miRNA合成途徑新因子的鑒定及其功能研究
發(fā)布時間：2019-06-05 14:26
【摘要】：microRNA(miRNA)是廣泛存在于真核生物體內(nèi)的一類長度約為21個核苷酸的非編碼RNA,其通過與靶基因的mRNA互補配對,降解靶標m RNA或抑制其翻譯過程,在生物生長發(fā)育過程起重要調(diào)控作用。miRNA自發(fā)現(xiàn)以來,一直是生命科學領(lǐng)域研究的熱點,關(guān)于miRNA的生物合成及作用機制已有相當多的報道。近幾年研究發(fā)現(xiàn),miRNA是非細胞自主性的,它可以在細胞間及組織間進行運動。為了研究miRNA的運動,前人構(gòu)建了擬南芥SUC2:amiR-SUL轉(zhuǎn)基因株系,該株系利用韌皮部伴胞細胞特異表達性啟動子SUCROSE-PROTON SYMPORTER 2(SUC2)驅(qū)動表達人工mi RNA,靶向煙草SULPHUR(SUL)基因在擬南芥中同源的葉綠素合成基因CHLORINA42(CH42),導致葉脈10-15層細胞變白。本研究以SUC2:amiR-SUL株系為材料,采用EMS誘變正向遺傳篩選葉脈白斑增大或減小的突變體,發(fā)現(xiàn)新的影響miRNA通路的因子。我們選取了一個葉脈白斑減小的突變體SUP-B65,通過stem-loop RT-PCR檢測突變體中mi RNA的含量,以及利用熒光定量PCR檢測突變體中miRNA的初級轉(zhuǎn)錄本(pri-miRNA)與mi RNA靶標基因的表達量。同時將SUP-B65與SUC2:amiR-SUL回交,從F2代中選出與SUP-B65表型一致的植株約60棵,提取它們的基因組樣品,將樣品混合后送公司進行全基因組重測序以確定突變位點。根據(jù)d-CAPs原理針對突變位點設(shè)計引物進行基因型鑒定。并且構(gòu)建回復突變載體,通過回復SUP-B65表型以確定基因型鑒定結(jié)果。熒光定量PCR及stem-loop RT-PCR結(jié)果表明SUP-B65中的pri-miRNA和miRNA水平相對于SUC2:amiR-SUL均降低,靶標基因的表達水平普遍升高。這說明SUP-B65突變體中受到影響的是miRNA的生物合成,而非miRNA的運動。全基因組重測序共獲得4個候選突變位點基因,分別為AT4G11610、AT4G12560、AT5G67030和AT5G67280,基因型鑒定證實突變位點基因為AT4G12560和AT5G67280,這兩個基因在擬南芥數(shù)據(jù)庫分別對應CONSTITUTIVE EXPRESSER OF PR GENES 1(CPR1)和ABA DEFICIENT 1(ABA1),說明SUP-B65為cpr1aba1雙突變體,進一步的回復突變實驗驗證了基因型鑒定的結(jié)果。已有研究報道,在抗病通路中CPR1負調(diào)控SNC1的表達,CPR1突變導致SNC1表達上調(diào),另外ABA1的缺失會導致SNC1積累在核內(nèi),因此推測cpr1aba1雙突變體表型是由SNC1在細胞核內(nèi)過量積累造成,并且CPR1基因突變對SUP-B65的表型起到關(guān)鍵作用,ABA1基因突變起到增強的作用。據(jù)此推測cpr1aba1雙突變體的表型是由SNC1的核定位導致的。后續(xù)研究將構(gòu)建帶核定位信號的SNC1載體獲得轉(zhuǎn)基因植株,通過進一步的實驗確認本研究的推測。
[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.
【學位授予單位】：深圳大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
本文編號：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.
【學位授予單位】：深圳大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
【參考文獻】
相關(guān)期刊論文 前1條
1 崔清志;劉曉虹;陳惠明;;EMS誘變技術(shù)研究進展[J];湖南農(nóng)業(yè)科學;2013年05期
，本文編號：2493594
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