Kloning promoter P-actin ikan mas, Cyprinus carpio Lin. 1758 dan analisis fungsionalnya menggunakan gen target protein pendaran hijau (GFP) [P-actin promoter cloning of common carp, Cyprinus carpio Lin. 1758 and its functional analysis using targeted Green Fluorescent Protein (GFP) gene]


Andi Aliah Hidayani, Odang Carman, nFN Alimuddin
 DOI  https://doi.org/10.32491/jii.v13i2.101

Abstract

Promoter in the expression vector plays an important role on regulating of gene expression in transgenic fish. In fish transgenesis, researcher convinced that the use of all-fish expression vector is safety and prospective. The study was performed to isolate P-actin promoter, the promoter which has ubiquitous, constitutive, housekeeping characteristics, of common carp as a first step to construct all-common carp expression vector. Common carp P-actin promoter (ccBA) was isolated using PCR method with FBP1, RBP1, and RBP2 primers. Sequencing was performed using ABI PRISM 3100 machine, and analysis of sequences was conducted using GENETYX version 7 software. The results of sequence analysis showed that the length of DNA fragment obtained was approximately 1.5 kb. Results of homology with P-actin promoter sequence of a gene bank database (Accession No.: M24113) was 97.5%. The evolutionary conserved of transcription factor for P-actin promoter including CCAT, CArG, and TATA boxes were found in the sequence. Ubiquitous and higher expression of green fluorescent protein driven by ccBA promoter in muscle of common carp larvae was detected. It is most likely that the isolated sequence is a common carp P-actin promoter.

 

Abstrak

Promoter dalam vektor ekspresi berperan penting dalam mengatur ekspresi gen pada ikan transgenik. Dalam transgenesis ikan, peneliti yakin bahwa penggunaan vektor ekspresi semua ikan aman dan prospektif. Penelitian ini dilakukan untuk mengisolasi promoter P-aktin, promoter yang memiliki karakteristik ubiquitous, constitutive, house keeping, dari ikan mas sebagai langkah awal untuk mengkonstruksi vektor ekspresi semua ikan mas. Promoter P-aktin ikan mas (ccBA) diisolasi menggunakan metode PCR dengan primer FBP1, RBP1, dan RBP2. Sequensing dilakukan dengan menggunakan mesin ABI PRISM 3100, dan analisis sekuen dilakukan dengan menggunakan perangkat lunak GENE-TYX versi 7. Hasil analisis sekuen menunjukkan bahwa panjang fragmen DNA yang diperoleh adalah sekitar 1,5 kb. Hasil homologi dengan sekuen promoter P-aktin dari pangkalan data bank gen (No. Aksesi: M24113) adalah sebesar 97,5%. Faktor transkripsi yang tetap secara evolusioner untuk promoter P-aktin promoter termasuk CCAT, CArG, dan boks TATA ditemukan dalam sekuen. Ubiquitous dan ekspresi tertinggi protein pendaran hijau (GFP) dikendalikan oleh promoter ccBA dalam otot larva ikan mas yang dideteksi. Dengan demikian, kemungkinan besar bahwa sekuen yang terisolasi adalah promoter P-aktin ikan mas.



Keywords

expression; GFP; cloning; P-actin promoter; common carp transgenic.

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