擬南芥中miRNA通路新因子篩選系統(tǒng)的驗證與應(yīng)用
發(fā)布時間：2018-12-30 16:16
【摘要】：microRNAs(miRNAs)是一類長度為20-24 nt的非編碼小RNA,在植物生長發(fā)育過程中具有重要作用。miRNA基因(MIR)在RNA聚合酶II(Pol II)的作用下轉(zhuǎn)錄生成莖環(huán)結(jié)構(gòu)的primary mi RNA(pri-miRNA),并加上5’帽子結(jié)構(gòu)和3’多聚腺苷尾巴,隨后pri-miRNA由DICER-LIKE1(DCL1)切割首先形成miRNA前體(pre-miRNA),進(jìn)一步形成miRNA/miRNA*二聚體。該二聚體的3’端被HUA ENHANCER1(HEN1)甲基化,miRNA鏈被選擇性的載入ARGONAUTE1(AGO1)蛋白,從而形成miRNA介導(dǎo)的沉默復(fù)合體(RNA induce silencing complex,RISC),指導(dǎo)該復(fù)合體對靶基因的mRNA進(jìn)行切割或抑制其翻譯,調(diào)節(jié)靶基因的表達(dá),而另外一條鏈miRNA*通常會被降解掉。成熟的miRNA除了在合成部位發(fā)揮功能,還能夠從合成部位運輸?shù)桨l(fā)揮作用的器官來發(fā)揮功能。miRNA行使完功能后會被HEN1 SUPPRESSOR1(HESO1)或者SMALL RNA DEGRADING NUCLEASE 1(SDN1)降解。為了進(jìn)一步篩選和鑒定參與植物miRNA合成、降解和運輸?shù)韧返囊蜃?我們利用擬南芥轉(zhuǎn)基因株系(SUC2:amiR-SUL)作為篩選系統(tǒng),該轉(zhuǎn)基因株系利用韌皮部特異表達(dá)的啟動子SUC2驅(qū)動人工miRNA,特異降解影響葉綠素合成的靶基因CH42(SUL的同源基因),導(dǎo)致植物葉片葉脈處呈現(xiàn)葉斑的表型。通過對該株系進(jìn)行甲基磺酸乙酯(EMS)誘變,并篩選葉脈處葉斑擴大或縮小的突變體,我們獲得突變體SUP-E45,該突變體葉片葉脈處葉斑明顯縮小。實驗結(jié)果顯示突變的基因所編碼的蛋白質(zhì)是參與miRNA途徑的AGO1蛋白。AGO1蛋白是miRNA通路中至關(guān)重要的蛋白,成熟的miRNA與AGO1結(jié)合形成RISC沉默復(fù)合體從而對靶基因的表達(dá)進(jìn)行負(fù)調(diào)控。篩選獲得的另一個突變體ab58中,葉片葉脈處葉斑也是明顯縮小,而且植株矮小,葉片卷曲,對溫度敏感。實驗結(jié)果顯示ab58突變體中發(fā)揮作用的突變基因是BON1。BON1屬于copine基因家族成員。bon1突變體中第三、四、五對真葉中內(nèi)源miRNA的表達(dá)量相對于SUC2:amiR-SUL植株降低,說明BON1可能是擬南芥miRNA通路中的新因子。后續(xù)研究將進(jìn)一步探索BON1基因在擬南芥miRNA通路中的功能和作用機制。在本研究中不僅篩選到已知的AGO1蛋白,而且也篩選到了新的BON1蛋白,不僅驗證了該篩選體系的可行性,而且通過該篩選體系還篩選出了可能參與miRNA通路的新因子,為后續(xù)的研究奠定一定的基礎(chǔ)。
[Abstract]:MicroRNAs (miRNAs) is a class of non-coding small RNA, with a length of 20-24 nt, which plays an important role in plant growth and development. MiRNA gene (MIR) is transcribed to form stem ring structure primary mi RNA (pri-miRNA under the action of RNA polymerase II (Pol II). Then pri-miRNA was cut by DICER-LIKE1 (DCL1) to form miRNA precursor (pre-miRNA) and then formed miRNA/miRNA* dimer. The 3 'end of the dimer is methylated by HUA ENHANCER1 (HEN1), and the miRNA chain is selectively loaded into the ARGONAUTE1 (AGO1) protein to form a miRNA mediated silencing complex (RNA induce silencing complex,RISC). It directs the complex to cleavage or inhibit the translation of the mRNA of the target gene, and regulates the expression of the target gene, while the other strand of miRNA* is usually degraded. Mature miRNA not only functions in synthetic sites, but also functions from synthetic sites to functioning organs. MiRNA is degraded by HEN1 SUPPRESSOR1 (HESO1) or SMALL RNA DEGRADING NUCLEASE 1 (SDN1) when it functions. In order to further screen and identify the factors involved in plant miRNA synthesis, degradation and transport pathways, we used Arabidopsis transgenic strain (SUC2:amiR-SUL) as a screening system. The target gene CH42 (homologous gene of SUL), which is a target gene for chlorophyll synthesis, is driven by phloem specific promoter SUC2, which is specifically expressed in phloem, resulting in the phenotype of leaf spot at the leaf vein of the plant. By mutagenesis with ethyl methanesulfonate (EMS) and screening mutants with enlarged or smaller leaf spots at the vein, we obtained the mutants SUP-E45, the mutants of which were obviously reduced at the leaf veins. The results show that the mutant gene encodes AGO1 protein which is involved in the miRNA pathway. AGO1 protein is a crucial protein in the miRNA pathway. Mature miRNA binds with AGO1 to form a RISC silencing complex which negatively regulates the expression of the target gene. In another mutant, ab58, the leaf spot at the leaf vein was also significantly reduced, and the plant was short, curly and sensitive to temperature. The results showed that the mutant gene