微藻高效基因枪转化方法的建立

Establishment of a Highly Efficient Transformation Method for Microalgae by the PDS-1000/He Gene Gun

基因枪法是外源基因导入微藻细胞的重要手段。然而,发展至今,微藻细胞基因枪转化效率一直偏低(10~50个转化子/μg DNA),高价低效的转化方法阻碍了基于高通量转化子的基因功能分析。为了提高基因枪的转化效率,本研究以三角褐指藻为材料,从抗生素选择培养基的改良,微载体的选择、制备、包埋、点膜和轰击参数的优化,以及受体细胞的处理等方面进行了系统研究。结果显示,采用50%海水盐度f/2培养基可以提高博来霉素的效价,f/2固体培养基中2216E营养物质的加入能缩短1/3的平板筛选时间。微载体制备应选择对金(钨)粉没有吸附作用的离心管,制备量/管应少于3.5 mg。微载体轰击量每次大约为0.75 mg,过量将会造成一个轰击死亡圈,过少将导致轰击成本上升。当轰击间距A为6.35 mm,间距B为11 mm,间距C为6 cm时,可以获得最多的转化细胞。109个受体细胞铺成较厚的多细胞层能显著提高转化效率。经过上述优化与改进,本研究将现有文献报道的转化效率提高了4.7~30倍,达到295±60个转化子/μg DNA。该方法除适用于三角褐指藻外,也可广泛应用于其他微藻(杜氏盐藻、小球藻)的基因枪转化研究,可以为微藻基因工程研究提供快速,高效和可靠的操作技术。

Microprojectile bombardment is an important means to introduce foreign genes into microalgae cells. However,the efficiency of microalgae transformation by the gene gun method has been low（ 10-50 transformants/μg DNA）. This high-cost and inefficient transformation method hinders gene functional analysis based on high-throughput transformants. Using Phaeodactylum tricornutum,a silicon alga,as a transgenic acceptor cell,we carried out systematic studies on the improvement of antibiotic selection medium,selection,preparation,coating,loading of microcarriers and optimization of bombardment parameters and processing of recipient cells. The results showed that 50% salinity f/2 medium can increase the potency of zeocin,and the addition of 2216 E nutrients in the f/2 solid medium can shorten the plate selection time by 1/3. The preparation of microcarriers should be performed on centrifuge tubes with no adsorption of gold（ tungsten） powders and the amount per tube should be less than 3. 5 mg. Theamount of microcarrier bombardment per shot is about 0. 75 mg, because too much will cause a bombardment death circle,while too little will increase the bombardment cost. When the bombardment spacing A is 6. 35 mm,the spacing B is 11 mm and the spacing C is 6 cm,the largest number of transformed cells can be obtained. In addition,a thicker multicellular layer of 109 receptor cells can significantly increase transformation efficiency. After the optimization and improvement above, the transformation efficiency was increased by 4. 7 to 30 times than that reported in the previous literature,reaching 295 ± 60 transformants/μg DNA. Moreover,the optimized method could also be widely used for other microalgae（ Dunaliella Salina and Chlorella sorokiniana） transformation. Hopefully,this gene gun transformation method could provide a first-hand,intuitive,detailed and easy-to-operate procedure for scientists in the field of microalgae genetic engineering.