Optimasi Ekstraksi RNA dan Teknik Kloning: Studi Kasus Kloning Gen Heading Date 3a pada Kelapa Sawit

Flowering holds significant importance in the plant kingdom as it facilitates genetic mixing, thereby supporting crucial genetic diversity. The phosphatidylethanolamine binding proteins (PEBP) protein family plays a pivotal role in regulating the timing of flowering and seed dormancy across diverse plant species. This research aimed to design a binary vector by constructing pCAMBIA1300, which incorporates the EgHd3a gene sequence from oil palm. The gene construction process involves RNA extraction, cDNA synthesis, amplification of the EgHd3a gene, cloning the gene into a cloning vector, subcloning into the pCAMBIA1300 binary vector, and concluding with gene validation through sequence analysis. During RNA extraction, the PCL-Chisam method has proven effective through iterative extraction, enhancing both the quality and quantity of total RNA. In the cloning process, conventional methods face challenges in selecting appropriate cleavage sites. To overcome this hurdle, the use of enzymes with compatible overhangs is proposed as a potential solution. Specifically, the substitution of BglII for BamHI has effectively addressed this challenge. The confirmation of the integration of gene fragments into the pCAMBIA1300 plasmid was achieved through sequencing. Although differences were identified in the sequence of EgHd3a-2, these alterations did not impact the encoded amino acids, thereby preserving the integrity of the protein sequence.