擬南芥類受體激酶RIPK與FERONIA共響應(yīng)RALF信號(hào)的遺傳與生化分析
發(fā)布時(shí)間：2018-10-17 13:28
【摘要】：植物細(xì)胞的生長發(fā)育受到多種信號(hào)因子的調(diào)控,其分子機(jī)制的揭示和闡明有助于深化對細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)領(lǐng)域的理解,也將極大克服制約我國農(nóng)業(yè)、林業(yè)等應(yīng)用領(lǐng)域發(fā)展的一些亟需解決的技術(shù)瓶頸。細(xì)胞快速堿化因子(Rapidalkalinizationfactor,RALF)作為一類植物界保守的多肽激素,因其能快速抑制質(zhì)膜氫泵(PM-H+ATPase)活性導(dǎo)致細(xì)胞壁堿化從而抑制細(xì)胞伸長而得名。近來研究表明擬南芥中的蛋白激酶FERONIA(FER)作為RALF1多肽信號(hào)的受體可通過胞外域與RALF1直接結(jié)合來感受RALF1信號(hào)刺激并將該信號(hào)傳遞至細(xì)胞內(nèi),最終調(diào)控質(zhì)膜的氫泵活性(PM-H+ATPase,e.g.AHA2)來控制細(xì)胞伸長,但響應(yīng)RALF1配體信號(hào)的早期精細(xì)分子機(jī)制并不十分清楚。例如蛋白激酶FER受體接受RALF1信號(hào)刺激后,如何將RALF1信號(hào)傳遞至細(xì)胞內(nèi)?是否存在一些下游成員,如胞質(zhì)類受體激酶(RLCK)參與了該信號(hào)通路?如果存在,這些胞內(nèi)的下游成員又以何種方式參與了該信號(hào)通路呢?因此深入研究能夠與蛋白激酶FER直接相互作用的下游分子如胞質(zhì)類受體激酶RLCK,并闡明其信號(hào)傳導(dǎo)的分子機(jī)制將有助于我們揭示受體蛋白激酶FER接受、傳遞RALF1小肽信號(hào)并最終調(diào)控細(xì)胞生長的早期精細(xì)分子機(jī)制。本論文依據(jù)此研究思路開展了一系列的生化與遺傳學(xué)實(shí)驗(yàn),論文的具體研究結(jié)果如下:(1)利用質(zhì)譜,酵母雙雜交(Y2H),雙熒光互補(bǔ)(BiFC),GST-pull down及Co-IP等多種分析蛋白相互作用的技術(shù)手段證明了 一個(gè)擬南芥的胞質(zhì)類受體激酶(RLCK-RIPK)能夠與FER受體蛋白激酶特異性的相互作用,且相互作用的強(qiáng)弱依賴于彼此的激酶活性。(2)遺傳學(xué)表型分析表明RIPK突變后表現(xiàn)出與FER突變類似的遺傳學(xué)表型,如根毛變短,根基酸化速率增強(qiáng),植株形態(tài)及葉片變小,對生長素NAA不敏感而對ABA敏感等。表型回復(fù)實(shí)驗(yàn)表明將RIPK過表達(dá)入FER突變體fer-4植株中能夠部分恢復(fù)FER的功能,證明FER與RIPK存在遺傳上的上下游關(guān)系。(3)利用大腸桿菌原核表達(dá)系統(tǒng)高效表達(dá)了融合GST標(biāo)簽的RIPK重組蛋白GST-RIPK,利用純化的GST-RIPK重組蛋白作為抗原免疫小白鼠制備了特異性較好的RIPK多克隆抗體。以野生型Col.0和ripk突變體為材料,利用Western blot技術(shù)結(jié)合堿性磷酸酶(Calf intestinal alkaline phosphatase;CIP)處理,證明了胞質(zhì)類受體激酶RIPK在擬南芥體內(nèi)存在兩種形式,分子量介于70 kDa和55 kDa之間。一種為非磷酸化的形式,我們命名為RIPK;另一種為磷酸化的形式,我們命名為P-RIPK。(4)通過分析野生型Co1.0與ripk突變體中FER的磷酸化水平變化及野生型Co1.0與fer-4突變體中RIPK的磷酸化水平變化并結(jié)合FER激酶和RIPK激酶相互磷酸化的體外實(shí)驗(yàn)證明了 FER與RIPK的相互作用依賴促進(jìn)彼此的磷酸化水平變化。(5)分析了 RIPK磷酸化水平的變化對RALF1小肽時(shí)間及濃度梯度的依賴性,結(jié)合RIPK突變體對RALF1小肽的響應(yīng)情況證明了 RIPK直接響應(yīng)RALF1小肽,且磷酸化水平受到RALF1小肽的調(diào)控。RIPK突變后降低了對RALF1小肽的敏感程度。(6)通過添加外源RALF1小肽誘導(dǎo)與否,利用雙熒光互補(bǔ)(BiFC)及Co-IP等技術(shù)進(jìn)一步證明了 FER與RIPK在細(xì)胞膜上的相互作用受RALF1小肽增強(qiáng)。此外我們利用酵母雙雜交技術(shù)在單子葉模式植物水稻中也證明了 FER的同源蛋白OsFLR2與RIPK的同源蛋白OsRIPK-A存在相互作用。生物信息學(xué)及Q-PCR分析進(jìn)一步證明了 RALF1,FER與RIPK在植物界比較保守且存在表達(dá)模式上的重疊,因此RALF1-FER-RIPK可能代表了植物界一種普遍的RALF1小肽信號(hào)傳遞模式。綜上所述,本研究結(jié)果將極大豐富對RALF1-FER信號(hào)網(wǎng)絡(luò)調(diào)節(jié)細(xì)胞伸長分子機(jī)制的理解。
[Abstract]:The growth and development of plant cells are regulated by a variety of signal factors, and the revealing and elucidation of molecular mechanism helps to deepen the understanding of the field of cell signal transduction, and will greatly overcome some urgent technical bottlenecks that restrict the development of the application fields of agriculture and forestry in China. Rapid alkalization factor (RALF), which is a conserved polypeptide hormone in the plant community, is named because it can rapidly inhibit the activity of PM-H + ATPase, which can cause cell wall alkalization and inhibit cell elongation. Recent studies have shown that the receptor of protein kinase FERONIA (FER) in Arabidopsis can be directly combined with RALF1 as a receptor of the RALF1 polypeptide signal to sense the RALF1 signal stimulation and transfer the signal to the cell, finally controlling the hydrogen pump activity (PM-H + ATPase, e.g. AHA2) of the plasma membrane to