摘要本研究旨在通过对根癌农杆菌侵染洋葱表皮细胞的条件进行优化,从而建立一种新的瞬时表达系统,并将其应用于玉米In5-2启动子的功能区域的分析中，明确In5-2启动子的乙酰苯胺类化合物诱导元件的具体位置。在本研究中采用根癌农杆菌(Agrobacterium tumefaciens)侵染洋葱(Allium cepa)表皮细胞,对转β-葡糖醛酸酶(GUS)基因的瞬时表达进行研究,并分析了侵染液中乙酰丁香酮(As)的浓度、侵染时间、菌液浓度、共培养时间对GUS基因的瞬间表达的影响。结果显示，在OD600值为0.8 的农杆菌液中感染15 min ,共培养3 d ,能够得到较高的GUS 基因瞬间表达，从而建立了一种新的瞬间表达系统。同时，构建了含不同长度的玉米(Zea mays)In5-2启动子片段缺失载体，利用新的瞬时表达系统分析其功能区域，推测出乙酰苯胺类化合物诱导元件位于ATG上游 -220~-143 bp 之间。结果表明新的瞬时表达系统可以快速有效地进行启动子的分析。
Abstract：This study was designed to optimize the factors about onion (Allium cepa) epidermal cells infected by Agrobacterium tumefaciens so as to establish a new transient expression system. Then the new transient expression system was applied to analyze maize In5-2 promoter functional area to clear the position of cis-acting elements responsive to chloroacetanilide. In this experiment onion(Allium cepa) epidermal cells were transformed with beta-glucuronidase(GUS) gene via Agrobacterium tumefaciens. The effects of different concentration of acetosyringone (As), infection period, concentration of Agrobacterium tumefaciens and days of co-culture on the transient expression of GUS gene were analyzed to establish a new transient expression system. The result indicated that a higher transient express of GUS gene could be acquired from onion epidermal cells which were dipped into Agrobaterium suspension (OD600 adjusted to 0.8, concentration of As adjusted to 20 μmol/L) for 15 min, and co-cultured for 3 days. So a new transient expression system was established. Then different length of maize(Zea mays) In5-2 promoter deletions were cloned to vector pCAMBIA1301 and transformed into the new transient expression system to analyze its functional regions. Cis-acting responsive elements to chloroacetanilide could be located within the range of -220 to -143 bp upstream of ATG. The results indicated that the new transient expression system is a fast and effective way to analyze promoter function.
