Isolation and Characterisation of Chitin Elicitor Binding Protein (CEBiP) gene in Oryza sativa variety UKMRC9

摘要

Chitin is a microbe-associated molecular pattern (MAMP) molecule in fungal cell wall that functions as elicitor which induces immune response in plants. Plants have its own defence system to protect itself from any invasion of pathogens. Chitin Elicitor Binding Protein (CEBiP) is one of the plant pattern recognition receptors (PRRs) that will detect the presence of the pathogen-associated molecular patterns (PAMPs) and activate plant defence system. In this study, we used Oryza sativa variety UKMRC9 to study the function of Chitin Elicitor Binding Protein (CEBiP) gene in plant immune system. The sample was extracted and amplified using the primer that were specifically designed for CEBiP based on the full gene sequence obtained from MSU Rice Genome Annotation Project database. The sample then was transformed into pGEM-T cloning vector. The plasmid construct of vector pCAMBIA 1305.2 was completed using Pmll and BstEII as the restriction enzyme. The sequencing results of pGEM-T-CEBiP-RC9 obtained are subjected to multiple sequence alignment and the generation of a phylogenetic tree. Analysis obtained from BLAST shows that CEBiP UKMRC9 has 100% identity with four sequences from NCBI database with high query coverage. Multiple sequence alignment also shows that there are some nucleotides that have been inserted and deleted (indels) in the amplified sequence. Pairwise alignment indicates high similarity between CEBiP UKMRC9 and Chitin elicitor binding protein [Oryza sativa Japonica Group], PREDICTED: chitin elicitor-binding protein [Oryza sativa Japonica Group], Chain A, Crystal Structure of Oscebip, Chain A, and Crystal Structure of Oscebip Complex in BLAST. Results from phylogeny analysis places CEBiP UKMRC9 under the same clade with Oryza sativa cv Nipponbare CEBiP MSU. Even within the graminaceae, there is cladal separation of organisms based on genus and species.