两个缺陷型TMV粒子在烟草中的互补表达

根据对ＴＭＶ高效复制和基因表达的顺式作用元件的分析，在体外重组包装了２个缺失型ＴＭＶ粒子：ＴＭＶＲＰ和ＴＭＶＣＰ。前者缺失了ＴＭＶ外壳蛋白ＣＰ基因的３′端及后序区域，后者缺失了大部分复制酶基因。把两者分别或共同电击感染烟草原生质体：１．用ＣＰ抗体进行免疫印渍检测，单独感染的原生质体内的ＣＰ在１６小时内无增加，而在共同感染的原生质体内，ＣＰ在感染２小时后就开始明显增加。２．用ＲＴ一两次ＰＣＲ法专一地检测新生负链ＲＮＡ的合成情况，在单独感染的原生质体内没有检测到，但在混合感染的原生质体内在感染１小时后就检测到ＣＰ基因特异的负链ＲＮＡ的形成，并用Ｓｏｕｔｈｅｒｎ杂交得到进一步验证。这些结果表明，复制酶缺失型ＴＭＶＣＰ内的ＣＰ基因不能表达，但可以在ＴＭＶＲＰ存在时，通过其所表达的复制酶互补作用得到复制从而有效表达．

The Construction and Complementary Expression of two TMV Defective Particles in Tobacco

Two defective mutants of the tobacco mosaic virus(TMV),TMVRP and TMV CP were constructed and assembled in vitro.In TMVRP,he 3'-end and in part of the TMV coat pretein(CP)gene was deleted;In TMRCP,most of the replicase genes were deleted.These mutant particles were seperately or complementally inoculated nit,the tobacco protoplasts by electroporation.The synthesis of TMV coat protein was detected by immuno dot-blot technique at 2h after inoculation only in those complementally infected protoplasts. In addition,using the RT/PCR technique,the minus-strand viral RNA of the TMV 3'-end including cp gene was also only detected in those protoplasts which wore complementally infected with both of the mutant particles.The synthesis of the minus-strand RNA started to be detectable at 1h after inoculation and it was confirmed by Southern blot analysis.