导　　师： 董金皋;王锡锋

学科专业： G1001

授予学位： 硕士

作　　者： ;

机构地区： 河北农业大学

摘　　要： 　　本试验的只要目的是以主要烟草产区的pvy分离物为材料，对其进行血清学和生物学鉴定，明确不同地区分离物的分子差异、株系种类和进化关系。同时制备高质量的pvy抗血清，为以抗体为基础的检测和实验方法提供保证。　　将pvy毒原摩擦接种于白肋烟扩繁后，采用peg沉降及超离心的方法进行病毒的提纯。提纯的病毒经电镜观察到大量线状病毒粒体，sds-聚丙烯酰氨电泳结果显示提纯病毒的分子量为22kd，且杂蛋白较少。提纯病毒免疫动物，进行pvy抗血清的制备，经间接elisa法测定血清效价为8192。新制备的血清与健康烟草和rsv，scmv均无血清学反应且经western检测验证有较好的特异性。　　 Potato virus Y /(PVY/) is a very important plant virus that can infect tobacco and cause serious econimic loses. The research of molecular variability of PVY have long history, but only a few PVY CP gene from CHINA have been sequenced, so the main objective of our experiments was to iditify the biological symptoms, molecular variability, strain type and phylogenetic relationship of different PVY CP genes using the materials of 108 field tobacco isolates which were infected by virus. At the same time, the other objective is the production of PVY antiserum that is the foundation of experiment based on antibody.PVY were purified with the procedure including PEG precipitation and differential centrifugation. The purified PVY that have typical flexuous rods particles were seen by electron microscopy. Virus coat protein migrated as a single component in SDS-PAGE with MW of 20 KD.Rabbit was immunized using the purified virus and the antiserum was obtained after the fifth immunization.Indirect ELISA assay showed the antiserum had titer of 1: 8192 with high specificity to PVY.Western bloting results combined with Indirect ELISA results showed that the PVY antiserum have no serological reaction with RSV、 SCMV and health tobacco extract. The highly specificity PVY polyclonal antibody lay foundation for the serological reseach methods.Thirty-three tobacco samples showing tobacco necrosis diseases were obtained from tobacco field representative of ten regions in China. Indirect-ELISA were done using the PVY antiserum to detect the all the 108 PVY field isolates. The results shown 33 isolates have positive reaction with PVY antiserum. The health tobacco were inoculated by those 33 isolates and shown systemic symptoms including two main symptoms: vein necrosis and mosaic. All the 33 isolates sequenced in this study had the capacity to infect tobacco systemically in greenhouse, causing two main symptoms: vein necrosis and mosaicThe result of Indirect ELISA showed they were all infected by Potato virus Y /(PVY/), and then the nucleotide sequences and predicted amino acid of the coat protein /(CP/) genes of PVY tobacco isolates from different regions of China were determined. Comparison of all sequenced isolates revealed high conservation of the CP gene. The CP gene nucleotide sequence showed more than 83.2/% identity with all the PVY isolates sequenced and the amino acid sequence identity is between 93.7 to 100/%. The highest degree of identity was observed in the C-terminal half where only 11 amino acid substitutions could be observed in contrast to the N-terminal half with 22 substitutions.

关 键 词： 马铃薯 病毒 外壳蛋白 多序列比对 系统进化树

分 类 号： [S432.41 S435.72]

领　　域： [农业科学] [农业科学] [农业科学] [农业科学] [农业科学]