Indonesian Journal of Biotechnology (Dec 2014)
Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii
Abstract
Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select a suitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Green fl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycus alvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP), medaka β-actin (pmBA-GFP) and Japanese fl ounder keratin (pJfKer-GFP) promoters were introduced by electroporation method. Electroporation was performed using a gene pulser (BIORAD) with voltage of 300 V, pulse length of 0.5 ms, pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expression level using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus (34.10%±1.49) expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activity with CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expression at medium level and similar number of fi lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoter had lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCR analysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressing fi lament callus in the same size (about 0.6 kb) with positive control of plasmid. Thus, CMV and CaMV promoters was an appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combining this achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K. alvarezii can be feasible.
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