Constructing and Cloning of a Vector Containing Partial EPFL9 Fragment Gene from Indica-Type Rice Oryza sativa IR64

Abstract

Rice, Oryza sativa, is the third-highest worldwide agricultural production. However, the current and future state of rice is becoming unpredictable due to climate change. Climate change affects plant growth and development especially in photosynthetic process. Stomata is the crucial part in photosynthetic process. In molecular level, stomata is regulated by stomagen, which is divided into 2: positive regulator EPFL9 gene and negative regulator EPF1 and EPF2. This study focuses more on EPFL9 that is responsible for increasing stomata density. Yet, an advance understanding of EPFL9 is still needed to provide more knowledge. Many studies have been conducted to modify EPFL9 gene through genetic engineering. This study aimed to construct a vector containing partial EPFL9 gene for cloning purpose. First, the upstream cloning part includes preparing the partial EPFL9 as gene of interest, digestion of pTC184-4 shuttle backbone vector, ligation, and transformation while the downstream process includes colony PCR, digestion analysis, and sequencing. The result showed that cloning was successful. The colony PCR confirms recombination plasmid pRep1263 contains partial EPFL9 gene, digestion analysis confirms the insert orientation while sequencing result stated 100% homology between recombinant plasmid pRep1263 and Oryza sativa containing EPFL9 gene and replicon derived from wheat dwarf virus. As future work, this cloned recombinant plasmid is subjected for homologous recombination-related gene targeting study.