control the cell elongation, However, the early fine molecular mechanism of the RALF1 ligand signal is not very clear. For example, when the protein kinase FER receptor accepts the RALF1 signal stimulation, how to transfer the RALF1 signal to the cell? Is there a number of downstream members such as cytoplasmic receptor kinases (RLCK) involved in the signaling pathway? What is the way downstream members in these cells participate in the signal path if present? Therefore, in-depth study of downstream molecules capable of interacting directly with protein kinase FER, such as cytoplasmic receptor kinase RLCK, and elucidate the molecular mechanism of its signaling will help us to reveal receptor protein kinase FER acceptance, Early fine molecular mechanism that delivers the RALF1 small peptide signal and ultimately regulates the cell growth. Based on this study, a series of biochemical and genetic experiments were carried out. The specific results of this paper are as follows: (1) using mass spectrum, yeast two-hybrid (B1H), double fluorescence complementary (BiFC), GST-pull down and Co-IP have been used to demonstrate the interaction of RCK-RIPk with the protein kinase of FER receptor, and the interaction strength is dependent on the kinase activity of each other. (2) Genetic phenotype analysis indicated that the mutation of RIPK showed a similar genetic phenotype as that of FER mutation, such as shorter root hair, enhanced root acidification rate, reduced plant morphology and leaf size, insensitive to IAA NAA, sensitive to ABA and so on. Phenotypic response experiments show that RIPK has been expressed in the FER mutant fer-4 plant, which can partially recover FER function, which proves that the relationship between FER and RIPK exists in the upstream and downstream. (3) using the prokaryotic expression system of E. coli to efficiently express the RIPK recombinant protein GST-RIIPK of the fusion GST tag, and using the purified GST-RIPK recombinant protein as an antigen immune mouse to prepare a specific RIPK polyclonal antibody. Using the wild-type Col. 0 and ripk mutants as materials, Western blot techniques were used to bind alkaline phosphatase (CIP) to prove that there are two types of cytoplasmic receptor kinase RIPK in Arabidopsis, with a molecular weight of between 70 kDa and 55 kDa. One is in the form of non-phosphorylated, we are named RIPK; the other is in phosphorylated form, and we are named P-RIPK. (4) By analyzing the changes of the phosphorylation levels of FER in wild-type Co1. 0 and pk mutants and the phosphorylation level of RIPK in wild-type Co1. 0 and fer-4 mutants, the interaction between FER and RIPK in vitro demonstrated that the interaction between FER and RIPK is dependent on promoting the phosphorylation level of each other. Parameterization. (5) The dependence of the change of RIPK phosphorylation level on the time and concentration gradient of RALF1 was analyzed. The response of RIPK mutant to RALF1 was proved to be a direct response to RALF1 small peptide, and the phosphorylation level was regulated by RALF1 small peptide. and the sensitivity of the RALF1 small peptide is reduced after the RIPK mutation. (6) By adding exogenous RALF1 small peptide to induce or not, the interaction of FER and RIPK on cell