Cloning and sequence analysis of Avr-Pik gene from Magnaporthe oryzae isolates from Sarawak

• Main Author: Martina Azelin, Dirum
• Format: Final Year Project Report
• Language: English; English
• Published: Universiti Malaysia Sarawak, (UNIMAS) 2016
• Subjects: Q Science (General)
• Online Access: http://ir.unimas.my/id/eprint/15445/1/Martina.pdf; http://ir.unimas.my/id/eprint/15445/4/Martina%20full.pdf; http://ir.unimas.my/id/eprint/15445/

Avr-Pik gene found in Magnaporthe oryzae is one of the specific effector protein that interact with host resistance (R) gene that result in effective activation of innate immunity. M. oryzae is an ascomycete fungal pathogen that has been found infecting cultivated rice (Oryzae sativa) and become the major constraint in rice global production. The aim of this research is to isolate, clone and sequence Avr-Pik gene from M. oryzae originated from Sarawak. Prior to isolation and cloning, a set of primer consist of forward primer (5'- ATGCGTGTTACCACTTTTAACA-3') and reverse primer (5'- TTAAAAGCCGGGCCTTTT-3') was designed based on Avr-Pik sequences obtained from database. Gradient Polymerase Chain Reaction (PCR) was performed to optimize the annealing temperature. DNA fragments of -342 bp were obtained from the amplification and were purified. Blue and white screening was conducted to grow colonies and distinguish between the recombinant and non-recombinant colonies. Subsequently, plasmid miniprep was conducted to obtain plasmid and it was sent for sequencing. The sequencing result was corroborated by using BLAST in which showed highest similarity with M. oryzae isolates from Japan. Based on this study, future identification and sequence analysis of this gene in understanding gene-for-gene resistance can be carried out, hence establishing Moryzae as the fungal model to study fungal pathogenicity.