超声辅助法促进水曲柳悬浮细胞的遗传转化探究  

Ultrasound-aided Genetic Transformation into Suspended Fraxinus mandshurica Cells

作　　者：汤明硕 王雯萱 马子越 刘林[1,2] 孔繁秋 詹亚光 齐凤慧[1,2] Tang Mingshuo;Wang Wenxuan;Ma Ziyue;Liu Lin;Kong Fanqiu;Zhan Yaguang;Qi Fenghui(State Key Laboratory of Tree Genetics and Breeding,Northeast Forestry University,Harbin,150040;College of Life Sciences,Northeast Forestry University,Harbin,150040)

机构地区：[1]东北林业大学,林木遗传育种国家重点实验室,哈尔滨150040 [2]东北林业大学生命科学学院,哈尔滨150040

出　　处：《分子植物育种》2024年第23期7757-7763,共7页Molecular Plant Breeding

基　　金：黑龙江省应用技术研发项目(GA19B201);中央高校基础研究经费(2572020DR09);东北林业大学大学生创新项目(202110225521)共同资助。

摘　　要：使用水曲柳悬浮细胞结合超声辅助处理,开发了一种有效且可重复的根癌农杆菌介导的遗传转化方法。以GUS和GFP为目标基因,悬浮细胞为受体材料,以不同时间的超声加真空处理辅助农杆菌介导法进行水曲柳悬浮细胞遗传转化研究。结果显示:以对数生长期的水曲柳悬浮细胞作为转化受体,根癌农杆菌浓度OD600为0.6时,与2 g悬浮细胞进行混合,并在超声波中超声90 s,真空下10 min进行侵染转化,共培养之后接种在添加50 mg/L卡那霉素和500 mg/L头孢霉素的固体或液体培养基中选择培养,所得筛选后细胞可观察到明显的GUS染色与GFP荧光,PCR能得到两条清晰的目的基因条带,说明获得了水曲柳转基因悬浮细胞。试验证明使用超声加真空辅助的农杆菌介导法可以有效地对水曲柳悬浮细胞进行遗传转化。An effective and reproducible genetic transformation method mediated by Agrobacterium tumefaciens was developed using Fraxinus mandshurica suspension cells combined with ultrasound-assisted treatment.With GUS and GFP as the target genes,and Fraxinus mandshurica suspension cells as the receptor materials,the genetic transformation of Fraxinus mandshurica was studied using the Agrobacterium-mediated method and assisted by ultrasonic and vacuum treatment at different times.The Fraxinus mandshurica suspension cells in the logarithmic growth phase were used as the transformation receptor.When the concentration of Agrobacterium tumefaciens was OD600=0.6,bacteria were mixed with 2 g of the suspension cells and sonicated for the 90 s under ultrasound and 10 min under vacuum for infection and transformation.After co-cultivation,cells were inoculated in a solid or liquid medium supplemented with 50 mg/L kanamycin and 500 mg/L cephalosporins.After the screening,the cells obtained could be observed with obvious GUS staining and GFP fluorescence,and PCR could get two clear target gene bands which proved that the transgenic Fraxinus mandshurica suspension cells were obtained.Studies prove that the use of the Agrobacterium-mediated method that is assisted by ultrasonic and vacuum can effectively genetically transform Fraxinus mandshurica suspension cells.

关 键 词：水曲柳 超声 悬浮细胞 遗传转化 

分 类 号：S792.41[农业科学—林木遗传育种]