DEVELOPMENT OF A SYSTEM FOR INTRODUCTION AND EXPRESSION OF PLANT REOVIRUS GENES USING OR INSECT TISSUE CULTURE CELLS.

尝试引入有或没有辅助病毒的病毒基因组rna都是不成功的。因此，分离最小的感染单位，即RNA聚合酶和基因组RNA复合物，进行了研究。水稻矮缩病毒RNA聚合酶基因组RNA复合体的分离。纯化后的病毒粒子经高浓度MgCl_2处理后，在CsCl中进行平衡密度梯度离心，去除外衣壳多肽P8。分离的核心颗粒由P1、P3和P7多肽和基因组rna组成。核心颗粒保留RNA聚合酶活性。进一步对岩心进行CsTFA平衡密度梯度离心，将P1和P7从岩心中去除。所得空颗粒的主要多肽是P3。P7在梯度管中呈高密度带状，表明它与基因组rna结合。在西北印迹分析中，基因组dsRNAs特异性结合P7，而不是其他结构蛋白。基于这些结果，假设P7在活性RNA聚合酶基因组RNA复合物中起关键作用。成功地建立了飞虱的细胞培养。在与叶蝉相同的培养基中转染61 ~ 73次，获得5个细胞系。细胞大小约为叶蝉的一半，生长缓慢。接种水稻黑条矮缩病毒后，用间接免疫荧光染色证实侵染。这是在植物病毒研究中首次使用飞虱。

Attempts to introduce viral genomic RNAs with or without a helper virus were unsuccessful. Therefore, isolation of the minimum infective units, that is an RNA polymerase and genomic RNA complex, was investigated.RNA polymerase-genomic RNA complex of rice dwarf virus was isolated. After treatment of high concentration of MgCl_2, purified virions were subjected to equilibrium density gradient centrifugation in CsCl, resulting in removal of outer capsid polypeptide P8. The isolated core particle is consisted of P1, P3, and P7 polypeptides and genomic RNAs. The core particles retain an RNA polymerase activity. The core is further subjected to CsTFA equilibrium density gradient centrifugation to remove P1 and P7 from the core. The major polypeptide of the resultant empty particles was P3. P7 banded at higher density in the gradient tube, suggesting that it binds to genomic RNAs. In North-Western blotting analyzes, genomic dsRNAs specifically bound to P7, but not other structural proteins. Base on these results, it was hypothesized that P7 playd a critical role in an active RNA polymerase-genomic RNA complex.Cell culture of planthopper was successfully established. After 61-73 transfers in the same medium as that for leafhoppers, 5 cell lines were established. Cell are about a half size of leafhoppers and grow slowly. Upon inoculation with rice black-streaked dwarf virus, infection is demonstrated by indirect immuno fluorescent staining. This is the first use of planthoppers in plant virus research.