Isolation of Kenaf leaf peroxisomes to investigate the localization of Hibiscus chlorotic ringspot virus (HCRSV) coat protein (CP) Setiawan F. and Wong S.M

Coat protein (CP) of Hibiscus chlorotic ringspot virus (HCRSV) had been found to be responsible for the spread of infection. Kenaf (Hibiscus cannabinus) was used as a model organism in this study. As CP is most likely localized in the peroxisomes, there is a need to produce a protocol adapted solely for the isolation of peroxisomes from the leaf of Kenaf as peroxisomal characteristics differ greatly within and amongst plants. At the moment, Sucrose and Percoll density gradient are the two most commonly used method to isolate peroxisomes. Modifications were made to the protocols to optimize the yield of peroxisomes from the leaves of both healthy and virus infected Kenaf. Hydroxypyruvate reductase assay, Western blot analysis and transmission electron microscopy (TEM) were carried out to check the presence and the intactness of the peroxisomes eluded. The presence of CP inside the peroxisomes was also investigated through Western blot after thermolysin treatment to remove outer proteins. The results of this study showed that the protocols yield only low amount of intact peroxisomes. The identification of CP through Western blot also did not yield satisfactory result. As such, this study will serve as a platform for further investigation in the similar direction. INTRODUCTION CP of HCRSV was responsible for the spread of infection in the plant it infected. In this study, Kenaf was used as a model organism. As CP was expected to be localized in the peroxisomes, peroxisomes study thus need to be carried out. Firstly, this study will optimize the two protocols used to isolate peroxisomes, namely the sucrose density gradient method and the Percoll density gradient method. The isolated product will then be tested for presence and intactness of peroxisomes. Western blot analysis will also be used to check whether CP is localized in the peroxisomes.