拟南芥中miRNA通路新因子筛选系统的验证与应用
发布时间：2018-12-30 16:16
【摘要】：microRNAs(miRNAs)是一类长度为20-24 nt的非编码小RNA,在植物生长发育过程中具有重要作用。miRNA基因(MIR)在RNA聚合酶II(Pol II)的作用下转录生成茎环结构的primary mi RNA(pri-miRNA),并加上5’帽子结构和3’多聚腺苷尾巴,随后pri-miRNA由DICER-LIKE1(DCL1)切割首先形成miRNA前体(pre-miRNA),进一步形成miRNA/miRNA*二聚体。该二聚体的3’端被HUA ENHANCER1(HEN1)甲基化,miRNA链被选择性的载入ARGONAUTE1(AGO1)蛋白,从而形成miRNA介导的沉默复合体(RNA induce silencing complex,RISC),指导该复合体对靶基因的mRNA进行切割或抑制其翻译,调节靶基因的表达,而另外一条链miRNA*通常会被降解掉。成熟的miRNA除了在合成部位发挥功能,还能够从合成部位运输到发挥作用的器官来发挥功能。miRNA行使完功能后会被HEN1 SUPPRESSOR1(HESO1)或者SMALL RNA DEGRADING NUCLEASE 1(SDN1)降解。为了进一步筛选和鉴定参与植物miRNA合成、降解和运输等通路的因子,我们利用拟南芥转基因株系(SUC2:amiR-SUL)作为筛选系统,该转基因株系利用韧皮部特异表达的启动子SUC2驱动人工miRNA,特异降解影响叶绿素合成的靶基因CH42(SUL的同源基因),导致植物叶片叶脉处呈现叶斑的表型。通过对该株系进行甲基磺酸乙酯(EMS)诱变,并筛选叶脉处叶斑扩大或缩小的突变体,我们获得突变体SUP-E45,该突变体叶片叶脉处叶斑明显缩小。实验结果显示突变的基因所编码的蛋白质是参与miRNA途径的AGO1蛋白。AGO1蛋白是miRNA通路中至关重要的蛋白,成熟的miRNA与AGO1结合形成RISC沉默复合体从而对靶基因的表达进行负调控。筛选获得的另一个突变体ab58中,叶片叶脉处叶斑也是明显缩小,而且植株矮小,叶片卷曲,对温度敏感。实验结果显示ab58突变体中发挥作用的突变基因是BON1。BON1属于copine基因家族成员。bon1突变体中第三、四、五对真叶中内源miRNA的表达量相对于SUC2:amiR-SUL植株降低,说明BON1可能是拟南芥miRNA通路中的新因子。后续研究将进一步探索BON1基因在拟南芥miRNA通路中的功能和作用机制。在本研究中不仅筛选到已知的AGO1蛋白,而且也筛选到了新的BON1蛋白,不仅验证了该筛选体系的可行性,而且通过该筛选体系还筛选出了可能参与miRNA通路的新因子,为后续的研究奠定一定的基础。
[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.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：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.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
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