摘要番茄是研究肉果果实生长发育和成熟的模式植物。目前对番茄果实成熟的遗传调控机制上已取得了重要进展，但是对于miRNA介导的番茄果实成熟调控的分子机制的研究非常缺乏。近期的研究表明microRNA在植物花的发育和花粉的萌发等生长阶段都起重要的调控作用，在果实后期的成熟阶段，microRNA的作用逐渐明确。在拟南芥中，microRNA AtmiR157通过抑制AtSPL3 mRNA的翻译以防花期提前，在成年的叶片花序生长时期AtmiR157的水平降到最低水平。然而到目前为止，尽管有许多报道在番茄果实发育过程中发现众多miRNAs，但是没有直接的试验证据支持这些miRNA在番茄果实成熟过程中发挥重要作用。利用同源克隆，我们得到番茄miRNA SlymiR157基因，但关于SlymiR157在番茄果实成熟中的作用尚不明确。本试验利用病毒学技术，通过构建马铃薯X病毒PVX载体在野生型番茄AC果实中超表达SlymiR157可直接延缓果实成熟，初步表明SlymiR157在番茄果实成熟中发挥重要作用。47813

Tomato has been long model plant for fruit fleshy fruit development and ripening. Although significant progress has been made toward unraveling ripening-associated genetic mechanisms in tomato, the mechanism involved in miRNA-mediated regulation of tomato fruit ripening remains largelyunknown. Recent studies have shown that microRNA played an important role in floral development and pollen germination, as well as in development of fruit ripening. In Arabidopsis thaliana, microRNA AtmiR157 increased significantly and prevented abnormal early flowering through miRNA-induced AtSPL3 translation repression, whereas AtmiR157 expression decreased to a very low level after the flowering stage. In tomato, however, no miRNA has been experimentally documented to be functionally involved in fruit ripening although many miRNAs have been profiled in fruits. Here, using homologous cloning, we found tomato miRNA SlymiR157 gene, however, the biological role of SlymiR157 in tomato fruit ripening is unclear. In this experiment, via a viral ectopic expression of SlymiR157 using potato virus X PVX, we found that PVX-induced SlymiR157 overexpression resulted in delayed fruit ripening within 2-3 weeks after infection in tomato cultivar AC. Our findings preliminarily reveal that slymiR157 plays a critical role in modulating fruit ripening in tomato.

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毕业论文关键词：番茄； 果实成熟； SlymiR157； PVX病毒技术

Keyword: tomato; fruit ripening; SlymiR157; PVX virus technology

目    录

1、引言4

  1.1、番茄简介 4

  1.2、背景意义 4

  1.3、研究现状 4

  1.4、研究内容及技术路线 5

    1.4.1、研究内容 5

    1.4.2、技术路线 5

2、实验材料与方法6

  2.1、实验材料 6

  2.2、实验方法 6

2.2.1、番茄果实RNA提取7

2.2.2、cDNA的合成 7

2.2.3、SlymiR157前体pre-SlymiR157克隆  7

2.2.4、PCR产物回收 8

2.2.5、PCR产物连入T载体 8

2.2.6、转化E.coli8

2.2.7、提取质粒 8

2.2.8、构建PVX/pre-SlymiR157载体 9

    2.2.9、PVX/SlymiR157载体构建 10

    2.2.10、载体线性化与体外转录 10

2.2.10.1、准备线性化的病毒超表达载体10

    2.2.10.2、已线性化的病毒超表达载体的体外转录10

3、实验结果及分析11

4、结论18

5、展望18

参考文献 19

致谢 20

1、引言 

  1.1、番茄简介

    番茄（Solanum lycopersicum）原产于南美洲和中美洲，是茄科茄属番茄亚属的多年生草本植物。番茄果实口感酸甜独特，并富含维生素、胡萝卜素、叶酸和钾等多种微量元素，尤其含有番茄红素，具有降低罹患心血管疾病和癌症的风险1。由于番茄富含多种营养元素和番茄红素，其果实及其加工品的消费需求越来越高，因此，番茄现作为食用果蔬在全球大量种植。 利用PVX病毒技术初步探索SlymiR157延缓番茄成熟的作用:http://www.youerw.com/shengwu/lunwen_50051.html