番茄WRKY22与WRKY25基因沉默载体的构建
毕业论文关键词：转录因子；WRKY基因；实时定量PCR；VIGS
Silencing Vector Construction of Tomato WRKY22 and WRKY25
Abstract：This research focuses on construction of virus induced gene silencing (VIGS) vectors of WRKY genes. Firstly, total RNA was extracted from tomato, and then WRKY22, WRKY25 gene were cloned by reverse transcription PCR technology with the specific primers. At the same time thedifferent treatments were applied on theMicro-Tom(MT) to analysis the WRKY22, WRKY25 expression pattern by real-time quantitative PCR. The results showed that WRKY22and WRKY25 were successfully constructed into the vector, meanwhile both WRKY22and WRKY25 may inpidually participate in the early stage and late stage of Verticillium dahliae and Oidium neolycopersici resistance signaling pathways. Spatial expression indicated that the relative expression of WRKY22 and WRKY25 were higher in the root and mature fruit respectively. The above results displayed that the tomato WRKY22 and WRKY25 may participate in tomato defense responses against biological stresses. It has laid the foundation for the transformation of Agrobacterium and functional verification of the tomato WRKY22 and WRKY25.
Key words：Transcription factor；WRKY gene；real-time PCR；virus induced gene silencing
目 录
摘 要 1
引言 1
1. 材料与仪器 2
1.1 供试材料 2
1.2 主要试剂 2
1.3 主要仪器 2
2. 实验方法 3
2.1 实时定量PCR分析WRKY22、WRKY25的表达模式 3
2.2 番茄 RNA 的提取及反转录 3
2.2.1 番茄 RNA 的提取 3
2.2.2 cDNA的合成 4
2.3 目的基因的扩增及胶回收 4
2.3.1 目的基因的扩增 4
2.3.2 目的片段胶回收 4
2.4 pYY13质粒预处理 5
2.4.1 pYY13质粒的提取 5
2.4.2 质粒pYY13单酶切及切胶回收纯化 5
2.5 重组载体pYY13-WRKY22和pYY13-WRKY25的构建 5
2.5.1 质粒pYY13与目的基因WRKY22、WRKY25的连接反应 5
2.5.2 重组质粒的转化 6
2.6 重组质粒验证 6
3. 结果与分析 6
3.1 实时荧光定量光谱图 6
3.2 目的片段扩增 9
3.3 质粒pYY13的Pst I酶切 9
3.4 沉默载体构建分析 10
4. 讨论 10
参考文献 12
致谢 14
番茄WRKY22与WRKY25基因沉默载体的构建引言
转录因子是一种结合在特定DNA序列的蛋白质分子，通过增强或抑制基因的转录效率来控制基因的表达。WRKY蛋白最早于1994年提出，由Ishiguro和Nakamura在甜土豆中分离得到的[1]。WRKY作为植物特有的一类转录因子家族，因其N端由保守序列7个氨基酸WRKYGQK组成的而命名。随着不断深入的研究，探明WRKY转录因子通过与W-box顺式作用元件发生特异性互作，参与植物的生长发育及植物对各种生物、非生物胁迫防御反应[2-4]。
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