Transformasi Gen Kappa (K)-Carrageenase pada Rumput Laut Kappaphycus alvarezii

Abstract

Foreign gene transformation technique have been applied to improve the valuable traits of farmed organism. Carrageenan content determines quality of seaweed Kappaphycus alvarezii. As the first step towards increasing of kappa(κ)-carrageenan content in K. alvarezii, in this study kappa(κ)-Carrageenase gene (κ-Car) from Pseudoalteromonas was transformed into K. alvarezii mediated by Agrobacterium tumefaciens method. The κ-Car gene involves in κ-carrageenan biosynthesis. This research consisted of three steps. The first steps of research purpose was to produce a cloning vector and an expression vector for transformation process. The κ-Car gene sequence was artificially synthesized, an then inserted in pMSH plasmid to generate a binary pMSH/κ-Car plasmid. The pMSH/κ-Car was controlled by 35S CaMV promoter (35S) and Nos terminator (tNos). Transformation of pMSH/κ-Car to Escherichia coli as cloning vector was performed using a heat-shock method, while into Agrobacterium tumefaciens was using triparental mating method. The result showed that the transformed E. coli and A. tumefaciens colonies could grow on the selective media. PCR analysis with DNA template from bacterial colonies using 35S-Forward primer (35S-F) and tNos-Reverse (tNos-R) produced DNA fragment with size of 2,000 bp, the same size with the total length of 35S promoter, κ-Car gene, and tNos sequences. Thus pMSH/κ-Car construct and A. tumefaciens transformant carrying pMSH/κ-Car were successed to be produced. The second steps of research purpose was to produce a transgenic K. alvarezii carrying κ-Car gene. Transfomation of κ-Car gene to K. alvarezii was conducted using A. tumefaciens mediation method. Analysis of K. alvarezii transgenic carrying κ-Car was performed by PCR method with two set primers. The result showed that survival percentage posttransformation of K. alvarezii explants was 36% and sprout was 88.9% while the percentage of transgenic was 100% out of the sprout explants samples. The size of DNA band of PCR product from K. alvarezii transformants using 35S-F and tNos-R set primers was 2,000 bp, the same size as PCR product of the positive control of pMSH/κ-Car plasmid as template, while no amplification product in non-transformated as negative control was found. Furthermore, PCR analysis using 35S-F and 35S-R set primers showed amplification product of 300 bp, the same size as the length of 35S promoter sequence. Thus, K. alvarezii transgenic carrying κ-Car gene had been generated..dst