番茄WRKY31和WRKY33沉默载体构建及其功能验证
毕业论文关键词: 番茄；WRKY 转录因子；烟草脆裂病毒；实时定量 PCR
Silencing Vectors Construction and Function Analysis of tomato WRKY31 and WRKY33
Abstract：This research focuses on the functional study of tomato WRKY31 and WRKY33. The target transcription factor gene WRKY31 and WRKY33 can be obtained by PCR with the specific primers, and then constructed to tobacco rattle virus (TRV) vector pYY13, which were transformed into Agrobacterium GV3101. The real-time quantitative PCR was used to detect the relative expression pattern under various pathogen and chitin. The results showed that both WRKY31 and WRKY33 were involved in the early stage and late stage of Verticillium dahliae and Oidium neolycopersici resistance signaling pathways. Meanwhile WRKY33 had been found it took part in the symbiosis of tomato and Glomus mosseae under the low concentration of phosphate. Spatial expression revealed that the relative expression of WRKY31 and WRKY33 were higher in the red fruit and root inpidually. The above results showed that tomato WRKY31 and WRKY33 may be involved in the signal transduction mechanism of tomato and different fungus and symbiotic fungal.
Key words: tomato; WRKY transcription factor; tobacco rattle virus; real-time quantitative PCR
目 录
摘要 1
引言 2
1. 材料与方法 3
1.1 试验材料与仪器 3
1.1.1 材料与试剂 3
1.1.2 主要仪器 3
1.2 试验方法 3
1.2.1 番茄总RNA的提取及cDNA的合成 3
1.2.2 目的基因的扩增及载体构建 3
1.2.3 大肠杆菌的转化及阳性菌株的鉴定 4
1.2.4 农杆菌转化及阳性菌株的鉴定 4
1.2.5 Real-Time qPCR检测表达量变化 4
2. 结果与分析 5
2.1 RNA的提取及目的基因的扩增 5
2.2 重组载体的构建及验证 6
2.3 阳性重组载体农杆菌转化及验证 6
2.4 实时定量结果分析 7
3. 讨论与展望 10
参考文献 11
致谢 13
番茄 WRKY31 和 WRKY33 沉默载体构建及其功能验证引言
WRKY 转录因子是一类植物所特有的转录因子家族，迄今为止，在酵母和动物（包括线虫、果蝇）中都没有发现，因在其N一端含有由 WRKYGQK 组成的高度保守的氨基酸序列而得名。自 1994 年从甘薯（sweet potato）中分离出第一个 WRKY 转录因子 SPF1（sweet potato factor1） 以来，在其他不同种类植物中也陆续发现了大量同类转录因子[1]。目前在拟南芥中发现大约有 72 个 WRKY 转录因子，水稻中已经报道的大约有 106 个转录因子，番茄中大致有 81 个转录因子，在烟草、野生燕麦、小麦、大麦、棉花、等近十多种植物中都有报道[2-5]。
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