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Isolation and Cloning of Bisabolene Synthase Gene from Abies Grandis for Biofuel Production

Faezeh Haerizadeh, Asad Masoumiasl

Abstract


The rise of petroleum cost and environmental pollutions which are the result of fossil fuels utilization causes use of biofuel to be taken into account as a sustainable and renewable alternative to fossil fuels. The bark of conifer trees produces different compounds, such as terpenoid bisabolene which are like biodiesel in physiochemical aspects. On the other hand, bisabolene synthase (one of the key enzymes in bisabolene production pathway) is expressed in bark of conifer trees. Therefore, this study was designed and conducted with the aim of isolation and cloning of this gene form Abies grandis. In this study by modification in existed methods, RNA was extracted from the bark of Abies grandis successfully. The present extraction method is needless phenol utilization, low-cost and more efficient in comparison with other methods. After cDNA synthesis and using designed primers, polymerase chain reaction was done and expected gene was multiplied. Then the enzymatic digestion reaction of gene and pBluescript plasmid was done using BamHI and XbaI enzymes and for cloning above gene was inserted in plasmid and was transferred to E. coli. To determine the subgroup of cloned gene, the sequence of cloned gene was compared with the sequence of the same gene in Picea abies and Pseudotsuga menziesii and as a result the similarity of mRNA sequence in cloned gene and recorded genes in Genebank was 70.368%. This study is the first report about isolation and cloning of this gene in Iran and based on we can have a plan for biodiesel production.


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