菊花淹水响应基因CmRAP2.3的克隆与功能分析
毕业论文关键词：淹水胁迫；菊花；ERF类转录因子；基因克隆
CLONING AND FUNCTIONAL VERIFICATION OF RAP2.3 GENE IN CHRYSANTHEMUM
Abstract: Chrysanthemum is one of Chinese top ten traditional flowers, which has a long history of cultivation. It has a very high value in garden ornamental enjoying and greening. Chrysanthemum is very strong, but its flooding tolerance is poor, which often causes serious losses in production or landscape planning. The aim of this thesis was to study on the gene sequence and function of CmRAP2.3 gene in chrysanthemum and explained the response mechanism to flooding stress in chrysanthemum. In this study, we used cut flower variety ‘NanNongXueFeng’ as the experimental materials. We extracted the total RNA and obtained the reverse transcription cDNA , the full length of CmRAP2.3 gene was also obtained by high-fidelity PCR. The real-time quantitative PCR method was used to study the expression of CmRAP2.3 gene in the flooded environment. Furthermore, the transcriptional activation of CmRAP2.3 gene was verified by the yeast one-hybrid assay and its subcellular localization was analyzed by transient expression in onion epidermal cells. The results showed that the open reading frame of CmRAP2.3 gene was 702 bp, which encoded 234 amino acids. This gene had a typical DNA domain of the ERF transcription factors. The real-time quantitative PCR showed that the CmRAP2.3 gene was over-expressed under the flooding condition. The yeast one-hybrid assay showed that the CmRAP2.3 gene had the transcriptional activation function. The subcellular localization demonstrated that the gene was located in the nucleus. It can be concluded that CmRAP2.3 gene involves the process of plant’s response under the flooding stress and plays an important role in regulation of plant which is in the flooded environment.
Key words: flooding stress; Chrysanthemum morifolium; ERF transcription factor; gene cloning
目录
摘要 4
关键词 4
Abstract 4
Key words 4
第一章 引言 5
第二章 CmRAP2.3基因克隆与序列分析 6
1 材料与方法 6
1．1 植物材料及处理 6
1．1．1 植物材料 6
1．1．2 处理 6
1．2 实验方法 6
1．2．1 总RNA提取 6
1．2．2 第一链cDNA的合成 6
1．2．3 高保真PCR扩增 7
1．2．4 目的片段连接 8