CNGC2和PEPR2调节拟南芥细胞内外钙动态变化的分子机理及生理功能研究

发布时间：2018-06-24 10:02

  本文选题：拟南芥 + 环化核苷酸门控通道2　； 参考：《内蒙古大学》2016年博士论文

【摘要】：钙(Calcium,Ca)是植物生长发育的大量元素,也是重要的第二信使。本实验以模式植物拟南芥(Arabidopsis thaliana, At)为实验材料,研究Ca2+分布和Ca2+信号相关的两方面内容。第一部分研究内容是：在高钙环境下,缺失环核苷酸门控离子通道2(Cyclic nucleotide-Gated Channel 2, CNGC2)基因cngc2突变体的叶片质外体Ca2+积累对其生理功能的影响。第二部分研究内容是：拟南芥短肽受体2(AtPeptide Receptor2, AtPEPR2)介导拟南芥短肽(AtPeptide 1, AtPep1)诱导的根中细胞质Ca2+升高,进而抑制谷氨酰胺外排基因(Glutamine Dumper, GDU)表达。(1)Ca是所有细胞生长发育的必需矿质元素,对于植物细胞而言,植物根细胞吸收Ca2十,并通过木质部向上运输到叶片中,进而在叶片中分布,对其分布机理的研究甚少。我们对缺失CNGC2基因的cngc2突变体进行研究,发现在0.1mM Ca2+浓度下,cngc2与野生型植物(Columbia ecotype, Col-0)长势一样。在10 mM Ca2+浓度下,当cngc2从根系吸收更多的Ca2+后,cngc2叶片细胞外空间Ca2+外溢,而后导致活性氧积累,成熟叶片边缘黄化,出现死亡斑点和生长受抑制的现象,并且死亡斑点主要分布在小叶脉周围区域。通过构建启动子连接p-葡萄糖苷酶报告基因(Glucuronidase,GUS)进行组织特异性分析,发现拟南芥CNGC2基因主要表达在叶片小叶脉周围区域。通过原子吸收方法测定总Ca含量,发现cngc2突变体比Col-0野生型叶片总Ca含量低,但细胞壁中与果胶结合的Ca含量高于Col-0,这也说明cngc2更多的Ca2+积累在质外体。当前的报道表明cngc2突变体在非毒力病原菌侵染下没有超敏反应(Hypersensitive Response,HR),我们使用OD 600=0.02的avr DC3000 RPM1+菌液侵染生长在不同Ca2+浓度的水培液中的植物,发现在0.1 mM Ca2+浓度下cngc2有HR反应；10 mM Ca2+浓度处理3天,cngc2没有HR反应,表明cngc2没有HR反应是由于Ca2+外溢积累在质外体导致的。Cg2+外溢还导致了cngc2暗周期结束后淀粉的积累,说明Ca2+外溢影响了光合作用暗周期淀粉的降解。(2)拟南芥新型短肽受体1(AtPeptide Receptorl, AtPEPR1)和AtPEPR2是富集亮氨酸受体蛋白激酶家族的成员,能够结合一组由短肽前体(AtPROPEP)基因编码的内源肽AtPep So前人的研究发现AtPEPR2在AtPep1介导的拟南芥叶片初级免疫反应中扮演着重要角色。在本研究中,我们发现缺失AtPEPR基因的缺失型突变体atpeprl和atpepr2有短根表型,AtPEPR1和AtPEPR2部分介导了AtPep1抑制根生长的表型,且在此过程中AtPEPR2的作用强于AtPEPR1. AtPep1引起的细胞质Ca2+浓度的升高部分由AtPEPR1和AtPEPR2介导,AtPEPR2在介导Ca2+浓度升高的过程起主要作用。为了研究AtPEPR2参与的受AtPep1诱导而转录的基因,我们对Col-0和atpepr2进行含有AtPep1处理一个小时和不含有AtPep1处理的根转录组表达谱分析,结果显示AtPep1诱导表达的基因其中75%是由AtPEPR2介导的,2个显著诱导的基因部分依赖于细胞外Ca2+的升高。在筛选基因过程中,谷氨酰胺外排基因引起了我们的重视,拟南芥基因组编码7个AtGD Us,该基因编码氨基酸外排转运体。AtPepl完全依赖AtPEPR2调控的下调基因程度最大的10个基因中,AtGDU2,3,5占其中3个。通过AtGDU3 GUS活性分析,AtPep1处理强烈抑制了根中AtGDU3的活性,螯合细胞外Ca2+能够缓解AtPep1对AtGDU3的抑制作用。过量表达AtGDU3拟南芥具有短根表型,但对AtPep1抑制短根现象不敏感。总体来说,本研究揭示了AtPEPR2在AtPep1根延伸以及根信号转导作用中扮演着重要角色。综上所述,本实验一方面研究植物叶片对Ca2十的分布,为Ca2十分布与Ca2+吸收的关系研究打下基础；另一方面,AtPep1是从拟南芥体内提取出来的内源短肽,外源加入纳摩尔级浓度的AtPep1通过AtPEPR2抑制拟南芥根生长,说明AtPep1可能是一种新型激素,参与植物生长和发育。
[Abstract]:Calcium (Ca) is a large element of plant growth and development, and is also an important second messenger. In this experiment, the experimental material of Arabidopsis thaliana (At) was used as experimental material to study the two aspects of the Ca2+ distribution and Ca2+ signal. The first part of this study was: the absence of cyclic nucleotide gated ion channel 2 (Cyc) under high calcium environment. The effect of LIC nucleotide-Gated Channel 2, CNGC2) gene cngc2 mutants on the physiological function of the leaf extracellular Ca2+. The second part of the study is that Arabidopsis short peptide receptor 2 (AtPeptide Receptor2, AtPEPR2) mediates the increase of cytoplasm Ca2+ induced by Arabidopsis short peptide (AtPeptide 1, AtPep1), and then inhibits glutamine The expression of Glutamine Dumper (GDU). (1) Ca is an essential mineral element for the growth and development of all cells. For plant cells, plant root cells absorb Ca2 ten, and are transported up to leaves through xylem and then distributed in leaves, and few studies on the distribution mechanism. We have a cngc2 mutant of the missing CNGC2 gene. Under the concentration of 0.1mM Ca2+, cngc2 was found to be the same as that of wild type plants (Columbia ecotype, Col-0). At the concentration of 10 mM Ca2+, when cngc2 absorbed more Ca2+ from the root system, the outer space Ca2+ spilt in the outer space of cngc2 leaves, and then the accumulation of active oxygen, the yellowing of the edges of the mature leaf slices, the occurrence of death spots and inhibition of growth. And the death spots were mainly distributed around the area around the small veins. Through the construction of the promoter linked p- glucosidase reporter gene (Glucuronidase, GUS) for tissue specific analysis, it was