Matousek J, Schröder A R, Trnĕná L, Reimers M, Baumstark T, Dĕdic P, Vlasák J, Becker I, Kreuzaler F, Fladung M
Institute of Plant Molecular Biology, Czech Academy of Sciences, Ceské Budĕjovice.
Biol Chem Hoppe Seyler. 1994 Nov;375(11):765-77. doi: 10.1515/bchm3.1994.375.11.765.
Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.
利用温度梯度凝胶电泳以及针对双链RNA的特异性抗体进行免疫化学检测，研究了针对马铃薯纺锤块茎类病毒（PSTVd）正链或负链序列的不同反义RNA之间的复合物形成情况。短负链序列针对正链类病毒复制中间体的上部中央保守区（UCCR），一条对应于杆状二级结构左半部分的正链（VL +）针对负链复制中间体。结果表明，反义RNA仅在低温（生理）孵育期间以低产率与相应的靶RNA形成复合物，但当反义RNA已存在于溶液中时，在靶RNA的体外转录过程中以高产率形成复合物。通过根癌农杆菌转化将反义RNA序列整合到马铃薯中。通过Northern分析体内分析反义RNA的表达。使用转基因马铃薯品系进行感染试验，以评估它们对PSTVd感染的抗性程度。尽管一些品系显示出对类病毒积累的显著抑制，但在不同转基因马铃薯品系中获得了类病毒感染的高度变异性。由于在接种后6至8周在所有转基因品系中都观察到了严重感染的植株，因此必须假定存在一个超过反义效应的类病毒阈值浓度。当使用叶盘上的农杆菌感染试验测试类病毒积累率时，可以实现更强的反义效应。
