BINARY VECTOR CONSTRUCTION OF KAPPA(κ)-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

Muh Alias L. Rajamuddin (Jurusan Budidaya Perikanan, Politeknik Pertanian Negeri Pangkep, Pangkep.)
. Alimuddin (Departemen Budidaya Perairan, FPIK, Institut Pertanian Bogor, Bogor.)
Utut Widyastuti (Departemen Biologi, FMIPA, Institut Pertanian Bogor, Bogor.)
Enang Harris (Departemen Budidaya Perairan, FPIK, Institut Pertanian Bogor, Bogor.)
Emma Suryati (Balai Riset dan Pengembangan Budidaya Air Payau, Maros.)

Jurnal Ilmu dan Teknologi Kelautan Tropis
Vol. 8 No. 1 (2016): Elektronik Jurnal Ilmu dan Teknologi Kelautan Tropis

Increasing of kappa (κ)-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of  κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes.  The κ-Carrageenase (κ-Car) gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced.                                                                                   Keywords:  Agrobacterium tumefaciens, kappa(κ)-Carrageenase gene, transgenesis, vector