Example of the Research

Plum scab virus coat protein coding gene cloning

and protein synthesis in E. coli cells

Plum scab disease, caused by plum pox potyvirus (PPV), represents of the most prevalent and damaging diseases of large fruit.

Successful fight against spreading of the virus requires effective virus testing means and possibility to create varieties resistant to this virus. Resistance of plants to a pathogen may be induced by transformation of genes of the pathogen. Clonning of virus protein genes and introduction of these genes into plants allows for creation of transgene plants resistant to this virus.

A gene coding coat protein of the PPV virus was cloned. The RT-PCR method was used for cloning of the CP sequence. Elements, corresponding to the end of regions 5’ and 3’ of the mature protein coded by CP genes, were synthesized. After amplification, CP cDNA was cloned. The identified sequence of amino acids contained 993 bp CP amino acids sequence. This sequence was compared to sequences of other plum pox virus coat proteins.

Expression of a specific virus coat protein gene in transgenic plants protects the plant from later infection by the virus, from which the CP gene has been obtained and, in some cases, it protects from infections by related viruses.

Structures containing the CP gene were transformed into the binary system of vectors of Nicotiana benthamiana via Agrobacterium tumefaciens. After transformation, calluses were grown on the medium with canamicin. Upon obtaining model transgenic Nicotiana benthamiana plants, expressing differently modified coat protein gene, the structure providing the most optimum resistance result is selected. Results of this experiment are used for development of transgenic plants resistant to the plum pox virus.

Expression of the CP in E.coli cells. For this purpose, the CP was expressed in E.coli cells. Upon insertion of the whole region coded by the CP into pET16b, the recombination vector was transferred to E.coli BL21 cells. Quick extraction of protein from E.coli cells was performed. Electrophoresic analysis of cell extracts and Western imunoblot testing revealed expression of this protein in E. coli cells.