摘要
为了验证转基因烟草中表达的外壳蛋白（ＣＰ）能够重新包被侵入的烟草花叶病毒（ＴＭＶ）的假设，利用抗原表位标记的方法观察ＣＰ亚单位在病毒５′端的交换。通过ＰＣＲ 方法将来源于鼠肝炎病毒（ＭＨＶ） Ｓ蛋白的两个小肽段（１１ ａ．ａ．和１５ ａ．ａ．）的ＤＮＡ序列分别插入ＴＭＶ－Ｕ１ ＣＰ基因邻近３′端的两个位点，并构建了带有外源序列的ＴＭＶ 侵染克隆Ｖ９ （１１ ａ．ａ．）和Ｅ１５ （１５ ａ．ａ．）。通过体外转录反应，得到Ｖ９ ＲＮＡ 及Ｅ１５ ＲＮＡ。突变病毒ＲＮＡ 侵染烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）后表现不同特性。Ｖ９ 和Ｅ１５ 侵染ＸａｎｔｈｉＮＮ烟草后同野生型ＴＭＶ一样产生枯斑。但是，当它们侵染Ｘａｎｔｈｉｎｎ 烟草时，Ｖ９ 产生同侵染ＸａｎｔｈｉＮＮ 烟草相同的枯斑，而Ｅ１５的特性同ＴＭＶ－Ｕ１几乎完全相同，能对Ｘａｎｔｈｉｎｎ 烟草进行系统侵染并在叶片中聚集大量的带有外源片段的外壳蛋白，而且病毒的结构极其稳定。Ｖ９ 和Ｅ１５
To test the hypothesis and prediction that transgenic coat protein (CP) could recoat the uncoated virions, an epitope tag approach was exploited to look for the exchange of coat protein subunits at the 5′ end of the challenged virus. By mutagenesis using a PCR based method, DNA sequences, encoding peptide epitopes (11 and 15 amino acids in length) of the murine hepatitis virus (MHV) S protein were separately inserted into one of the two sites near the 3′ end of the coded region of the TMV CP gene. Two TMV infections clones with each of the tagged coat protein genes, V9 (11 amino acids) and E15 (15 amino acids) were generated. Viral RNA was produced by in vitro transcription with T7 RNA polymerase and used to infect Xanthi nn and Xanthi NN tobacco plants. Both V9 and E15 caused local necrotic lesion in NN tobacco plants similar to those induced by wild type TMV. Systemic symptoms identical to those caused by TMV U1 infection and equivalent amounts of coat protein with the MHV epitope (E15) were found in Xanthi nn tobacco plants. Unexpectedly, similar types of local lesions were observed on the inoculated leaves of Xanthi nn tobacco plants infected by V9. The different sites of insertion of each MHV epitope may be responsible for the different characteristics of V9 and E15.
关键词
烟草
转基因烟草
外壳蛋白
烟草花叶病毒
Transgenic plants
Coat protein
Tobacco mosaic virus
Epitope tag
Murine hepatitis virus
