miRNA合成途径新因子的鉴定及其功能研究
发布时间：2019-06-05 14:26
【摘要】：microRNA(miRNA)是广泛存在于真核生物体内的一类长度约为21个核苷酸的非编码RNA,其通过与靶基因的mRNA互补配对,降解靶标m RNA或抑制其翻译过程,在生物生长发育过程起重要调控作用。miRNA自发现以来,一直是生命科学领域研究的热点,关于miRNA的生物合成及作用机制已有相当多的报道。近几年研究发现,miRNA是非细胞自主性的,它可以在细胞间及组织间进行运动。为了研究miRNA的运动,前人构建了拟南芥SUC2:amiR-SUL转基因株系,该株系利用韧皮部伴胞细胞特异表达性启动子SUCROSE-PROTON SYMPORTER 2(SUC2)驱动表达人工mi RNA,靶向烟草SULPHUR(SUL)基因在拟南芥中同源的叶绿素合成基因CHLORINA42(CH42),导致叶脉10-15层细胞变白。本研究以SUC2:amiR-SUL株系为材料,采用EMS诱变正向遗传筛选叶脉白斑增大或减小的突变体,发现新的影响miRNA通路的因子。我们选取了一个叶脉白斑减小的突变体SUP-B65,通过stem-loop RT-PCR检测突变体中mi RNA的含量,以及利用荧光定量PCR检测突变体中miRNA的初级转录本(pri-miRNA)与mi RNA靶标基因的表达量。同时将SUP-B65与SUC2:amiR-SUL回交,从F2代中选出与SUP-B65表型一致的植株约60棵,提取它们的基因组样品,将样品混合后送公司进行全基因组重测序以确定突变位点。根据d-CAPs原理针对突变位点设计引物进行基因型鉴定。并且构建回复突变载体,通过回复SUP-B65表型以确定基因型鉴定结果。荧光定量PCR及stem-loop RT-PCR结果表明SUP-B65中的pri-miRNA和miRNA水平相对于SUC2:amiR-SUL均降低,靶标基因的表达水平普遍升高。这说明SUP-B65突变体中受到影响的是miRNA的生物合成,而非miRNA的运动。全基因组重测序共获得4个候选突变位点基因,分别为AT4G11610、AT4G12560、AT5G67030和AT5G67280,基因型鉴定证实突变位点基因为AT4G12560和AT5G67280,这两个基因在拟南芥数据库分别对应CONSTITUTIVE EXPRESSER OF PR GENES 1(CPR1)和ABA DEFICIENT 1(ABA1),说明SUP-B65为cpr1aba1双突变体,进一步的回复突变实验验证了基因型鉴定的结果。已有研究报道,在抗病通路中CPR1负调控SNC1的表达,CPR1突变导致SNC1表达上调,另外ABA1的缺失会导致SNC1积累在核内,因此推测cpr1aba1双突变体表型是由SNC1在细胞核内过量积累造成,并且CPR1基因突变对SUP-B65的表型起到关键作用,ABA1基因突变起到增强的作用。据此推测cpr1aba1双突变体的表型是由SNC1的核定位导致的。后续研究将构建带核定位信号的SNC1载体获得转基因植株,通过进一步的实验确认本研究的推测。
[Abstract]:MicroRNA (miRNA) is a kind of non-coding RNA, with a length of about 21 nucleotides, which widely exists in eukaryotes. It degrades the target mRNA or suppresses its translation by complementary pairing with the target gene mRNA. MiRNA has been a hot topic in the field of life science since its discovery, and there have been many reports on the biosynthesis and mechanism of miRNA. In recent years, it has been found that miRNA is non-cellular autonomy and can move between cells and tissues. In order to study the movement of miRNA, Arabidopsis SUC2:amiR-SUL transgenic strain was constructed, which was driven by phloem associated cell specific expression promoter SUCROSE-PROTON SYMPORTER 2 (SUC2) to express artificial miRNA. CHLORINA42 (CH42), a homologous chlorophyll synthesis gene targeting tobacco SULPHUR (SUL) gene in Arabidopsis thaliana, caused the whitening of 10 脳 15 layers of cells in the vein. In this study, SUC2:amiR-SUL lines were used as materials to screen mutants with increased or decreased white spot in leaf veins by EMS mutagenesis, and new factors affecting miRNA pathway were found. We selected a mutant SUP-B65, with reduced white spot in vein to detect the content of mi RNA in the mutant by stem-loop RT-PCR. The expression of primary transcript (pri-miRNA) and miRNA target gene of miRNA in mutant was detected by fluorescence quantitative PCR (FQ-PCR). At the same time, about 60 plants with the same phenotype as SUP-B65 were selected from F2 generation, and their genomic samples were extracted. The samples were mixed and sent to the company for genome resequencing to determine the mutation sites. According to d-CAPs principle, primers were designed for genotyping of mutation sites. The recovery mutation vector was constructed, and the genotypic identification results were determined by replying SUP-B65 phenotype. The results of fluorescence quantitative PCR and stem-loop RT-PCR showed that the levels of pri-miRNA and miRNA in SUP-B65 were lower than those in SUC2:amiR-SUL, and the expression level of target gene was generally increased. This indicates that the biosynthesis of miRNA, not the movement of miRNA, is affected in SUP-B65 mutants. A total of four candidate mutation sites were obtained by genome resequencing. AT4G11610,AT4G12560,AT5G67030 and AT5G67280, genotyping confirmed that the mutation sites were AT4G12560 and AT5G67280, respectively. These two genes correspond to CONSTITUTIVE EXPRESSER OF PR GENES 1 (CPR1) and ABA DEFICIENT 1 (ABA1) in Arabidopsis thaliana database, indicating that SUP-B65 is a double mutant of cpr1aba1. Further reply mutation experiments confirmed the results of genotypic identification. It has been reported that CPR1 negatively regulate the expression of SNC1 in the disease resistance pathway, CPR1 mutation leads to the up-regulation of SNC1 expression, and the deletion of ABA1 will lead to the accumulation of SNC1 in the nucleus. Therefore, it is speculated that the phenotype of cpr1aba1 double mutant is caused by the excessive accumulation of SNC1 in the nucleus. Moreover, CPR1 gene mutation plays a key role in the phenotype of SUP-B65, and ABA1 gene mutation plays an enhanced role. It is speculated that the phenotype of cpr1aba1 double mutant is caused by the nuclear localization of SNC1. The transgenic plants will be obtained by constructing SNC1 vector with nuclear localization signal, and the conjecture of this study will be confirmed by further experiments.
