目录
摘要Ⅱ
关键词Ⅱ
AbstractⅢ
引言
1引言1
2 材料与方法2
2.1 实验材料 2
2.1.1水稻材料2　
2.1.2水稻培养2　
2.1.3水稻镉处理2
2.2实验方法 2
2.2.1　水稻基因组DNA的提取2
2.2.2　酵母报道载体的构建3
2.2.3　酵母单杂交实验6
2.2.4　转录组测序结果分析7
3 结果与分析8
3.1 启动子序列分析8
3.2 构建酵母筛选载体 9
3.3 酵母单杂交筛选OsNRAMP5启动子结合蛋白9
3.4 互作基因的验证11
3.5 转录组测序分析Cd响应基因 12
4 讨论15
致谢15
参考文献15
附录一17
附录二17
附录三18
酵母单杂交筛选水稻基因NRAMP5上游转录因子
摘 要
OsNRAMP5是水稻镉吸收的主要转运体，为了研究调控OsNRAMP5表达的机制，本实验利用酵母单杂交技术，依据OsNRAMP5启动子序列，分析目的顺式作用元件，构成酵母报道载体（pBaitAbAi），并转化进酵母细胞Y1H Gold，生成BaitReporter酵母菌株，再共转化诱饵菌株，通过同源重组筛选OsNRAMP5上游调控因子。酵母单杂交筛库结果经测序及Blast分析，获得了如OsHSFA4A、OsHSFA4D等一共8个转录因子。进一步的互作实验证实了OsHSFA4A、OsHSFA4D等其6个转录因子能与OsNRAMP5启动子结合。同时对镉处理水稻根进行转录组测序，发现共计4622个差异表达基因，其中2816个基因上调，1806个基因显著下调。将RNASeq分析与酵母筛库结果进行比对，发现筛库所得8个转录因子中有5个表达量上调，2个略微下调。综合结果表明，OsHSFA4A、OsHSFA4D等6个转录因子可能参与OsNRAMP5转录的调控，这一发现为更加明确水稻对镉的吸收转运机制打下基础。 *51今日免费论文网|www.51jrft.com +Q: &351916072& 

SCREENING OF UPSTREAM TRANSCRIPTION FACTORS OF RICE NRAMP5 BY YEAST ONE HYBRID
ABSTRACT
OsNRAMP5 has been confirmed that is a major transporter of cadmium absorption of rice, in order to study the mechanisms that control OsNRAMP5 expression, this experiment using yeast single hybrid technology, on the basis of OsNRAMP5 promoter sequences, cis effect element analysis purpose, constitute a yeast carrier reported (pBaitAbAi), and conversion into yeast cells Y1H Gold, generate the BaitReporter yeast strains, then cotransformation Bait strains, screening by homologous recombination OsNRAMP5 upstream regulatory factors. After sequencing and Blast analysis, a total of 8 transcription factors, such as OsHSFA4A and OsHSFA4D, were obtained. Further interaction experiments confirmed that OsHSFA4A, OsHSFA4D and other six transcription factors could bind to the promoter of OsNRAMP5. At the same time, through transcriptome sequencing of cadmiumtreated rice roots, a total of 4622 differentially expressed genes were found, among which 2816 genes were upregulated and 1806 genes were significantly downregulated. The RNAseq analysis was compared with the results of the yeast sieve bank, and it was found that 5 of the 8 transcription factors obtained from the sieve bank were upregulated and 2 were slightly downregulated. In summary, OsHSFA4A, OsHSFA4D and other six transcription factors may be involved in the regulation of OsNRAMP5 transcription, which lays a foundation for a clearer understanding of the mechanism of cadmium uptake and transport in rice.
KEY WORDS：Cadmium; Yeast One Hybrid; OsNRAMP5; Transcription factors

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