Subviral RNAs of TCV: Symptom Induction and the Generation of Discontinous RNAs

芜菁皱缩病毒(TCV)将被用作研究症状表现、缺陷干扰(DI)RNA形成和RNA重组的模型。TCV系统是唯一适合于这些研究的系统；它是唯一与无毒和有毒的sat-RNAs相关的动植物病毒，它可以重组它们的基因组和DI RNAs，后者可以从体外合成的TCV转录本产生从头开始。此外，TCV的两个分离物具有不同的生物学特性：亚病毒RNA仅在与TCV-M分离物相关时才会产生症状，而DI RNA仅从TCV-B分离物中产生。将构建TCV嵌合基因组RNA，以确定TCV RNA的哪个区域参与了不同症状的产生和DI的形成。还将研究DI RNAs和sat-RNA重组体的形成机制。Sat-RNAC中假定的内部复制酶识别序列位于所有重组Sat-RNAs的右手端，它将被删除、改变和/或从其当前位置移动，以确定该序列在重组中的重要性。对于后代RNA的形成，还将测试复制选择模型与断裂和连接模型。具有明确突变的SAT-RNA转录本，允许正链或负链的差异复制，将被添加到原生质体和植物提取物中。RNA重组将通过聚合酶链式反应技术进行检测。

Turnip crinkle virus (TCV) will be used as a model to study symptom manifestation, defective interfering (DI) RNA formation and RNA recombination. The TCV system is uniquely suited for these studies; it is the only plant or animal virus associated with avirulent and virulent sat-RNAs which can recombine their genomes and DI RNAs which can be generated de novo from in vitro synthesized TCV transcripts. Furthermore, two isolates of TCV have distinct biological properties: subviral RNAs only produce symptoms when associated with the TCV-M isolate and DI RNAs have only been generated from the TCV-B isolate. Chimeric TCV genomic RNAs will be constructed to ascertain which region of TCV RNA is involved in differential symptom production and DI formation. The mechanism by which DI RNAs and sat-RNA recombinants are formed will also be investigated. The putative internal replicase recognition sequence in sat-RNA C, found at the right hand junction of all recombinant sat-RNAs, will be deleted, altered and/or moved from its current location to determine the importance of the sequence in recombination. A copy choice model versus a breakage and ligation model for the formation of descendance RNAs will also be tested. Sat-RNA transcripts with defined mutations, allowing differential replication of plus or minus strands, will be added to both protoplasts and plant extracts. RNA recombination will be detected by polymerase chain reaction techniques.***//