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利用烟草坏死病毒A中国大豆分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)的全长侵染性cDNA克隆构建系列突变体进行体外转录.在枯斑寄主苋色藜(Chenopodium amaranticolor)上的侵染性检测结果表明,TNV-AC亚基因组RNA1的转录起始位点位于基因组RNA的G2184,亚基因组RNA2的转录起始位点位于基因组RNA的G2460.进一步分析表明,亚基因组RNA1中p8和p6基因的翻译灭活突变可导致病毒在苋色藜上的侵染症状消失,但不影响病毒在烟草原生质体中的复制,说明p8和p6基因产物可能影响病毒在细胞之间的移动,是病毒造成枯斑所必需的基因.通过原核表达产物分析,证明了亚基因组RNA2中外壳蛋白(coat protein,CP)基因的可读框起始于基因组RNA的2612~2614位AUG.CP可读框核苷酸替换或缺失突变分析表明,完整的CP蛋白虽不是病毒建立侵染所必需的,但其翻译起始密码子区域的核苷酸序列保守性及可读框核酸序列的完整性对病毒侵染苋色藜后产生枯斑的数量、显症时间和病毒RNA的积累量具有明显影响.综合以上结果,对TNV-AC的cp基因在侵染枯斑寄主苋色藜过程中的其他功能进行了讨论.
A series of mutants were constructed using full-length infectious cDNA clones of Tobacco necrosis virus A Chinese isolate (TNV-AC) for in vitro transcription. On the Chenopodium amaranticolor The results of infectivity test showed that the transcription start site of TNV-AC subgenomic RNA1 was located in G2184 of genomic RNA and the transcription initiation site of subgenomic RNA2 was located in genomic RNA of G2460. Further analysis showed that the subgenomic RNAs p8 and Mutation inactivation of the p6 gene leads to the disappearance of the virus’s infection on the amaranth, but does not affect the replication of the virus in the tobacco protoplasts, suggesting that the p8 and p6 gene products may affect the virus’s movement between the cells and is The result of prokaryotic expression analysis showed that the open reading frame of the coat protein (CP) gene in subgenomic RNA2 originated from AUG.CP open reading frame of 2612 ~ 2614 of genomic RNA Analysis of nucleotide substitutions or deletions revealed that the intact CP protein, although not essential for the virus to establish an infestation, has conserved nucleotide sequence conserved within the translational start codon region and an open reading frame The completeness of the acid sequence has a significant effect on the number of dead spots, the time of symptom onset and the amount of viral RNA accumulated in the infected amaranth.According to the above results, the cp gene of TNV- Other functions in the process of quinoa are discussed.