High quality cDNA synthesis and amplification of chalcone synthase gene (CHS) from justicia gendarussa burm. F.

摘要

Chalcone synthase (CHS) (EC 2.3.1.74) catalyzes the first committed step of flavonoid biosynthesis pathway. It combines 4-coumaroyl-CoA with three C2 units from malonyl-CoA to produce naringenin chalcone which transforms to various numbers of flavonoids. In this work, we attempt to isolate the CHS gene from Gendarusa, which could serve as a preliminary study to elucidate the CHS gene regulation in the flavonoid biosynthetic pathway of Malaysian medicinal plants generally, and in Justicia gendarussa Burm. F. species specifically. The total RNA was isolated from the mature leaves of Justicia gendarussa Burm. F. using the modified CTAB method. Extracted RNA, treated with RQ1 RNase-free DNasekit and cDNA synthesis in order to prepare the rich template of DNA for PCR by using M-MLV Reverse Transcriptase kit. High concentration of cDNA and A260/280 ratio reversely transcribed cDNAs guarantee the quality and quantity of synthesized cDNA. The CHS gene was amplified using the Phusion DNA Polymeraseand showed the same size of the previously amplified CHS gene from Melastoma, 1100bp.