found that the Arabidopsis CNGC2 gene was mainly expressed in the area around the leaf vein. The total Ca content was measured by atomic absorption method, and the cngc2 mutant was found to be more than Col-0. The total Ca content of the wild type leaves is low, but the content of Ca in the cell wall with pectin is higher than that of Col-0, which also indicates that more Ca2+ of cngc2 is accumulated in the extracellular body. The current report shows that the cngc2 mutant has no hypersensitivity reaction (Hypersensitive Response, HR) under the infection of non virulence pathogens (Hypersensitive Response, HR), and we use OD 600=0.02 AVR contamination liquid infection. Plants growing in hydroponic fluid with different Ca2+ concentrations found cngc2 HR reaction at the concentration of 0.1 mM Ca2+; 10 mM Ca2+ concentration for 3 days and cngc2 without HR reaction, indicating that cngc2 no HR reaction was caused by the accumulation of Ca2+ spillover in the extrasomatic body and the accumulation of starch after the end of the dark cycle, indicating the overflow shadow. The degradation of dark periodic starch of photosynthesis. (2) the new short peptide receptor 1 (AtPeptide Receptorl, AtPEPR1) and AtPEPR2 of Arabidopsis are members of the rich leucine receptor protein kinase family, which can be combined with a group of predecessors of the endogenous peptide AtPep So encoded by the short peptide precursor (AtPROPEP) gene to find AtPEPR2 in AtPep1 mediated Arabidopsis leaf In this study, we found that the deletion mutant atpeprl and atpepr2 missing AtPEPR gene have short root phenotypes, and AtPEPR1 and AtPEPR2 partially mediate the phenotype of AtPep1 inhibiting root growth, and in this process, the role of AtPEPR2 is stronger than the increase of cytoplasmic Ca2+ concentration caused by AtPEPR1. AtPep1. The high part is mediated by AtPEPR1 and AtPEPR2, and AtPEPR2 plays a major role in the process of mediating Ca2+ concentration. In order to study the transcriptional genes involved in AtPep1 induced by AtPEPR2, we analyzed the expression profiles of Col-0 and atpepr2 in the root transcriptional group containing AtPep1 processing for one hour and without AtPep1. The results showed the AtPep1 induction table. Of which 75% of the genes are mediated by AtPEPR2, the 2 significant inducible genes are partly dependent on the increase of extracellular Ca2+. In the process of screening genes, the glutamine outer row gene has aroused our attention. The Arabidopsis genome encodes 7 AtGD Us, and the gene encoding the amino acid efflux transporter.AtPepl is completely dependent on the down regulation of AtPEPR2 regulation. Of the 10 genes with the largest gene level, AtGDU2,3,5 accounted for 3 of them. Through the analysis of AtGDU3 GUS activity, AtPep1 treatment strongly inhibited the activity of AtGDU3 in the root, and Ca2+ could alleviate the inhibitory effect of AtPep1 on AtGDU3. Excessive expression of AtGDU3 Arabidopsis has a short root phenotype, but it is not sensitive to AtPep1 for the inhibition of short roots. Generally speaking, it is not sensitive to AtPep1. This study revealed that AtPEPR2 plays an important role in AtPep1 root extension and root signal transduction. On the one hand, this experiment studies the distribution of plant leaves to Ca2 ten, which lays the foundation for the relationship between Ca2 ten distribution and Ca2+ absorption; on the other hand, AtPep1 is an endogenous short peptide extracted from the body of Arabidopsis thaliana. The AtPep1 concentration at the nanomolar level inhibited the growth of Arabidopsis thaliana roots by AtPEPR2, indicating that AtPep1 might be a new hormone involved in plant growth and development.
【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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