类受体激酶CRPK1调控拟南芥低温应答的分子机制研究

发布时间：2018-06-27 11:32

本文选题：类受体激酶CRPK1 + 磷酸化　；参考：《中国农业大学》2017年博士论文

【摘要】：低温作为一种重要的环境因子,对植物的生长、发育、代谢和地理分布有重要的影响。为了适应环境,植物进化出了复杂的应对机制来抵抗低温胁迫。首先,植物遭受低温胁迫时,细胞膜可能是作为初级信号感受器,细胞膜流动性变化使植物率先感受到低温信号。但是感受信号的精确机制以及信号传递机制还不清楚。其次,在低温信号转导途径研究中,目前主要是围绕转录因子CBF/DREB1(C-repeat-bindingfactor/drought responsive element binding protein 1)展开的。低温迅速诱导CBF基因大量表达,高量的CBF进一步激活其下游冷响应基因COR(cold responsive)的大量表达,从而使植物抵抗低温胁迫。但是研究多集中在CBBF基因的转录水平,发现了一系列调控CBF基因的转录因子,而在CBF蛋白水平调控机制研究方面则是空白。此外,由于CBF基因过量表达抑制植物生长,植物如何精细地调控低温应答和生长发育的平衡也不清楚。本研究通过冻处理实验筛选类受体激酶突变体,克隆了一个新的类受体激酶CRPK1(cold-responsive protein kinase 1),其缺失突变体具有明显的抗冻表型,突变体中CBFs受冷诱导水平微弱地高于野生型,而冷响应基因受冷诱导水平显著高于野生型。并且,野生型的CRPK1基因能够回复突变体的抗冻表型,CBF及冷响应基因表达水平也回复到野生型水平。证明CRPK1参与了低温调控,并且调控过程依赖于CBF信号途径。通过显微镜观察CRPK1-GFP定位和组分分离实验证明CRPK1定位在细胞膜上。另一方面,体内体外的磷酸化实验证明CRPK1具有激酶活性,并且低温处理能够激活其激酶活性。将CRPK1关键的保守位点K69突变后,激酶活性完全消失,并且将突变型的CRPK1K69E转入crpk1-1突变体中,无法回复突变体的抗冻表型,证明CRPK1激酶活性对于其执行低温应答功能至关重要。进一步通过酵母双杂交实验,筛选到CRPK1互作蛋白14-3-3蛋白。体内体外的磷酸化实验均证明,在低温胁迫下14-3-3λ能够被CRPK1磷酸化,并且证明S70、S112、S193和T214是14-3-3λ上主要的磷酸化位点,当这四个位点突变成丙氨酸后,CRPK1磷酸化14-3-3λ的信号几乎丧失。并且14-3-3kλ双突变体同样具有抗冻表型,野生型的14-3-3λ能够回复双突变体的表型,而突变型的14-3-3λ4A不能回复双突变体的表型。证明14-3-3s也参与了低温调控,并且调控过程依赖于CRPK1对其磷酸化。一方面,研究发现低温处理导致细胞质中14-3-3λ被CRPK1磷酸化,被磷酸化的14-3-3λ进入细胞核中。另一方面,植物体内体外的实验证明14-3-3λ与CBF蛋白互作,并且互作后会促进CBF1和CBF3蛋白的降解。进一步研究发现,14-3-3s入核过程依赖于CRPK1对其磷酸化,并且突变成非磷酸化形式的14-3-34A不能促进CBF1和CBF3蛋白降解。遗传实验进一步证明,CRPK1和14-3-3s参与低温调控依赖于CBF途径。CRPK1和14-3-3s精细调控植物体内CBF的蛋白水平,影响CBF下游冷响应基因的表达。综合上述结果,新的细胞膜类受体激酶CRPK1在低温信号转导通路中是一个负调控因子。低温胁迫激活CRPK1激酶活性,磷酸化14-3-3蛋白,细胞质中被磷酸化的14-3-3s进入细胞核中,促进细胞核中的CBF1/CBF3蛋白的降解,从而影响CBF下游冷响应基因的表达,调控植物对低温胁迫的响应。CRPK1介导的负调控途径作为一个"刹车"精细地调控植物冷响应的强度,避免过度冷响应造成的生长抑制,从而平衡植物生长发育和抵抗逆境间的关系。上述研究不仅揭示了低温信号从细胞膜传递至细胞核中的负调控机制,也阐释了 CBF蛋白翻译后修饰的调控机理,为阐明植物低温信号转导途径提供了重要的信息。
[Abstract]:As an important environmental factor, low temperature has an important influence on the growth, development, metabolism and geographical distribution of plants. In order to adapt to the environment, plants have evolved a complex coping mechanism to resist low temperature stress. First, when plants suffer from low temperature stress, the cell membrane may be used as a primary signal receptor and the cell membrane fluidity changes It is the first to feel the low temperature signal. But the precise mechanism of the sensory signal and the mechanism of signal transmission are not clear. Secondly, in the study of the low temperature signal transduction pathway, the current mainly focuses on the transcription factor CBF/DREB1 (C-repeat-bindingfactor/drought responsive element binding protein 1). The low temperature rapidly induces a large number of CBF genes. The high amount of CBF further activates the heavy expression of the downstream cold response gene COR (cold responsive), which makes plants resist low temperature stress. However, the study focused on the transcriptional level of the CBBF gene, and found a series of transcriptional factors regulating the CBF gene, while the study on the regulation mechanism of the CBF protein level is blank. In addition, C is due to C. BF gene overexpression inhibits plant growth, and it is not clear how plants regulate the balance of low temperature response and growth and development. In this study, a new receptor kinase CRPK1 (cold-responsive protein kinase 1) was cloned through the freeze treatment experiments, and the missing mutant had obvious antifreeze phenotype. The cold induced level of CBFs in the mutant was slightly higher than that in the wild type, while the cold response gene was significantly higher than that of the wild type. And the wild type CRPK1 gene could revert to the antifreeze phenotype of the mutant, and the expression level of CBF and the cold response gene reverted to the wild type. It was confirmed that CRPK1 was involved in low temperature regulation, and