摘要以番茄（Solanum lycopersicum）是研究肉质果实发育和成熟的模式植物。番茄中的Cnr突变体是自发的不成熟突变体。它的突变是由于LeSPL-CNR基因发生突变，但不是核苷酸序列的突变，是启动子上游发生高度甲基化。番茄中有9个影响甲基化的甲基化转移酶，本课题研究的是其中的SlMET1基因。76687

通过构建番茄SlMET1沉默的RNAi载体，我们利用农杆菌介导的转基因技术诱导SlMET1基因沉默。该过程主要包括番茄无菌苗培育、子叶预培养、农杆菌选择和侵染、愈伤组织培养、生芽生根诱导等几个阶段。然后选择重组体进行移栽培养,即T0代株系。经过数代的人工选育，从而获得具有SlMET1基因稳定沉默的T2代株系对T2代纯合株系进行表型观察，记录SlMET1基因沉默所引起的植株表型，初步阐明SlMET1在番茄果实成熟中的作用。

Abstract Tomato has long been accepted as the model for fleshy fruit development and ripening。。 Cnr mutant in tomato is a spontaneous immature mutant with colorless fruits。 The nature of the mutant is due to the gene silencing of LeSPL-CNR gene, which does not result from the absence of any nucleotide sequence difference at the LeSPL-CNR locus, but results from the highly methylation status of cytosine in the LeSPL-CNR promotor region。 In tomato, there are 9 DNA methyltransferases related with cytosine methylation, this project is mainly foucsed on the role of gene SlMET1 in tomato。

By construcing the RNA interfering construct carrying DNA methyltransferase gene SlMET1, we found that transgenic tomato lines using transgenic technology mediated by Agrobacterium。 The process includes Seeds sterilisation, cotyledon culture, agrobacterium culture, cotyledons transformation, regeneration and rooting。 When roots appear and plants grow large enough to be transferred to soil in larger pots and grown in a greenhouse, these plants are called To plants。 After several generations of artificial selection, T2 plants with stable gene silencing of SlMET1 are selected as the homozygous ones。 On the basis of observation and data record of the phenotype of ripened fruits in T2 plants, the present project aims to preliminarily clarify the regulation of SlMET1 induced epigenetic state in fruit ripening in tomato。 

毕业论文关键词：番茄； 基因沉默；SlMET1; 转基因技术；表观遗传

Keyword: tomato；gene silencing；SlMET1; transgenetic technology；epigenetics

目录。4

引言。5

1 材料与方法5

  1。1 实验材料5

  1。2 实验方法5

1。2。1 番茄果实总RNA提取。5

    1。2。2 RNA反转录成cDNA。6

1。2。3 PCR反应6

    1。2。4  PCR产物纯化回收。。7

2 构建SlMET1基因沉默的RNAi载体8

  2。1  实验材料。8

  2。2  载体构建实验方法8

2。2。1 pRNAi-LIC 质粒的提取。8

2。2。2 pRNAi-LIC 载体的线性化9

2。2。3 线性化载体的纯化。9

2。2。4 T4DNA聚合酶处理PCR产物以及载体酶切片段。9

2。2。5 处理后片段连接并转化感受态大肠杆菌细胞10

2。2。6 转化及检测。。10

  2。3  实验结果及分析。。11

2。3。1 目的基因PCR产物。11

2。3。2 重组质粒双酶切检测。11

2。3。3 重组质粒转化农杆菌并检测。12

  2。4  讨论。13

3 番茄转基因。13

  3。1  实验材料。。13

3。1。1 植物材料13

3。1。2 分子生物学试剂13

  3。2  农杆菌介导的转基因14

  3。3  实验结果及分析。。14

  3。4  讨论。15