用不同方法把烟草花叶病毒RNA基因导入植物原生质体的研究

应用电激法和聚乙二醇法以及脂质体协调的上述两种方法对烟草和青菜原生质体进行烟草花叶病毒ＴＭＶ－ＲＮＡ的导入试验，并应用酶标免疫技术、电镜观察、半叶接种和十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）等方法对在原生质体中增殖的ＴＭＶ进行鉴定。实验证明，虽然电激法和聚乙二醇法均能有效地将外源病毒基因导入植物原生质体，但经阳离子脂质体处理后的ＴＭＶ－ＲＮＡ，其转染效率可提高１０倍以上。ＴＭＶ在原生质体转染４８小时后达到复制高峰。ＳＤＳ－ＰＡＧＥ显示，原生质体转染４８小时后，除出现ＴＭＶ外壳蛋白明显条带外，尚有１条分子量在５０～５５ｋｄ蛋白质条带也明显增强。这些研究结果对植物遗传工程和抗病毒基因有种研究提供重要的数据和基础。

Transfection of Plant Protoplasts with Tobacco Mosaic Virus RNA by Using Electroporation, PEC and Cationic Liposome-ediated Methods

Tobacco mosaic virus RNA was introduced into protoplasts from tobacco (Nicotiana tabacum L. cv. bright yellow)and Chinese cabbage (Brassica chinensis)by eleotroporation, PEG treatments and cationio liposome-mediated both of the above methods.The results indicated that although both of eleetroporation and PEG treatment methods could introduce TMV-RNA into protoplasts, but cationic liposome-mediated these two methods could enhance infection significantly. The minimum amount of TMV-RNA necessary for transfeotion of Protoplasts decreased down to 10 times, when the TMV-RNA molecules were eneapsulated before introduction into protoplasts by eleotroporation or PEG treatment. The results also showed that TMV could multiply inside protoplasts and reach the maximum after 48 hours of introduction of TMV-RNA. SDS-PAGE of the roughly extraeted solutions of transfected protoplasts at 48 hours after introduction of TMV-RNA showed that a 17.5 kd band, the molecular weight of which is equal to TMV coat protein clearly appeared and besides, there also a 50-55 kd protein band could be observed from these transfected protoplasts. The nature of this protein band which was absent from control protoplasts would be of interest to us in further studies.