Aktivitas ACCase mesokarp kelapa sawit dan kloning fragmen gen penyandi ACCase subunit biotin karboksilase

Abstract

Genetic engineering to produce high yielding oil palm might be done by over expressing gene encoding key enzyme for oil biosynthesis in the oil palm mesocarp, one of which is ACCase. The objective of this research was to analyze ACCase activity of mesocarp from several developmental stages of fruit and to clone conserved region cDNA of gene encoding biotin carboxylase subunit of ACCase (BC-htACCase) from oil palm mesocarp. Activity of ACCase was analyzed by HPLC. Amplification of cDNA was done by means of reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate heterologous primer on several annealing temperature and MgCl2 concentration. The cDNA fragment of RT-PCR product was cloned, sequenced and analyzed to confirm that the cloned cDNA was conserved region of BChtACCase. The result showed that ACCase activity increased from the 14 week to the 20 week-old fruit, and then decreased. Using heterologous degenerate primers, cDNA fragments of BC-htACCase conserved region (469 bp) can be specifically amplified at 60 oC annealing temperature with 2 mM MgCl2 concentration. The result of BlastX analysis showed that the sequence of cloned cDNA fragment was highly homologous with the conserved region of BC-htACCase from Glycine max, Arabidopsis thaliana, Nicotiana tabacum, and Brassica napus with 243, 237, 236, 231 bit score, and E. value 2e-63, 1e-61, 2e-61 and 5e-60, respectively.