拟南芥液泡蔗糖转化酶基因沉默植物表达载体的构建

摘要本文主要研究拟南芥液泡蔗糖转化酶基因沉默植物表达载体的构建。通过对拟南芥液泡蔗糖转化酶基因片段的克隆，基因重组，构建植物表达载体，转化农杆菌，并加以用来转化拟南芥，并获得敲除液泡蔗糖转化酶基因的转基因拟南芥。并以此为材料，研究液泡蔗糖转化酶的生物学功能。我们通过对拟南芥液泡蔗糖转化酶基因的分析，采用PCR方法克隆了该基因。进而通过使用限制性内切酶、连接酶等，经过T-载体、pHannibal等中间载体，最终成功构建了含有拟南芥液泡蔗糖转化酶基因片段的RNAi载体，其目标是构建一个敲除基因的转基因拟南芥。
实验结果表明：将cDNA用限制性酶NotⅠ从PART27载体上酶切下来，连接到经同样酶切到的含有invertase的RNAi核酸片段，转化大肠杆菌。通过菌液PCR检测和质粒酶切鉴定，证明植物表达构建成功。20167
毕业论文关键词：拟南芥液泡蔗糖转化酶基因；基因沉默；基因重组；表达载体；
Arabidopsis vacuolar invertase gene silencing plant expression vector
Abstract: The Arabidopsis vacuolar invertase gene silencing plant expression vector was studied here. The aim of this experiment is to transform Arabidopsis and to get vacuolar invertase knockout transgenic Arabidopsis plants and thus to explain the biological function of vacuolar invertase enzyme. Arabidopsis vacuolar invertase gene was cloned by PCR after gene sequence analysis. The restriction enzymes, ligase, T-vector, pHannibal other intermediate vectors were used to constructed RNAi expression vector， pART27, which contains Arabidopsis vacuoles invertase gene fragment.and T-DNA.
The results here showed that: The cDNA was clonged from RT-PCR method and then was digested with restriction enzymes NotI down from PART27 expression vector, which contain T-DNA,after connecting to the same restriction enzyme to invertase-intron-invertase gene fragement and then transformed into E. coli bacteria and confirmed by PCR detection and plasmid restriction analysis.
Keywords: Arabidopsis vacuolar invertase；Gene silencing; gene recombination; expression vector
目录
第一章绪论1
1.1拟南芥物种背景1
1.1.1拟南芥的物种简介1
1.1.2拟南芥形态特征 1
1.1.3拟南芥模式生物 1
1.2 RNAi干扰背景2
1.2.1RNAi干扰的简介 2
1.2.2RNAi现象的发现 2
1.2.3基因沉默的简介 3
1.2.4反义RNA及Dicer等酶的简介3
1.3植物表达载体的构建的研究目的和意义 4
1.4实验相关原理介绍 4
1.4.1 CTAB法原理4
1.4.2聚合酶链反应（PCR）原理5
1.4.3琼脂糖凝胶原理 5
1.4.5 氯化钙法转化质粒DNA原理6
1.5本课题实验原理 6
第二章实验材料和方法7
2.1 利用PCR克隆invertase基因 7
2.1.1实验仪器和材料 7
2.1.2 实验试剂7
2.1.3 实验步骤7
2.2 做连接体T-invertase 10
2.2.1反应条件 10
2.2.2 反应体系 11
2.2.3 回收11
2.2.4 转化11

2.2.5 挑单克隆 11
2.3构建载体T-invertase 11
2.3.1实验仪器和材料12
2.3.2 实验试剂 12
2.3.3 实验步骤 12
2.4构建载体pHannibal -invertase14
2.4.1实验仪器和材料14
2.4.2 实验试剂 14
2.4.3实验步骤 15
2.5构建载体invertase- pHannibal -invertase17
2.5.1实验仪器和材料17
2.5.2 实验试剂 17
2.5.3实验器具和仪器设备17
2.6构建载体PART27-（F+R)-invertase 18
2.6.1实验仪器和材料 18
2.6.2 实验试剂 19
2.6.3 实验器具和仪器设备 19
第三章重要的实验结果及其分析 20
第四章讨论 24
4.1讨论 24
致谢 25
参考文献 26
第一章：绪论拟南芥液泡蔗糖转化酶基因沉默植物表达载体的构建:http://www.751com.cn/shengwu/lunwen_11835.html