發(fā)布時(shí)間：2019-02-16 05:43

【摘要】：micro RNA(miRNA)是一類保守的、長(zhǎng)度為19~24個(gè)堿基的小分子非編碼RNA。在植物中,miRNA通過(guò)對(duì)靶基因進(jìn)行剪切、抑制蛋白翻譯因子對(duì)靶基因的識(shí)別或?qū)Π谢虻幕蚪M進(jìn)行甲基化等,從而在轉(zhuǎn)錄或轉(zhuǎn)錄后水平特異調(diào)控靶基因的表達(dá)。miRNA合成及作用途徑中的重要因子,如AGO蛋白、DCL1及HEN1等在植物的發(fā)育過(guò)程中發(fā)揮著重要的作用。AGO2是RNA Induced silencing complex(RISC)的核心元件之一,有研究報(bào)道AGO2為miR403的靶基因;然而,在重要的果實(shí)模式生物——番茄中,AGO2與miR403的生理功能尚不明確。因此,為了闡明番茄miR403的生理功能,以micro-Tom番茄為研究對(duì)象,利用生物信息學(xué)方法和分子生物學(xué)技術(shù),獲得并驗(yàn)證番茄miR403序列,通過(guò)現(xiàn)代生物技術(shù)手段構(gòu)建表達(dá)載體、遺傳轉(zhuǎn)化獲得miR403超表達(dá)和缺失表達(dá)的轉(zhuǎn)基因植株,觀察其表型并對(duì)其靶基因進(jìn)行分析,得到如下主要研究結(jié)果:1.通過(guò)擬南芥miR403(MI0001072)成熟序列在番茄siRNA數(shù)據(jù)庫(kù)進(jìn)行blast比對(duì),找出潛在的番茄miR403(S14362631);通過(guò)在番茄基因組數(shù)據(jù)庫(kù)中進(jìn)行低閾值對(duì)比,找出與番茄miR403匹配的基因組位點(diǎn),其所在位置即為miR403的潛在前體序列;通過(guò)RNA結(jié)構(gòu)軟件分析,確定其是能夠形成完整莖環(huán)結(jié)構(gòu)的Sly-miR403前體序列;2.用番茄miR403成熟序列對(duì)比,找出潛在靶基因AGO2,采用5’RACE mapping技術(shù)檢測(cè)AGO2的匹配位點(diǎn),證實(shí)Sly-miR403在番茄中可發(fā)揮作用,能有效剪切AGO2基因的mRNA;同時(shí)用定量PCR檢測(cè)Sly-miR403及其靶基因在番茄中的表達(dá)模式,驗(yàn)證成熟序列的正確性并分析其在番茄生長(zhǎng)發(fā)育中潛在的作用;3.根據(jù)獲得的Sly-pre-miR403序列,設(shè)計(jì)基因特異性引物,對(duì)Sly-pre-miR403進(jìn)行擴(kuò)增,并克隆到植物表達(dá)載體p Bi121上,命名為pBi121-miR403;根據(jù)Sly-miR403的成熟序列,設(shè)計(jì)miR403-sponge片段,合成后構(gòu)建到pBi121上,命名為pBi121-miR403-sponge載體;將pBi121-miR403、pBi121-miR403-sponge用凍融法轉(zhuǎn)入農(nóng)桿菌C58中,并用葉盤法轉(zhuǎn)入野生型番茄,獲得其轉(zhuǎn)基因植株;4.通過(guò)觀察轉(zhuǎn)基因植株的表型并分析發(fā)現(xiàn),pBi121-miR403超表達(dá)的轉(zhuǎn)基因植株出現(xiàn)開花延遲、葉片異常、種子發(fā)芽過(guò)程中對(duì)ABA的抗性及頂端分生異常的現(xiàn)象;5.檢測(cè)靶基因在轉(zhuǎn)基因及非轉(zhuǎn)基因植株中的表達(dá)變化,明確miR403超表達(dá)后降低其靶標(biāo)基因SlAGO2的豐度,同時(shí)提高SlAGO1A/SlAGO1B的豐度,從而影響miR156、miR159及miR394等下游miRNA的表達(dá),進(jìn)而影響番茄的生長(zhǎng)發(fā)育。提出了miR403/AGO2、miR168/AGO1在生長(zhǎng)發(fā)育、病毒入侵條件下不同的平衡假設(shè)。課題以重要的果實(shí)模式生物番茄為研究材料,分離和鑒定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超表達(dá)后降低其靶標(biāo)基因SlAGO2的豐度,同時(shí)提高SlAGO1A/Sl AGO1B的豐度,影響miR156、miR159及miR394等下游miRNA的表達(dá),進(jìn)而影響番茄的生長(zhǎng)發(fā)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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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