番茄Sly-miR403的分离鉴定及在番茄发育过程中的功能研究

发布时间：2019-02-16 05:43

【摘要】：micro RNA(miRNA)是一类保守的、长度为19~24个碱基的小分子非编码RNA。在植物中,miRNA通过对靶基因进行剪切、抑制蛋白翻译因子对靶基因的识别或对靶基因的基因组进行甲基化等,从而在转录或转录后水平特异调控靶基因的表达。miRNA合成及作用途径中的重要因子,如AGO蛋白、DCL1及HEN1等在植物的发育过程中发挥着重要的作用。AGO2是RNA Induced silencing complex(RISC)的核心元件之一,有研究报道AGO2为miR403的靶基因;然而,在重要的果实模式生物——番茄中,AGO2与miR403的生理功能尚不明确。因此,为了阐明番茄miR403的生理功能,以micro-Tom番茄为研究对象,利用生物信息学方法和分子生物学技术,获得并验证番茄miR403序列,通过现代生物技术手段构建表达载体、遗传转化获得miR403超表达和缺失表达的转基因植株,观察其表型并对其靶基因进行分析,得到如下主要研究结果:1.通过拟南芥miR403(MI0001072)成熟序列在番茄siRNA数据库进行blast比对,找出潜在的番茄miR403(S14362631);通过在番茄基因组数据库中进行低阈值对比,找出与番茄miR403匹配的基因组位点,其所在位置即为miR403的潜在前体序列;通过RNA结构软件分析,确定其是能够形成完整茎环结构的Sly-miR403前体序列;2.用番茄miR403成熟序列对比,找出潜在靶基因AGO2,采用5’RACE mapping技术检测AGO2的匹配位点,证实Sly-miR403在番茄中可发挥作用,能有效剪切AGO2基因的mRNA;同时用定量PCR检测Sly-miR403及其靶基因在番茄中的表达模式,验证成熟序列的正确性并分析其在番茄生长发育中潜在的作用;3.根据获得的Sly-pre-miR403序列,设计基因特异性引物,对Sly-pre-miR403进行扩增,并克隆到植物表达载体p Bi121上,命名为pBi121-miR403;根据Sly-miR403的成熟序列,设计miR403-sponge片段,合成后构建到pBi121上,命名为pBi121-miR403-sponge载体;将pBi121-miR403、pBi121-miR403-sponge用冻融法转入农杆菌C58中,并用叶盘法转入野生型番茄,获得其转基因植株;4.通过观察转基因植株的表型并分析发现,pBi121-miR403超表达的转基因植株出现开花延迟、叶片异常、种子发芽过程中对ABA的抗性及顶端分生异常的现象;5.检测靶基因在转基因及非转基因植株中的表达变化,明确miR403超表达后降低其靶标基因SlAGO2的丰度,同时提高SlAGO1A/SlAGO1B的丰度,从而影响miR156、miR159及miR394等下游miRNA的表达,进而影响番茄的生长发育。提出了miR403/AGO2、miR168/AGO1在生长发育、病毒入侵条件下不同的平衡假设。课题以重要的果实模式生物番茄为研究材料,分离和鉴定Sly-miR403及其靶基因AGO2。研究表明,番茄miR403能有效剪切AGO2基因的mRNA,miR403超表达后降低其靶标基因SlAGO2的丰度,同时提高SlAGO1A/Sl AGO1B的丰度,影响miR156、miR159及miR394等下游miRNA的表达,进而影响番茄的生长发育。
[Abstract]:Micro RNA (miRNA) is a kind of conserved, 19- to 24-base non-coding RNA.. In plants, miRNA cleans target genes, inhibits the recognition of target genes by protein translation factors, or methylates the genomes of target genes. Thus specifically regulating the expression of target genes at the transcriptional or posttranscriptional level. Important factors in the synthesis and pathway of miRNA, such as AGO protein, DCL1 and HEN1 play an important role in plant development. AGO2 is one of the core components of RNA Induced silencing complex (RISC). It has been reported that AGO2 is the target gene of miR403. However, the physiological functions of AGO2 and miR403 in tomato, an important fruit model organism, are unclear. Therefore, in order to elucidate the physiological function of tomato miR403, the expression vector of micro-Tom tomato was constructed by using bioinformatics and molecular biology techniques to obtain and verify the miR403 sequence of tomato. Transgenic plants with miR403 overexpression and deletion expression were obtained by genetic transformation. The phenotypes were observed and their target genes were analyzed. The main results were as follows: 1. The potential tomato miR403 (S14362631) was identified by blast alignment of Arabidopsis miR403 (MI0001072) mature sequence in tomato siRNA database. By comparing the low threshold in tomato genome database, we find out the locus of tomato miR403, which is the potential precursor sequence of miR403. Through the analysis of RNA structure software, it is confirmed that it is the Sly-miR403 precursor sequence which can form the complete stem ring structure. 2. By comparing the mature sequence of tomato miR403, the potential target gene AGO2, was identified to detect the matching site of AGO2 by 5'RACE mapping technique. It was proved that Sly-miR403 could play a role in tomato and could effectively cut the mRNA; of AGO2 gene. At the same time, quantitative PCR was used to detect the expression pattern of Sly-miR403 and its target gene in tomato, to verify the correctness of mature sequence and to analyze its potential role in tomato growth and development. 3. According to the obtained Sly-pre-miR403 sequence, a gene specific primer was designed to amplify the Sly-pre-miR403 and cloned into the plant expression vector p Bi121, named pBi121-miR403;. According to the mature sequence of Sly-miR403, the miR403-sponge fragment was designed and synthesized into pBi121 and named as pBi121-miR403-sponge vector. The pBi121-miR403,pBi121-miR403-sponge was transferred into Agrobacterium tumefaciens C58 by freeze-thaw method, and the wild type tomato was transferred into tomato by leaf disc method. The phenotypes of transgenic plants were observed and analyzed. It was found that the transgenic plants with pBi121-miR403 overexpression had delayed flowering, abnormal leaves, resistance to ABA during seed germination and abnormal apical meristem. 5. The changes of target gene expression in transgenic and non-transgenic plants were detected, and it was determined that miR403 overexpression reduced the abundance of target gene SlAGO2 and increased the abundance of SlAGO1A/SlAGO1B, thus affecting the expression of downstream miRNA such as miR156,miR159 and miR394. And then affect the growth and development of tomato. The different equilibrium hypothesis of miR403/AGO2,miR168/AGO1 under the condition of growth, development and virus invasion was put forward. Sly-miR403 and its target gene AGO2. were isolated and identified by using important fruit model biological tomato as research materials. The results showed that miR403 could effectively cut the mRNA,miR403 expression of AGO2 gene and reduce the SlAGO2 abundance of its target gene, increase the abundance of SlAGO1A/Sl AGO1B, affect the expression of miRNA in the downstream of miR156,miR159 and miR394, and then affect the growth and development of tomato.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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