本文選題：擬南芥 + 環(huán)化核苷酸門控通道2��； 參考：《內(nèi)蒙古大學(xué)》2016年博士論文

【摘要】：鈣(Calcium,Ca)是植物生長發(fā)育的大量元素,也是重要的第二信使。本實(shí)驗(yàn)以模式植物擬南芥(Arabidopsis thaliana, At)為實(shí)驗(yàn)材料,研究Ca2+分布和Ca2+信號相關(guān)的兩方面內(nèi)容。第一部分研究內(nèi)容是：在高鈣環(huán)境下,缺失環(huán)核苷酸門控離子通道2(Cyclic nucleotide-Gated Channel 2, CNGC2)基因cngc2突變體的葉片質(zhì)外體Ca2+積累對其生理功能的影響。第二部分研究內(nèi)容是：擬南芥短肽受體2(AtPeptide Receptor2, AtPEPR2)介導(dǎo)擬南芥短肽(AtPeptide 1, AtPep1)誘導(dǎo)的根中細(xì)胞質(zhì)Ca2+升高,進(jìn)而抑制谷氨酰胺外排基因(Glutamine Dumper, GDU)表達(dá)。(1)Ca是所有細(xì)胞生長發(fā)育的必需礦質(zhì)元素,對于植物細(xì)胞而言,植物根細(xì)胞吸收Ca2十,并通過木質(zhì)部向上運(yùn)輸?shù)饺~片中,進(jìn)而在葉片中分布,對其分布機(jī)理的研究甚少。我們對缺失CNGC2基因的cngc2突變體進(jìn)行研究,發(fā)現(xiàn)在0.1mM Ca2+濃度下,cngc2與野生型植物(Columbia ecotype, Col-0)長勢一樣。在10 mM Ca2+濃度下,當(dāng)cngc2從根系吸收更多的Ca2+后,cngc2葉片細(xì)胞外空間Ca2+外溢,而后導(dǎo)致活性氧積累,成熟葉片邊緣黃化,出現(xiàn)死亡斑點(diǎn)和生長受抑制的現(xiàn)象,并且死亡斑點(diǎn)主要分布在小葉脈周圍區(qū)域。通過構(gòu)建啟動子連接p-葡萄糖苷酶報告基因(Glucuronidase,GUS)進(jìn)行組織特異性分析,發(fā)現(xiàn)擬南芥CNGC2基因主要表達(dá)在葉片小葉脈周圍區(qū)域。通過原子吸收方法測定總Ca含量,發(fā)現(xiàn)cngc2突變體比Col-0野生型葉片總Ca含量低,但細(xì)胞壁中與果膠結(jié)合的Ca含量高于Col-0,這也說明cngc2更多的Ca2+積累在質(zhì)外體。當(dāng)前的報道表明cngc2突變體在非毒力病原菌侵染下沒有超敏反應(yīng)(Hypersensitive Response,HR),我們使用OD 600=0.02的avr DC3000 RPM1+菌液侵染生長在不同Ca2+濃度的水培液中的植物,發(fā)現(xiàn)在0.1 mM Ca2+濃度下cngc2有HR反應(yīng)；10 mM Ca2+濃度處理3天,cngc2沒有HR反應(yīng),表明cngc2沒有HR反應(yīng)是由于Ca2+外溢積累在質(zhì)外體導(dǎo)致的。Cg2+外溢還導(dǎo)致了cngc2暗周期結(jié)束后淀粉的積累,說明Ca2+外溢影響了光合作用暗周期淀粉的降解。(2)擬南芥新型短肽受體1(AtPeptide Receptorl, AtPEPR1)和AtPEPR2是富集亮氨酸受體蛋白激酶家族的成員,能夠結(jié)合一組由短肽前體(AtPROPEP)基因編碼的內(nèi)源肽AtPep So前人的研究發(fā)現(xiàn)AtPEPR2在AtPep1介導(dǎo)的擬南芥葉片初級免疫反應(yīng)中扮演著重要角色。在本研究中,我們發(fā)現(xiàn)缺失AtPEPR基因的缺失型突變體atpeprl和atpepr2有短根表型,AtPEPR1和AtPEPR2部分介導(dǎo)了AtPep1抑制根生長的表型,且在此過程中AtPEPR2的作用強(qiáng)于AtPEPR1. AtPep1引起的細(xì)胞質(zhì)Ca2+濃度的升高部分由AtPEPR1和AtPEPR2介導(dǎo),AtPEPR2在介導(dǎo)Ca2+濃度升高的過程起主要作用。為了研究AtPEPR2參與的受AtPep1誘導(dǎo)而轉(zhuǎn)錄的基因,我們對Col-0和atpepr2進(jìn)行含有AtPep1處理一個小時和不含有AtPep1處理的根轉(zhuǎn)錄組表達(dá)譜分析,結(jié)果顯示AtPep1誘導(dǎo)表達(dá)的基因其中75%是由AtPEPR2介導(dǎo)的,2個顯著誘導(dǎo)的基因部分依賴于細(xì)胞外Ca2+的升高。在篩選基因過程中,谷氨酰胺外排基因引起了我們的重視,擬南芥基因組編碼7個AtGD Us,該基因編碼氨基酸外排轉(zhuǎn)運(yùn)體。AtPepl完全依賴AtPEPR2調(diào)控的下調(diào)基因程度最大的10個基因中,AtGDU2,3,5占其中3個。通過AtGDU3 GUS活性分析,AtPep1處理強(qiáng)烈抑制了根中AtGDU3的活性,螯合細(xì)胞外Ca2+能夠緩解AtPep1對AtGDU3的抑制作用。過量表達(dá)AtGDU3擬南芥具有短根表型,但對AtPep1抑制短根現(xiàn)象不敏感。總體來說,本研究揭示了AtPEPR2在AtPep1根延伸以及根信號轉(zhuǎn)導(dǎo)作用中扮演著重要角色。綜上所述,本實(shí)驗(yàn)一方面研究植物葉片對Ca2十的分布,為Ca2十分布與Ca2+吸收的關(guān)系研究打下基礎(chǔ)；另一方面,AtPep1是從擬南芥體內(nèi)提取出來的內(nèi)源短肽,外源加入納摩爾級濃度的AtPep1通過AtPEPR2抑制擬南芥根生長,說明AtPep1可能是一種新型激素,參與植物生長和發(fā)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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

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本文編號：2061148