拟南芥类受体激酶RIPK与FERONIA共响应RALF信号的遗传与生化分析
发布时间：2018-10-17 13:28
【摘要】：植物细胞的生长发育受到多种信号因子的调控,其分子机制的揭示和阐明有助于深化对细胞信号转导领域的理解,也将极大克服制约我国农业、林业等应用领域发展的一些亟需解决的技术瓶颈。细胞快速碱化因子(Rapidalkalinizationfactor,RALF)作为一类植物界保守的多肽激素,因其能快速抑制质膜氢泵(PM-H+ATPase)活性导致细胞壁碱化从而抑制细胞伸长而得名。近来研究表明拟南芥中的蛋白激酶FERONIA(FER)作为RALF1多肽信号的受体可通过胞外域与RALF1直接结合来感受RALF1信号刺激并将该信号传递至细胞内,最终调控质膜的氢泵活性(PM-H+ATPase,e.g.AHA2)来控制细胞伸长,但响应RALF1配体信号的早期精细分子机制并不十分清楚。例如蛋白激酶FER受体接受RALF1信号刺激后,如何将RALF1信号传递至细胞内?是否存在一些下游成员,如胞质类受体激酶(RLCK)参与了该信号通路?如果存在,这些胞内的下游成员又以何种方式参与了该信号通路呢?因此深入研究能够与蛋白激酶FER直接相互作用的下游分子如胞质类受体激酶RLCK,并阐明其信号传导的分子机制将有助于我们揭示受体蛋白激酶FER接受、传递RALF1小肽信号并最终调控细胞生长的早期精细分子机制。本论文依据此研究思路开展了一系列的生化与遗传学实验,论文的具体研究结果如下:(1)利用质谱,酵母双杂交(Y2H),双荧光互补(BiFC),GST-pull down及Co-IP等多种分析蛋白相互作用的技术手段证明了 一个拟南芥的胞质类受体激酶(RLCK-RIPK)能够与FER受体蛋白激酶特异性的相互作用,且相互作用的强弱依赖于彼此的激酶活性。(2)遗传学表型分析表明RIPK突变后表现出与FER突变类似的遗传学表型,如根毛变短,根基酸化速率增强,植株形态及叶片变小,对生长素NAA不敏感而对ABA敏感等。表型回复实验表明将RIPK过表达入FER突变体fer-4植株中能够部分恢复FER的功能,证明FER与RIPK存在遗传上的上下游关系。(3)利用大肠杆菌原核表达系统高效表达了融合GST标签的RIPK重组蛋白GST-RIPK,利用纯化的GST-RIPK重组蛋白作为抗原免疫小白鼠制备了特异性较好的RIPK多克隆抗体。以野生型Col.0和ripk突变体为材料,利用Western blot技术结合碱性磷酸酶(Calf intestinal alkaline phosphatase;CIP)处理,证明了胞质类受体激酶RIPK在拟南芥体内存在两种形式,分子量介于70 kDa和55 kDa之间。一种为非磷酸化的形式,我们命名为RIPK;另一种为磷酸化的形式,我们命名为P-RIPK。(4)通过分析野生型Co1.0与ripk突变体中FER的磷酸化水平变化及野生型Co1.0与fer-4突变体中RIPK的磷酸化水平变化并结合FER激酶和RIPK激酶相互磷酸化的体外实验证明了 FER与RIPK的相互作用依赖促进彼此的磷酸化水平变化。(5)分析了 RIPK磷酸化水平的变化对RALF1小肽时间及浓度梯度的依赖性,结合RIPK突变体对RALF1小肽的响应情况证明了 RIPK直接响应RALF1小肽,且磷酸化水平受到RALF1小肽的调控。RIPK突变后降低了对RALF1小肽的敏感程度。(6)通过添加外源RALF1小肽诱导与否,利用双荧光互补(BiFC)及Co-IP等技术进一步证明了 FER与RIPK在细胞膜上的相互作用受RALF1小肽增强。此外我们利用酵母双杂交技术在单子叶模式植物水稻中也证明了 FER的同源蛋白OsFLR2与RIPK的同源蛋白OsRIPK-A存在相互作用。生物信息学及Q-PCR分析进一步证明了 RALF1,FER与RIPK在植物界比较保守且存在表达模式上的重叠,因此RALF1-FER-RIPK可能代表了植物界一种普遍的RALF1小肽信号传递模式。综上所述,本研究结果将极大丰富对RALF1-FER信号网络调节细胞伸长分子机制的理解。
[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.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946
本文编号：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.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946
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