权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

开发使用昆虫组织培养细胞引入和表达植物呼肠孤病毒基因的系统。

基本信息

批准号：
05454129
负责人：
UYEDA Ichiro
金额：
$ 4.48万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454129/
关键词：
Plant reoviruses Vector insects Tissue culture イネ萎縮ウイルス媒介昆虫細胞

项目摘要

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.

在有或没有辅助病毒的情况下引入病毒基因组RNA的尝试都是不成功的。因此，本研究对水稻矮缩病毒的最小感染单位即RNA聚合酶和基因组RNA复合体的分离进行了研究。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，除去外壳多肽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。将芯进一步进行CsTFA平衡密度梯度离心以从芯中除去P1和P7。所得空颗粒的主要多肽是P3。P7在梯度管中以更高的密度条带化，表明其结合基因组RNA。在North-Western印迹分析中，基因组dsRNA特异性结合P7，但不结合其他结构蛋白。根据这些结果，推测P7在活跃的RNA聚合酶-基因组RNA复合物中起关键作用。在与叶蝉相同的培养基中经过61-73次转移后，建立了5个细胞系。细胞大小约为叶蝉的一半，生长缓慢。在接种水稻黑条矮缩病毒后，通过间接免疫荧光染色证明感染。这是首次将稻飞虱用于植物病毒研究。