[烟草亚细胞定位]农杆菌瞬时侵染烟草-激光扫描共聚焦法

[植物科学领域]利用农杆菌侵染烟草进行体内瞬时表达的方法
Method for transient expression in vivo by infecting tobacco with Agrobacterium
一、原理（Principle）
烟草叶片的瞬时表达系统常用来进行基因的亚细胞定位监测，监测蛋白在细胞中分布的位置，从而对其功能进行预测。

通过基因翻译的蛋白与绿色荧光蛋白构成融合蛋白，在激光共聚焦仪器下观测绿色荧光的分布，判断蛋白表达的部位。

同时，还可根据荧光的光强度检测蛋白的表达量。

该过程是经根癌农杆菌介导的，进而将目的基因整合到到烟草的细胞内的。

Tobacco leaf transient expression systems are often used to monitor the subcellular localization of genes, monitor the distribution of proteins in cells, and predict their function. The gene translated protein and green fluorescent protein constitute a fusion protein. The distribution of green fluorescence is observed under a laser confocal instrument to determine the protein expression site. At the same time, the expression level of the protein can also be detected based on the light intensity of the fluorescence. This process is mediated by Agrobacterium tumefaciens, which integrates the gene of interest into tobacco cells.
二、材料与试剂
1. 携带表达载体的农杆菌菌株（通常表达载体由35S启动子驱动）
2. 2-4周的烟草植株
3. LB培养基
4. 乙酰丁香酮
5. MES: 2-(N-吗啉代)乙磺酸
6. 抗生素
7. 注射器
Materials and reagents
1. Agrobacterium strain carrying an expression vector (usually the expression vector is driven by the 35S promoter)
2. Tobacco plants for 2-4 weeks
3.LB medium
4.Acetosyringone
5.MES: 2- (N-morpholino) ethanesulfonic acid
6.Antibiotics
7. syringe
三、仪器
1. 50 ml 离心管
2. 光谱仪
3. 紫外灯
4. 荧光显微镜
Third, the instrument
1. 50 ml centrifuge tube
2. spectrometer
3. UV lamp
4. fluorescence microscope
四、步骤
1. 挑取单克隆于5 ml LB液体培养中，28~30°C震荡培养。

通常，LB中加入100 µg/ml 庆大霉素（农杆菌株GV3101携带抗性），50 µg/ml 大观霉素（载体携带）。

2. 将1 ml 过夜培养的农杆菌转接到25 ml LB液体培养基中（加有与1相同的抗生素，另外加入高压灭菌的乙酰丁香酮）。

3. 检测过夜培养的菌液OD600的值。

4. 5000 g，15分钟集菌，用重悬液重悬菌体，最终OD600为0.4。

5. 室温放置2~3 h后注射烟草。

6. 将侵染液装入5 ml 注射器内，用拇指按压注射器反板将液体从叶片下表皮注射到烟草叶片内（勿使用子叶）。

注射后，烟草叶片会出现湿润的现象。

7. 注射后2-5天，在便携式长波长紫外灯下检测GFP荧光信号（只适用于荧光很强的叶片）。

8. 通过荧光显微镜或者激光共聚交荧光显微镜检测GFP信号。

同时，可以提取蛋白，检测蛋白的含量。

Steps
1. Pick a single clone in 5 ml LB liquid culture and shake culture at 28 ~ 30 °C. Generally, LB is added with 100 µg / ml gentamicin (agrobacterium strain GV3101 carries resistance) and 50 µg/ml spectinomycin (carried by carrier).
2. Transfer 1 ml of Agrobacterium cultured overnight to 25 ml of LB liquid medium (add the same antibiotic as 1 and add autoclaved acetylsyringone).
3. Check the OD600 value of the bacterial solution cultured overnight.
4. 5000 g, 15 minutes to collect bacteria, resuspend the bacteria with a resuspension, the final OD600 is 0.4.
5. Inject tobacco after placing at room temperature for 2 ~ 3 hours.
6. Fill the infectious solution into a 5 ml syringe, and press the back of the syringe with your thumb to inject the liquid from the lower epidermis of the leaf into the tobacco leaf (do not use cotyledons). After injection, the tobacco leaves will appear moist.
7. 2-5 days after injection, detect the GFP fluorescence signal under a portable long-wavelength UV lamp (only applicable to highly fluorescent leaves).
8. Detect the GFP signal using a fluorescence microscope or a laser copolymerized fluorescence microscope. At the same time, protein can be extracted and the content of protein can be detected.
五、配方
1. 加有相应抗生素的LB液体培养基（一种抗生素是菌株携带，一种为载体携带）。

2. 乙酰丁香酮（100 mM 溶于乙醇，-20°C储存）。

3. 1 M MgCl2
4. 重悬液（10 mM MgCl2，10 mM 2-(N-吗啉代)乙磺酸（pH
5.6）高温高压灭菌15分钟，100 uM 乙酰丁香酮，高温高压灭菌）。

Five, formula
1. LB liquid medium supplemented with corresponding antibiotics (one antibiotic is carried by the strain and one is carried by the carrier).
2. Acetylsyringone (100 mM in ethanol, stored at -20 °C).
3.1 M MgCl2
4. Resuspend (10 mM MgCl2, 10 mM 2-(N-morpholino) ethanesulfonic acid (pH=
5.6), autoclave for 15 minutes, 100 µM acetylsyringone, autoclave).。