【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
本文编号：2493594
[Abstract]:MicroRNA (miRNA) is a kind of non-coding RNA, with a length of about 21 nucleotides, which widely exists in eukaryotes. It degrades the target mRNA or suppresses its translation by complementary pairing with the target gene mRNA. MiRNA has been a hot topic in the field of life science since its discovery, and there have been many reports on the biosynthesis and mechanism of miRNA. In recent years, it has been found that miRNA is non-cellular autonomy and can move between cells and tissues. In order to study the movement of miRNA, Arabidopsis SUC2:amiR-SUL transgenic strain was constructed, which was driven by phloem associated cell specific expression promoter SUCROSE-PROTON SYMPORTER 2 (SUC2) to express artificial miRNA. CHLORINA42 (CH42), a homologous chlorophyll synthesis gene targeting tobacco SULPHUR (SUL) gene in Arabidopsis thaliana, caused the whitening of 10 脳 15 layers of cells in the vein. In this study, SUC2:amiR-SUL lines were used as materials to screen mutants with increased or decreased white spot in leaf veins by EMS mutagenesis, and new factors affecting miRNA pathway were found. We selected a mutant SUP-B65, with reduced white spot in vein to detect the content of mi RNA in the mutant by stem-loop RT-PCR. The expression of primary transcript (pri-miRNA) and miRNA target gene of miRNA in mutant was detected by fluorescence quantitative PCR (FQ-PCR). At the same time, about 60 plants with the same phenotype as SUP-B65 were selected from F2 generation, and their genomic samples were extracted. The samples were mixed and sent to the company for genome resequencing to determine the mutation sites. According to d-CAPs principle, primers were designed for genotyping of mutation sites. The recovery mutation vector was constructed, and the genotypic identification results were determined by replying SUP-B65 phenotype. The results of fluorescence quantitative PCR and stem-loop RT-PCR showed that the levels of pri-miRNA and miRNA in SUP-B65 were lower than those in SUC2:amiR-SUL, and the expression level of target gene was generally increased. This indicates that the biosynthesis of miRNA, not the movement of miRNA, is affected in SUP-B65 mutants. A total of four candidate mutation sites were obtained by genome resequencing. AT4G11610,AT4G12560,AT5G67030 and AT5G67280, genotyping confirmed that the mutation sites were AT4G12560 and AT5G67280, respectively. These two genes correspond to CONSTITUTIVE EXPRESSER OF PR GENES 1 (CPR1) and ABA DEFICIENT 1 (ABA1) in Arabidopsis thaliana database, indicating that SUP-B65 is a double mutant of cpr1aba1. Further reply mutation experiments confirmed the results of genotypic identification. It has been reported that CPR1 negatively regulate the expression of SNC1 in the disease resistance pathway, CPR1 mutation leads to the up-regulation of SNC1 expression, and the deletion of ABA1 will lead to the accumulation of SNC1 in the nucleus. Therefore, it is speculated that the phenotype of cpr1aba1 double mutant is caused by the excessive accumulation of SNC1 in the nucleus. Moreover, CPR1 gene mutation plays a key role in the phenotype of SUP-B65, and ABA1 gene mutation plays an enhanced role. It is speculated that the phenotype of cpr1aba1 double mutant is caused by the nuclear localization of SNC1. The transgenic plants will be obtained by constructing SNC1 vector with nuclear localization signal, and the conjecture of this study will be confirmed by further experiments.
【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
【参考文献】
相关期刊论文 前1条
1 崔清志;刘晓虹;陈惠明;;EMS诱变技术研究进展[J];湖南农业科学;2013年05期
，本文编号：2493594
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