the regulation process was dependent on the control process. Depending on the CBF signal pathway, the CRPK1-GFP localization and component separation experiments show that CRPK1 is located on the cell membrane. On the other hand, the phosphorylation experiment in vitro and in vivo shows that CRPK1 has kinase activity, and the low temperature treatment can activate its kinase activity. The kinase activity completely disappeared after the mutation of the key conserving site K69 of CRPK1. Moreover, the mutant CRPK1K69E was transferred into the crpk1-1 mutant and could not recover the antifreeze phenotype of the mutant. It was proved that the activity of CRPK1 kinase was crucial to its low temperature response function. Further, the yeast two hybrid experiment was used to screen the CRPK1 interaction protein 14-3-3 protein. The phosphorylation experiment in the body outside the body proved that 14- was under low temperature stress. 3-3 lambda can be phosphorylated by CRPK1, and it is proved that S70, S112, S193 and T214 are the main phosphorylation sites on 14-3-3 lambda. When these four loci become alanine, the signal of CRPK1 phosphorylated 14-3-3 lambda is almost lost. And the 14-3-3k lambda double mutant has the same antifreeze phenotype, and the wild type 14-3-3 lambda can respond to the phenotypes of the double mutants, and the mutations are mutated. The type of 14-3-3 lambda 4A does not respond to the phenotype of the double mutants. It is proved that 14-3-3s is also involved in low temperature regulation, and the regulation process depends on the phosphorylation of CRPK1. On the one hand, the study found that low temperature treatment leads to CRPK1 phosphorylation in the cytoplasm and the phosphorylated 14-3-3 lambda into the cell nucleus. On the other hand, the experimental evidence in vitro and in vivo is found in the plant. The interaction between the 14-3-3 lambda and CBF protein and the interaction between CBF1 and CBF3 proteins will promote the degradation of CBF1 and CBF3 proteins. Further studies have found that the 14-3-3s nucleation process relies on the phosphorylation of CRPK1 to them, and that 14-3-34A in the form of a non phosphorylated form does not promote the degradation of CBF1 and CBF3 proteins. Genetic experiments show that CRPK1 and 14-3-3s are involved in the low temperature regulation dependence. The CBF pathway.CRPK1 and 14-3-3s regulate the protein level of CBF in the body of the plant and affect the expression of cold response genes in the downstream of CBF. The results show that the new cell membrane receptor kinase CRPK1 is a negative regulator in the low temperature signal transduction pathway. The activity of CRPK1 kinase, phosphorylated 14-3-3 protein, and phosphoric acid in the cytoplasm are activated by low temperature stress. The transformed 14-3-3s enters the nucleus and promotes the degradation of the CBF1/CBF3 protein in the nucleus, thus affecting the expression of the cold response genes in the downstream CBF and regulating the negative regulation pathway mediated by the response of the plant to low temperature stress as a "brake" to regulate the intensity of the cold response of the plant and avoid the growth inhibition caused by the excessive cold response. To balance the relationship between plant growth and resistance to stress, the above studies not only reveal the negative regulation mechanism of the transmission of low temperature signals from the cell membrane to the nucleus, but also explain the regulatory mechanism of the posttranslational modification of CBF protein, which provides important information for clarifying the pathway of plant low temperature signal transduction.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

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