Evaluation of Different Promoters Driving the GFP Reporter Gene in Seaweed Kappaphycus Alvarezii  

Oleh:

Rajamuddin, Muh. Alias L. ; Alimuddin ; Widyastuti, Utut ; Faizal, Irvan

Jenis: Article from Journal - ilmiah nasional - terakreditasi DIKTI
Dalam koleksi: Indonesian Journal of Biotechnology vol. 19 no. 02 (2014), page 129-135.
Topik: transgenic; promoter; GFP; electroporation; ? lament callus; Kappaphycus alvarezii
Fulltext: 9304-17257_her.pdf (358.08KB)

Isi artikel

Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select a suitable promoter as the ? rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Green ? 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), cauli? ower mosaic virus (pCaMV-GFP), medaka ß-actin (pmBA-GFP) and Japanese ? 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 ? uorescent microscope. The results showed that CMV regulated highest number of ? lament callus (34.10%±1.49) expressing GFP at medium to strong ? uorescence levels. CaMV promoter had relatively similar activity with CMV, but lower number of ? lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expression at medium level and similar number of ? lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoter had lowest activity by means in number of ? lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCR analysis for transgenic con? rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressing ? 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 ? lament callus by electroporation method. Combining this achievement with developing a culture method of ? lament callus to be thallus, stable transgenic breeding in K. alvarezii can be feasible.

Opini Anda

Klik untuk menuliskan opini Anda tentang koleksi ini!

Kembali