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盐芥microRNA164基因功能研究

更新时间 2009-9-12 23:01:31 点击数:

盐芥microRNA164基因功能研究
Research on Function of miR164 in Thellungiella Salsuginea
【中文摘要】 microRNAs(miRNAs)是生物体内源长度约为20-23nt的非编码小RNA,广泛分布于真核细胞基因组中,是最大的基因家族之一,大约占到整个基因组的1%。在基因组上通常具有独立的基因座位。1993年,Lee和Wightman等在研究线虫胚胎发育过程时发现了第一个mircoRNA基因——lin-4。随后,在拟南芥、水稻、玉米、高粱、甘蔗以及苔藓植物中先后发现了大量的miRNA。miRNA是一种负调控因子。在植物体内,初级转录产物在Dicer-like酶的作用下切割并自身折叠成不完全配对的发卡结构,然后参与组装沉默复合体。通过与靶mRNA在剪切位点附近几乎完全的互补配对而在转录后水平上对基因的表达进行负调控,导致mRNA的降解或翻译抑制,从而参与调控植物器官的形态建成、生长发育、激素分泌、信号转导以及对外界环境胁迫应答等生物学过程。miR164由MIR164A、MIR164B、MIR164C三个基因座位编码,靶基因为NAC家族的六个转录因子。miR164通过调控这些靶基因影响生长素梯度、侧根的起始以及花瓣的数量。其中CUC1与CUC2控制条分生组织的形成和器官边界的发育。拟南芥中,miR164a缺失突变体和含有抗miR164的CUC2的突变体的叶片边缘缺刻加深;相反,过量表达miR164的突变体叶边缘平滑,过量表达抗miR164的CUC1也没有出现此表型。CUC2失活造成miR164a缺失突变体或野生型叶缘缺刻消失。并且发现miR164a与CUC2的表达区域重叠,最初在幼叶的整个边缘区表达,然后逐渐局限于叶片边缘凹进的区域。因此,拟南芥中miR164通过负调控CUC2控制叶边缺刻程度。2007年,Jose′Manuel Franco-Zorrilla等人在研究拟南芥磷饥饿诱导的miRNA——miR399时发现了一个非编码蛋白基因IPS1。IPS1含有与miR399互补配对的基序,但是这种互补配对被一个错配环打断,错配环正好位于miR399的剪切位点,导致IPS1不能被miR399剪切,反而螯合了miR399。并且这种结合比miR399与底物的结合更有竞争性。因此,当IPS1过量表达积累时,大量的miR399与IPS1配对,只有少数miR399与靶基因PHO2的mRNA配对,发挥了剪切靶基因mRNA的作用,最终导致靶基因mRNA的大量积累,表现为枝条中磷含量的降低。这种miRNA抑制机制称为靶基因模拟。靶基因模拟提供了一种新的分析miRNA功能的方法。盐芥与拟南芥近缘,属于十字花科植物。具有拟南芥很多同样的优点以作为实验系统:如类似的形态、小的基因组、短的生活史、丰富的种子和易于被转化等。但是,盐芥对多种胁迫的耐受能力远远高于拟南芥。是一种新型的盐生模式植物。本实验以盐芥为材料,设计两对引物分别从拟南芥中克隆获得MIR164基因和IPS1基因。一方面将MIR164基因连接本实验室设计的pCAMBIA-改造后的pCAMBIA 3301载体,构建了MIR164过量表达载体,转入农杆菌浸染野生型盐芥,获得过量表达转化株系。另一方面,将IPS1错配环两端与miR399互补配对的片段替换成与miR164互补配对的片段,连接pCAMBIA-改造后的pCAMBIA 3301载体,构建成MIR164沉默载体,转入农杆菌浸染野生型盐芥,获得MIR164沉默转化株系3株。同时,为了验证盐芥中CUC2也是miR164的靶基因,进行了盐芥CUC2同源基因的克隆。首先,提取盐芥RNA,反转录获得cDNA,然后设计一对简并引物,以盐芥cDNA为模板进行PCR,克隆得到盐芥中CUC2同源基因长268bp的片段,由于拟南芥中miR164与CUC2的结合位点位于774bp-794bp,因此又设计一个特异引物进行3' -RACE。克隆得到CUC2同源基因的3’端序列。

