Structure and Function of Genes Involved in Insect Transmission of Rice Ragged Stunt Virus.

1)电泳基因组dsRNAs时，通过迁移位移检测到两种类型的基因组片段9 （S9）。将迁移较快和较慢的波段分别记为L和U。虽然大部分带L的分离物的传播效率不高，但也有一些与带U的分离物具有相同的传播效率。2)从基因组dsrna中合成U和L的cdna，克隆到质粒载体pUC119中，并进行测序。两者都有相同的1132个核苷酸长度，因此迁移率的变化不是由于长度的差异。L与U相比有一些取代，在843核苷酸位置的A到C转换在所有L中都是常见的。为了证明这种转化导致了迁移率的转移，我们合成了仅在843位核苷酸不同的U和L。电泳分析表明，这种转化是迁移率变化的主要原因。取代产生了编码多肽的Asp到Ala转换。3)与L具有相同昆虫传播率的分离株，除843核苷酸位置外，在486核苷酸位置有另一个取代。这种变化导致编码多肽的Asn到Ser转化。4)在大肠杆菌中表达了S9编码的多肽，制备了该蛋白的抗血清。在蛋白抗体的western blotting分析中，5个结构多肽中只有位于病毒粒子表面的38.6 K多肽反应阳性。上述结果表明，位于病毒粒子表面的38.6K多肽可能与昆虫载体受体相互作用，参与昆虫传播。

1) When the genomic dsRNAs were electrophoresed, two types of genome segment 9 (S9) were detected by the mobility shift.The faster and slower migrating bands were designated as L and U,respectively.Although most of the isoates with L were not transmitted efficiently, some were in the same efficiency as those with U.2) cDNAs to U and L were synthesized from the genomic dsRNAs, cloned into a plasmid vector pUC119, and sequenced.Both had the same length of 1132 nucleotides, therefore the mobility shift was not due to the differencein in length.L had some substitutions compared with U and the A to C convertion at a nucleotide position of 843 was common in all Ls.In order to prove this convertion results in the mobility shift, synthetic U and L having different nucleotides only at the position 843 were made.The electrophoretic analysis showed the convertion is responsible to the mobility shift.The substitution created the Asp to Ala convertion of the encoded polypeptide.3) An isolate with L having an equal insect transmission rate was found to have another substitution at a nucleotide position 486 in addition to taht at the position 843.This change results in Asn to Ser convertion of the encoded polypeptide.4) A polypeptide encoded by S9 was expressed in E.coli and antisera to the protein was made.In western blotting analyzes using the antibodies to the protein, only 38.6 K polypeptide located on a virion surface reacted positively among five structual polypeptides.Above results indicated that 38.6K polypeptide located on the virion surface is involved in insect transmission interacting probably with receptors of an insect vector.