Reverse transcription-polymerase chain reaction (RT-PCR) amplification, cloning, expression and purification of tomato chlorosis virus p22 protein
2007
Farhan, K.
Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus are two whitefly-transmitted criniviruses with bipartite genomes causing indistinguishable chlorotic symptoms on tomato plants. Diagnostic reverse-transcription polymerase chain reaction (RT-PCR) determined that the observed chlorosis on tomato plants collected from the western part of Crete was due to ToCV infections. The complete RNA dependent RNA polymerase, coat protein, heat shock protein 70 and p22 genes of ToCV genes were RT-PCR amplified, cloned and sequenced, to reveal only minor nucleotide differences with the already available sequences of the American and Spanish isolates of the virus. The complete ToCV-p22 gene was cloned into the pMAL-c2x expression vector and the fusion protein pMAL-ToCV-p22 was overexpressed in Escherichia coli and purified to near homogeneity by affinity and ion exchange chromatography. These experiments constitute the initial steps towards the elucidation of the role of ToCV p22 in the replication of the virus.
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Эту запись предоставил Mediterranean Agronomic Institute of Chania