【英文摘要】 MicroRNAs (miRNAs) are a class of endogenous noncoding single-stranded RNA with about 22-23 nucleotides length which are widely distributed in genome of Eukaryotic cells. microRNAs are one of the largest gene familise with the proportion of about 1% in genome which locad in independent locus. The first gene recognized to encode microRNA, lin-4 of C. elegans, were identified on the basis of the developmental timing defects associated with loss-of-function mutations by Lee and Wightman in 1993. It is now known that there are hundreds of microRNAs genes in arabidopsis ,Oryza Sativa, Zea mays, Sorghum bicolo, Saccharum officenarum L and bryophyte. In plants, these nucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNA. microRNAs function as sequence-specific negative regulators in post-transcriptional gene silencing by almost perfect complementarity with target mRNA around the cleavage site , which leads to mRNA cleavage or translational repression.By this way,miRNA function in Biological process,such as morphogenesis,growth and development,hormone secretion, signal transduction and the response capacity to the external environmental stress.miR164 is encoded by MIR164A, MIR164B, and MIR164 C,whose targets are six members of the NAC family of transcription factors.miR164 function in auxin Auxin gradients ,the oritation of the Lateral root and the quantity of petal. CUC2 and CUC1 are required for embryonic shoot meristem formation and specification of the organ boundary.which are specificly regulated by miR164a. The mutations in the MIR164A gene deepen serration of the leaf margin. By contrast, leaves of plants overexpressing miR164 have smooth margins. Enhanced leaf serration was observed following the expression of an miR164-resistant CUC2 but not of an miR164-resistant CUC1. Furthermore, CUC2 inactivation abolished serration in mir164a mutants and the wild type. CUC2 and MIR164A are transcribed in overlapping domains at the margins of young leaf primordia, with transcription gradually restricted to the sinus, where the leaf margins become serrated. CUC2 specifically controls leaf margin development under the regulation of miR164.IPS1(INDUCED BY PHOSPHATE STARVATION1) is a non–protein coding gene which is dicovered in the research of miR399 by Jose′Manuel Franco-Zorrilla1 et al. in 2007.It contains a motif with sequence complementarity to the phosphate (Pi) starvation–induced miRNA--miR-399.The pairing is interrupted by a mismatched loop at the expected miR399 cleavage site. IPS1 mRNA is not cleaved but instead sequesters miR-399.IPS1 is a more efficient competitor for miR-399 compared with a cleavable substrate Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. We coin the term‘target mimicry’to define this mechanism of inhibition of miRNA activity. Target mimicry provide a new method for miRNA research。Thellungiella salsuginea is close relative to Arabidopsis which is also belong to Cruciferae and has good genetic features such as similar morphology, small genome size, short life cycle, high seed number and an efficient transformation method. However, T. salsuginea is able to withstand dramatic salinity shock.We cloned IPS1 and miR164 form the genome of Arabidopsis.On one hand,we completed the construction of silencing vector with miR164,on the other hand,we constructed overexpressing vector by partly replacing the sequence of IPS1 with the sequence which is complementary to miR164.In order to check whether CUC2 is also the target gene of miR164 in T. salsuginea,we cloned the CUC2 Homologous gene in T. salsuginea by using Degenerate PCR and 3' -race.

【中文关键词】 miRNA; miR164; IPS1; 缺刻; 盐芥
【英文关键词】 miRNA; miR164; IPS1; serration; Thellungiella salsuginea
毕业论文目录】
摘要 7-9
ABSTRACT 9-10
第一部分 文献综述 11-26
    1 MIRNA 简介 11-19
        1.1 MIRNA 的发现 12
        1.2 MIRNA 的生物合成及组装 12-14
            1.2.1 miRNA 前体的转录 12
            1.2.2 miRNA 的加工和外运 12-13
            1.2.3 miRNA 组装入沉默复合体 13-14
        1.3 MIRNA 的作用机制 14
        1.4 MIRNA 的功能 14-15
        1.5 MIRNA 的鉴定 15-16
        1.6 植物中保守的MIRNA 16
        1.7 植物中不保守的MIRNA 16-17
        1.8 MIRNA 与SIRNA 的比较 17
        1.9 植物MIRNA 靶基因的鉴定 17-19
            1.9.1 植物miRNA 靶基因的鉴定 17-18
            1.9.2 植物miRNA 调控范围 18-19
            1.9.3 植物miRNA 靶基因的实验确定 19
    2 MIR164 与植物缺刻 19-22
        2.1 植物叶片的发育及影响因子 19-20
        2.2 MIR164 与CUC2 的突变体 20-22
            2.2.1 miR164a 突变体缺刻加深 20-21
            2.2.2 miR164a 靶基因的确定 21
            2.2.3 CUC2 与MIR164A 的表达定位 21-22
            2.2.4 叶片缺刻形成的模型 22
    3 IP51 的发现及作用机制 22-23
        3.1 IP51 的研究 22
        3.2 IP51 的作用机制 22-23
    4 排水器的结构与发育 23-24
    5 简并PCR 技术 24-25
        5.1 简并引物的设计 24
        5.2 简并PCR 技术的局限性 24-25
        5.3 使用简并PCR 获得基因全长的方法 25
    6 耐盐模式植物盐芥 25-26
第二部分 实验论文 26-48
    1 实验材料 26-28
        1.1 植物材料 26
        1.2 菌种及质粒 26
        1.3 引物序列 26-27
        1.4 酶及试剂盒 27
        1.5 主要仪器 27
        1.6 培养基及化学试剂 27-28
        1.7 分析软件 28
    2 实验方法 28-35
        2.1 MIR164 沉默载体的构建 28-32
            2.1.1 CTAB 法提取植物基因组DNA 28-29
            2.1.2 克隆IP51 序列 29
            2.1.3 Gene Clean 方法从琼脂糖凝胶上回收DNA 片段 29
            2.1.4 改造的与miR164 互补的IP51 序列 29-30
            2.1.5 改造的IP51 连接载体 30-31
            2.1.6 大肠杆菌感受态的制备及转化 31
            2.1.7 质粒的提取 31-32
            2.1.8 酶切验证 32
            2.1.9 农杆菌感受态的制备及转化 32
        2.2 MIR164 过表载体的构建 32-33
            2.2.1 PCR 克隆MIR164 基因 33
            2.2.2 MIR164 基因连接载体 33
        2.3 靶基因的克隆 33-35
            2.3.1 植物总RNA 的提取及反转录 33-34
            2.3.3 简并引物克隆盐芥 CUC2 同源基因 34-35
        2.4 快速扩增CDNA 3'末端(3'-RACE) 35
    3 实验结果 35-42
        3.1 改造的IPS1 检测 35-36
        3.2 沉默载体的检测 36-37
        3.3 盐芥CUC2 同源基因的克隆 37-41
        3.4 MIR164 沉默转化株系 41-42
    4 讨论 42-48
参考文献 48-55
硕士期间发表的论文 55-56
致谢 56

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