冷害信號(hào)轉(zhuǎn)導(dǎo)的OsMKK6-OsMPK3途徑,參與水稻鹽害信號(hào)轉(zhuǎn)導(dǎo)的OsMKK1-OsMPK4途徑。但是,其下游靶蛋白及其作用機(jī)制還不明確。本研究利用Bi FC、酵母雙雜交體系和MAPK磷酸化檢測方法,證實(shí)了OsMKK1,OsMKK6分別與OsMPK3,OsMPK4和OsMPK6之間的互作特異性。而且,創(chuàng)建了OsMKK1和OsMKK6基因的超表達(dá)和RNAi抑制表達(dá)轉(zhuǎn)基因植株,初步研究了它們的抗性功能。本文主要結(jié)果如下:1.OsMKK1和OsMKK6在原生質(zhì)體中的亞細(xì)胞定位。將p D1301S-EGFP-MKK1、p D1301S-EGFP-MKK6融合蛋白基因表達(dá)載體轉(zhuǎn)化水稻原生質(zhì)體。熒光共聚焦顯微鏡觀察發(fā)現(xiàn),OsMKK1定位在內(nèi)質(zhì)網(wǎng)上,與內(nèi)質(zhì)網(wǎng)探針m Cherry-HDEL完全重合;OsMKK6定位在細(xì)胞質(zhì)中;而對(duì)照GFP蛋白在細(xì)胞質(zhì)、細(xì)胞膜和細(xì)胞核中都有表達(dá)。2.雙分子熒光互補(bǔ)實(shí)驗(yàn)證明,OsMKK1與OsMPK3,OsMPK4在水稻原生質(zhì)體中互作;OsMKK6與OsMPK3,OsMPK4在水稻原生質(zhì)體中互作。酵母雙雜交實(shí)驗(yàn)證明,OsMKK1與OsMPK4,OsMPK6互作,OsMKK6與OsMPK4互作。3.OsMKK1和OsMKK6蛋白的原核誘導(dǎo)和激酶活性檢測。在大腸桿菌中誘導(dǎo)表達(dá)His-OsMKK1和His-OsMKK6-SUMO融合蛋白。體外磷酸化檢測表明,OsMKK1和OsMKK6具有自磷酸化活性。利用體外點(diǎn)突變PCR構(gòu)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達(dá)載體,在大腸桿菌BL21(DE3)中誘導(dǎo)表達(dá)GST-MPK3-M,GST-MPK4-M和GST-MPK6-M融合蛋白。體外磷酸化與Phos-tag-gel SDS-PAGE技術(shù)證明了GST-MPK3-M,GST-MPK4-M和GST-MPK6-M融合蛋白沒有自磷酸化活性,可以用于與OsMKK1,OsMKK6激酶互作研究。4.利用農(nóng)桿菌介導(dǎo)法獲得了OsMKK1和OsMKK6基因超表達(dá)和抑制表達(dá)轉(zhuǎn)基因水稻。繁殖加代,潮霉素篩選獲得了T4轉(zhuǎn)基因純合株系。5.OsMKK1和OsMKK6轉(zhuǎn)基因水稻萌發(fā)期的耐冷性鑒定。12℃低溫處理12 d,OsMKK1超表達(dá)轉(zhuǎn)基因水稻幼芽生長速度比野生型快,可溶性糖積累大于野生型;OsMKK6超表達(dá)轉(zhuǎn)基因水稻幼芽生長速度大于野生型。6.OsMKK1轉(zhuǎn)基因水稻苗期的耐冷性鑒定。低溫處理三葉期水稻幼苗,OsMKK1超表達(dá)轉(zhuǎn)基因水稻幼苗存活率高于野生型,可溶性糖積累大于野生型。超表達(dá)OsMKK1增強(qiáng)水稻苗期的耐冷性。7.OsMKK6轉(zhuǎn)基因水稻苗期的耐鹽性鑒定。150 m M Na Cl脅迫處理下,OsMKK6超表達(dá)轉(zhuǎn)基因水稻幼苗存活率高于野生型水稻,OsMKK6抑制表達(dá)轉(zhuǎn)基因水稻存活率低于野生型,脯氨酸積累大于野生型。OsMKK6增強(qiáng)水稻苗期耐鹽性。
[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，
本文編號(hào)：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，
本文編號(hào)：2190346
本文鏈接：http://sikaile.net/shoufeilunwen/zaizhiyanjiusheng/2190346.html
最近更新
教材專著