membrane was enhanced by double fluorescence complementation (BiFC) and Co-IP technique. In addition, we use yeast two-hybrid technique to prove that the homologous protein OsFLR2 of FER interacts with the homologous protein OsRIPK-A of RIIPK in monocotyledon mode plant rice. Bioinformatics and Q-PCR analysis further demonstrate that RALF1, FER and RIPK are relatively conservative in the plant community and there is an overlap in the expression pattern, so RALF1-FER-RIPK may represent a universal RALF1 small peptide signaling pattern in the plant community. In conclusion, the results of this study will enrich the understanding of RALF1-FER signal network to regulate the molecular mechanism of cell elongation.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q946
本文編號(hào)：2276807
[Abstract]:The growth and development of plant cells are regulated by a variety of signal factors, and the revealing and elucidation of molecular mechanism helps to deepen the understanding of the field of cell signal transduction, and will greatly overcome some urgent technical bottlenecks that restrict the development of the application fields of agriculture and forestry in China. Rapid alkalization factor (RALF), which is a conserved polypeptide hormone in the plant community, is named because it can rapidly inhibit the activity of PM-H + ATPase, which can cause cell wall alkalization and inhibit cell elongation. Recent studies have shown that the receptor of protein kinase FERONIA (FER) in Arabidopsis can be directly combined with RALF1 as a receptor of the RALF1 polypeptide signal to sense the RALF1 signal stimulation and transfer the signal to the cell, finally controlling the hydrogen pump activity (PM-H + ATPase, e.g. AHA2) of the plasma membrane to control the cell elongation, However, the early fine molecular mechanism of the RALF1 ligand signal is not very clear. For example, when the protein kinase FER receptor accepts the RALF1 signal stimulation, how to transfer the RALF1 signal to the cell? Is there a number of downstream members such as cytoplasmic receptor kinases (RLCK) involved in the signaling pathway? What is the way downstream members in these cells participate in the signal path if present? Therefore, in-depth study of downstream molecules capable of interacting directly with protein kinase FER, such as cytoplasmic receptor kinase RLCK, and elucidate the molecular mechanism of its signaling will help us to reveal receptor protein kinase FER acceptance, Early fine molecular mechanism that delivers the RALF1 small peptide signal and ultimately regulates the cell growth. Based on this study, a series of biochemical and genetic experiments were carried out. The specific results of this paper are as follows: (1) using mass spectrum, yeast two-hybrid (B1H), double fluorescence complementary (BiFC), GST-pull down and Co-IP have been used to demonstrate the interaction of RCK-RIPk with the protein kinase of FER receptor, and the interaction strength is dependent on the kinase activity of each other. (2) Genetic phenotype analysis indicated that the mutation of RIPK showed a similar genetic phenotype as that of FER mutation, such as shorter root hair, enhanced root acidification rate, reduced plant morphology and leaf size, insensitive to IAA