of ab58 was a member of copine gene family of BON1.BON1. The expression of endogenous miRNA in the third, fourth and fifth pairs of bon1 mutants was lower than that in SUC2:amiR-SUL plants. BON1 may be a new factor in Arabidopsis miRNA pathway. Further studies will explore the function and mechanism of BON1 gene in Arabidopsis miRNA pathway. In this study, not only the known AGO1 protein was screened, but also the new BON1 protein was screened, which not only verified the feasibility of the screening system, but also screened out new factors that might be involved in the miRNA pathway. To lay a foundation for further research.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2
[Abstract]:MicroRNAs (miRNAs) is a class of non-coding small RNA, with a length of 20-24 nt, which plays an important role in plant growth and development. MiRNA gene (MIR) is transcribed to form stem ring structure primary mi RNA (pri-miRNA under the action of RNA polymerase II (Pol II). Then pri-miRNA was cut by DICER-LIKE1 (DCL1) to form miRNA precursor (pre-miRNA) and then formed miRNA/miRNA* dimer. The 3 'end of the dimer is methylated by HUA ENHANCER1 (HEN1), and the miRNA chain is selectively loaded into the ARGONAUTE1 (AGO1) protein to form a miRNA mediated silencing complex (RNA induce silencing complex,RISC). It directs the complex to cleavage or inhibit the translation of the mRNA of the target gene, and regulates the expression of the target gene, while the other strand of miRNA* is usually degraded. Mature miRNA not only functions in synthetic sites, but also functions from synthetic sites to functioning organs. MiRNA is degraded by HEN1 SUPPRESSOR1 (HESO1) or SMALL RNA DEGRADING NUCLEASE 1 (SDN1) when it functions. In order to further screen and identify the factors involved in plant miRNA synthesis, degradation and transport pathways, we used Arabidopsis transgenic strain (SUC2:amiR-SUL) as a screening system. The target gene CH42 (homologous gene of SUL), which is a target gene for chlorophyll synthesis, is driven by phloem specific promoter SUC2, which is specifically expressed in phloem, resulting in the phenotype of leaf spot at the leaf vein of the plant. By mutagenesis with ethyl methanesulfonate (EMS) and screening mutants with enlarged or smaller leaf spots at the vein, we obtained the mutants SUP-E45, the mutants of which were obviously reduced at the leaf veins. The results show that the mutant gene encodes AGO1 protein which is involved in the miRNA pathway. AGO1 protein is a crucial protein in the miRNA pathway. Mature miRNA binds with AGO1 to form a RISC silencing complex which negatively regulates the expression of the target gene. In another mutant, ab58, the leaf spot at the leaf vein was also significantly reduced, and the plant was short, curly and sensitive to temperature. The results showed that the mutant gene of ab58 was a member of copine gene family of BON1.BON1. The expression of endogenous miRNA in the third, fourth and fifth pairs of bon1 mutants was lower than that in SUC2:amiR-SUL plants. BON1 may be a new factor in Arabidopsis miRNA pathway. Further studies will explore the function and mechanism of BON1 gene in Arabidopsis miRNA pathway. In this study, not only the known AGO1 protein was screened, but also the new BON1 protein was screened, which not only verified the feasibility of the screening system, but also screened out new factors that might be involved in the miRNA pathway. To lay a foundation for further research.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2
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