Transformation of 3-hydroxy-3-methylglutaryl coenzyme-A reductase-1 (hmgr1) gene enhances accumulation of mevalonic acid and isoprenoid derivatives in transgenic of Andrographis paniculata (Burm.f.) Wallich ex Nees
Febby Florencia, Erly Marwani*, Yeyet Setiawati

School of Life Sciences and Technology, Institut Teknologi Bandung
Jalan Ganesha No. 10, Bandung 40132, Indonesia
*erly[at]sith.itb.ac.id

Abstract

Andrographis paniculata has long been used as a herbal medicine. The primary bioactive compound in this plant is andrographolide. In the biosynthesis of andrographolide through the mevalonic acid (MVA) pathway, the 3-Hydroxy-3 Methylglutaryl Coenzyme-A Reductase (HMGR) has been reported as the rate limiting enzyme in the MVA pathway to the biosynthesis of isoprenoid, a precursor of terpenes intermediate metabolites such as geranyl diphosphate (GPP) and geranyl geranyl diphosphate (GGPP), which are essential for andrographolide biosynthesis. Our previous study revealed that an increase in the expression level of the hmgr1 gene could increase the andrographolide in transformed A. paniculata. The aim of this study was to examine the effect of the transformation of the hmgr1 gene into A. paniculata on the MVA, GPP, GGPP, and andrographolide concentration. In addition, the correlation of MVA, GPP, GGPP, and andrographolide concentration was also examined. In this study, the cotyledonary explants were transformed with Agrobacterium tumefaciens GV3101 carrying pBI121-hmgr1 plasmid with a strong promoter CaMV 35S using agroinfiltration method for 60 minutes, followed by cocultivation on Murashige and Skoog (MS) medium supplemented with 100 &#956-M acetosyringone for three days, disinfection with 400 ppm of cefotaxime and selection on MS medium containing 20 ppm of kanamycin. The transformed tissues were transferred into the shoot regeneration medium, the MS containing 2.5 ppm BAP, 0.5 ppm IAA, 100 ppm of cefotaxime, and 20 ppm of kanamycin. The presence of the hmgr1 gene in the transformed shoots was confirmed by Polymerase Chain Reaction (PCR). Analysis of MVA, GPP, GGPP, and andrographolide was performed using High-Performance Liquid Chromatography. The results showed that the transformed shoots could grow and multiply on a medium containing 20 ppm kanamycin and the stable transformation was confirmed by the PCR product of 2,682 bp which correspond to hmgr1 gene. The transformed shoots accumulated MVA, GPP, GGPP, and andrographolide higher than that of the non-transformed shoots, respectively up to 4.28- 4.20- 3.10- and 3.00-fold. Pearson correlation coefficient test showed a positive correlation between MVA, GPP, GGPP, and andrographolide concentrations where the increase in MVA concentration led to an increase of GPP, GGPP, and andrographolide concentration.

Keywords: Mevalonic acid- geranyl diphosphate- geranyl geranyl diphosphate- andrographolide- HMGR

Topic: LIFE SCIENCES