NAA, sensitive to ABA and so on. Phenotypic response experiments show that RIPK has been expressed in the FER mutant fer-4 plant, which can partially recover FER function, which proves that the relationship between FER and RIPK exists in the upstream and downstream. (3) using the prokaryotic expression system of E. coli to efficiently express the RIPK recombinant protein GST-RIIPK of the fusion GST tag, and using the purified GST-RIPK recombinant protein as an antigen immune mouse to prepare a specific RIPK polyclonal antibody. Using the wild-type Col. 0 and ripk mutants as materials, Western blot techniques were used to bind alkaline phosphatase (CIP) to prove that there are two types of cytoplasmic receptor kinase RIPK in Arabidopsis, with a molecular weight of between 70 kDa and 55 kDa. One is in the form of non-phosphorylated, we are named RIPK; the other is in phosphorylated form, and we are named P-RIPK. (4) By analyzing the changes of the phosphorylation levels of FER in wild-type Co1. 0 and pk mutants and the phosphorylation level of RIPK in wild-type Co1. 0 and fer-4 mutants, the interaction between FER and RIPK in vitro demonstrated that the interaction between FER and RIPK is dependent on promoting the phosphorylation level of each other. Parameterization. (5) The dependence of the change of RIPK phosphorylation level on the time and concentration gradient of RALF1 was analyzed. The response of RIPK mutant to RALF1 was proved to be a direct response to RALF1 small peptide, and the phosphorylation level was regulated by RALF1 small peptide. and the sensitivity of the RALF1 small peptide is reduced after the RIPK mutation. (6) By adding exogenous RALF1 small peptide to induce or not, the interaction of FER and RIPK on cell membrane was enhanced by double fluorescence complementation (BiFC) and Co-IP technique. In addition, we use yeast two-hybrid technique to prove that the homologous protein OsFLR2 of FER interacts with the homologous protein OsRIPK-A of RIIPK in monocotyledon mode plant rice. Bioinformatics and Q-PCR analysis further demonstrate that RALF1, FER and RIPK are relatively conservative in the plant community and there is an overlap in the expression pattern, so RALF1-FER-RIPK may represent a universal RALF1 small peptide signaling pattern in the plant community. In conclusion, the results of this study will enrich the understanding of RALF1-FER signal network to regulate the molecular mechanism of cell elongation.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q946
【參考文獻(xiàn)】
相關(guān)期刊論文 前7條
1 何含杰;張黨權(quán);唐麗;楊丹;;植物RLCK的生物學(xué)功能與信號(hào)途徑研究進(jìn)展[J];植物生理學(xué)報(bào);2014年07期
2 Chad E.Niederhuth;Sung Ki Cho;Kati Seitz;John C·Walker;;Letting Go is Never Easy:Abscission and Receptor- Like Protein Kinases[J];Journal of Integrative Plant Biology;2013年12期
3 韓永峰;楊倩;張勝偉;孫大業(yè);孫穎;;植物類受體激酶CrRLK1-L亞家族及其生物學(xué)功能[J];生物化學(xué)與生物物理進(jìn)展;2011年10期
4 ;Rice Blast Resistance of Transgenic Rice Plants with Pi-d2 Gene[J];Rice Science;2010年03期
5 陳德西;陳學(xué)偉;雷財(cái)林;馬炳田;王玉平;李仕貴;;轉(zhuǎn)Pi-d2基因水稻對稻瘟病的抗性分析[J];中國水稻科學(xué);2010年01期
6 白輝;李莉云;劉國振;;水稻抗白葉枯病基因Xa21的研究進(jìn)展[J];遺傳;2006年06期
7 張蕾,呂應(yīng)堂;植物受體蛋白激酶的研究進(jìn)展[J];生命科學(xué);2002年02期
，本文編號(hào)：2276807
本文鏈接：http://sikaile.net/shoufeilunwen/jckxbs/2276807.html
最近更新
教材專著
