油菜NF-YA9的启动子的克隆以及功能片段筛查研究
毕业论文关键词：油菜；转录因子；顺式作用元件；启动子
Cloning of NF-YA9 promoter from Brassica napus and screening of its functional fragments
Student majoring in ecology Jin Zeyang
Tutor Liang Mingxiang
Abstract：Canola (Brassica napus) is a kind of highly adaptable crop, such as salt tolerance, drought, high temperature and so on. With the help of this kind of characteristics, modern biology and molecular biology has been able to obtain new varieties with stronger resistance to stress by transgenic methods. In this study, the sequence of BnNF-YA9 was based on the previous experiment and PLANTCARE were used to predict the cis acting element of BnNF-YA9 promoter from ‘Nanyanyou No. 1’. After analysis, the promoter was pided into 5 segments, and primers of each segment were designed by Primer Premier 5, and fragment cloning were completed. Finally, partial fragments are cut by double enzymes and linked by restriction enzyme and binary vector pCAMBIA-1300 expression vectors containing 536bp fragments were successfully constructed.
Key words: Canola；Transcription factor；Cis-acting element；Promoter
目录
摘要1
关键词1
Abstract1
Key words1
引言1
1相关背景1
1.1 NF-Y转录因子2
1.2启动子概述2
1.3顺式作用元件概述2
2材料与方法2
2.１材料2
2.1.1植物材料2
2.1.2菌种与载体2
2.1.3工具酶与试剂盒 2
2.2方法 3
2.2.1油菜DNA的提取3
2.2.2油菜BnNF-YA9启动子的克隆3
2.2.2.1引物设计3
2.2.2.2启动子的克隆 5
2.2.2.3生物信息学分析6
2.2.3 含BnNF-YA9启动子目的基因表达载体的构建6
2.2.3.1酶切位点的选择及含酶切位点引物的设计6
2.2.3.2 目的基因含酶切位点的高保真扩增8
2.2.3.3 构建表达载体8
3结果与分析9
3．1BnNF-YA9启动子的克隆9
3.1.1普通PCR扩增结果9
3.1.2高保真PCR扩增结果10
3.1.3菌落PCR验证及测序比对结果10
3．2含BnNF-YA9启动子目的基因表达载体的构建11
3.2.1含酶切位点的高保真PCR扩增结果11
3.2.2目的基因片段双酶切结果12
4讨论12
4.1 PCR法扩增启动子12
4.2后期研究和展望12
致谢13
参考文献14
油菜NF-YA9的启动子的克隆以及功能片段筛查研究
引言 油菜，十字花科芸薹属，根系笔直，茎直立，分枝少，主要包括芥菜型 (B．Juncea)、甘蓝型 (B．napus)和白菜型油菜(B．rapa)3个栽培种。油菜作为我国主要的油料作物之一，其特点是在逆境环境下生长，给人类带来环境效益的同时又带来经济效益。我国的沿海滩涂资源丰富，滩涂面积高达 21709 km2，其中江苏省沿海滩涂面积达 0.5×104 km2，占全国滩涂面积的24%[1,2]。随着现代盐土农业概念的提出，沿海滩涂耐盐作物种质的研究和挖掘在最近几年也已相继取得一些进展。此次实验用的是甘蓝型油菜‘南盐油1号’ ，它是由南京农业大学自主研究，并在滩涂上种植而选育出的具有很好耐干旱、耐盐等逆境胁迫的油菜品种。通过克隆油菜BnNF-YA9启动子，为后续研究启动子上的顺式作用元件提供条件，也为将来油菜的抗逆品种的培育提供一些帮助。