烟草根细胞原生质体的制备  

Protoplast preparation of tobacco root cells

作　　者：翟妞[1] 徐国云[1] 张慧[1] 郑庆霞[1] 陈千思[1] 刘萍萍[1] 王晨[1] 金立锋[1] 周会娜[1] ZHAI Niu;XU Guoyun;ZHANG Hui;ZHENG Qingxia;CHEN Qiansi;LIU Pingping;WANG Chen;JIN Lifeng;ZHOU Huina(Zhengzhou Tobacco Research Institute of CNTC,Zhengzhou 450001,China)

机构地区：[1]中国烟草总公司郑州烟草研究院,郑州450001

出　　处：《烟草科技》2022年第10期39-43,共5页Tobacco Science & Technology

基　　金：河南省科技攻关项目“基于LC-MS的烟草腺毛细胞代谢组学研究体系构建”(212102110480);国家烟草专卖局烟草基因组重大专项项目“香气前体物代谢特征在烟草香味品质模块材料筛选中的应用研究”[110202001026(JY-09)]。

摘　　要：根细胞是植物单细胞多组学研究的重要材料,烟草作为重要的模式生物之一,其根细胞原生质体的高效分离也越来越重要。为了获得产量和活性较高的烟草根细胞原生质体,以本氏烟草和栽培烟草K326为材料,采用酶解法制备了烟草根细胞原生质体,同时优化了影响原生质体分离纯化的酶解液组合、酶解时间和酶解渗透压等条件,并通过显微镜观察和台盼蓝染色鉴定其活性。结果表明:①以1.5%纤维素酶+1.5%果胶酶+0.4 mol/L甘露醇的酶解液组合,经过2h酶解、500 r/min离心10 min得到的根尖细胞原生质体产量和活性相对较高;②最终获得了活性大于70%、数量达到105数量级的较高质量烟草根原生质体。Root cells are important research materials for multi-omics of plant single-cell.As one of the main model species,the efficient isolation of protoplasts from tobacco root cells has becoming increasingly pivotal.To obtain protoplasts from tobacco root cells with high yield and vitality,protoplasts were prepared with enzymolysis method by taking Nicotiana benthamiana and tobacco cv.K326(Nicotiana tabacum)as sample materials.In the meantime,the factors affecting the separation and purification of the protoplasts from root cells,including enzymolysis solution combination,hydrolysis time,osmotic pressure and so on,were optimized.The vitalities of isolated protoplasts were examined under microscope by Trypan blue staining.The results showed that:1)The protoplast yield and vitality from the root cells were relatively higher after 2 hours of enzymatic hydrolysis with 1.5%cellulase+1.5%pectinase+0.4 mol/L mannitol and centrifugation at 500 r/min for 10 min.2)The obtained tobacco root protoplasts featured high quality with the vitality greater than 70%and reached 105orders of magnitudes.

关 键 词：烟草 根细胞 酶解法 原生质体 

分 类 号：S572.01[农业科学—烟草工业]