Isolasi dan Kloning pada Escherichia coli Gen AaWRKY1 Artemisia annua L

Abstract

Artemisia annua WRKY1 (AaWRKY1) gene encodes a protein WRKY a transcriptional activator of Amorpha-4,11-diene synthase (ADS). The overexpression farnesil diphosphate AaWRKY1 genes can increase levels of artemisinin. This study was aimed to isolate an AaWRKY1 gene from Artemisia annua L. Amplification of AaWRKY1 gene was done by PCR (Polymerase Chain Reaction) technique using specific primer pairs and cDNA as a template. Subsequently, AaWRKY1 gene fragment was cloned into a cloning vector pJET 1.2 using T4-ligase and transformed into competent cell Escherichia coli DH5α through heat shock method, and grown on LB agar medium containing ampicillin. Result showed that amplification with specific primers of AaWRKY1 gene generated an amplicon fragment of ± 1500 bp. The gene fragment was succesfully cloned into pJET 1.2 and transformed into cells of E. coli DH5α. Confirmation of recombinant clone was carried out using colony PCR to obtain recombinant colonies.