Mukherjee Krishanu, Verma Yogita, Chakrabarti Swarup Kumar, Singh M N, Khurana S M Paul
Division of Plant Pathology, Central Potato Research Institute, Shimla 171 001, H.P., India.
Virus Genes. 2003 May;26(3):247-53. doi: 10.1023/a:1024495129556.
An Indian strain of potato leaf roll virus (PLRV) was purified to generate complementary DNA corresponding to the coat protein (CP) gene. Virus cDNA was synthesized from purified viral RNA using oligo (dT)-anchor primer and virus specific primers. The viral sequence encoding the coat protein was specifically amplified by polymerase chain reaction (PCR), using specific primers bordering the CP gene. The unique amplified product thus obtained was A-T cloned into the pGEM-T Easy vector and the authenticity of the cloned gene verified by dot blot hybridization and sequence analysis. Run-way-transcripts of the cloned CP gene could detect PLRV in tissue imprints and tissue dilution. The nucleotide sequences and the deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99% identical at both the nucleotide and amino acid sequence level of other isolates. Multiple sequence alignment of deduced amino acid sequences revealed considerable homology to other luteoviruses. A nuclear localization signal located close to the N-terminus of the CP gene was predicted. This is the first report of PLRV coat protein sequence from an Indian strain.
一种印度马铃薯卷叶病毒(PLRV)毒株被纯化,以产生与外壳蛋白(CP)基因对应的互补DNA。使用寡聚(dT)-锚定引物和病毒特异性引物,从纯化的病毒RNA合成病毒cDNA。利用与CP基因相邻的特异性引物,通过聚合酶链反应(PCR)特异性扩增编码外壳蛋白的病毒序列。将如此获得的独特扩增产物A-T克隆到pGEM-T Easy载体中,并通过斑点杂交和序列分析验证克隆基因的真实性。克隆的CP基因的体外转录本可在组织印记和组织稀释液中检测到PLRV。将核苷酸序列和推导的氨基酸序列与其他PLRV分离株进行比较,发现在核苷酸和氨基酸序列水平上与其他分离株的一致性为97-99%。推导的氨基酸序列的多序列比对显示与其他黄症病毒有相当高的同源性。预测在CP基因N端附近有一个核定位信号。这是关于印度毒株PLRV外壳蛋白序列的首次报道。