Evaluation of different promoters driving the GFP reporter gene in seaweed Kappaphycus alvarezii

• Main Authors: Muh. Alias L. Rajamuddin, Alimuddin A, Utut Widyastuti, Irvan Faizal
• Format: Article
• Language: English
• Published: Universitas Gadjah Mada, Yogyakarta 2016-02-01
• Series: Indonesian Journal of Biotechnology
• Subjects: transgenic, promoter, GFP, electroporation, fi lament callus, Kappaphycus alvarezii
• Online Access: http://journal.ugm.ac.id/ijbiotech/article/view/9304
• ISSN: 0853-8654; 2089-2241

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