Posted July 19, 2016 by admin in Natural Resource Management | 310 Total Views
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Perliminary Evaluation Of Andrographis Paniculata Transgenic Expressing 3-Hydroxy-3-Methylglutaryl Coenzyme- A Reductase Grown In A Green House

Metabolic engineering of plant has potential to improve the quantity of  valuable secondary metabolites in plant. This approach can be  performed by several ways such as amplification of enzymes levels, addition of new enzymes pathway (introduce a new metabolic pathway into others (micro) organism),  inhibiting a specific metabolic flux or deletion of existing enzymatic pathways which competes with the expected pathways.  Most of these modification in the last decade are carried out with the use of genetic engineering technology (Verpoorte and Memelink, 2002). Moreover, addition of copies of a gene into the host genome can give advantageous as the repition of a section of DNA (multiple copy gene) may lead to overproduction of that gene’s protein /enzyme.

For these plant genetic modification, a certain metabolic pathways and genes that regulate the synthesis of certain secondary metabolites are necessary to be available. However, the avaibility of plant secondary metabolite pathways are very few thereby the characteristics of genes or enzymes which are involves in certain metabolic pathway are very poor. In our study with the intention to increase the secondary metabolites, androgrphollide (a diterpenen lactone found in A. paniculata), only hmgr gene (3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme) was available. HMGR   plays a role in the early stage of andrographollide biosynthesis, i.e. a  synthesis of mevalonic acid from 3-hydroxy-3-methylglutaryl.   Therefore in this study the hmgr gene was adopted as gene of interest.

HMGR is a gene family consisted of two hmgr genes, hmgr1 and hmgr2. HMGR is an enzyme which plays a role in the early stage of andrographolide biosynthesis.  Jha et al. (2011) reported that elicitor which was applied into Adrographis paniculata could increase hmgr1 gene expression followed by increasing the biosynthesis of  Andrographolide. Thus, we hypothesized that introducing additional hmgr gene with specific promoter  into A. paniculata cell or tissue would increase the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme.  So that, accumulation of andrographollide would also increase.

In present study, isolated hmgr1 was re-constructed into binary vector pBI121 with  CaMV 35S promoter and NOS terminator but without the presence of GUS gene. The recombinant plasmid was then introduced into E. coli for amplification followed by isolation of the plasmids by lysis method. The recombinant plasmids were then introduced into A. tumefaciens GV 3101by heat shock method. For transformation, a single colony of A. tumefaciens GV 3101 was suspended into 25 ml YEP medium supplemented with kanamycin (50 mg l-1)   and cultured overnight at 28oC. The Agrobacterium  cells,  approximately 106 cells/ml medium (OD600nm = 08),  were collected by centrifugation at 4,000 rpm for 20 min and  resuspended in liquid half-strength MS medium containing 100 μM acetosyringone.  The cotyledon explants  were dipped  for 60 minutes in the  A. tumefaciens suspension. Subsequently, the  cotyledon explants were blotted on sterile filter paper and co-cultivated for three days in dark at 28oC on agar-solidified (0.8% w/v) MS medium supplemented with 100 μM acetosyringone, 2.0 ppm BAP, 1.0 ppm IAA and 100 mg/L kanamycin. After three days, the presence of hmgr1 gene was confirmed by PCR analysis and visualization by agarose gel electrophoresis. This present study was also report the establishment for efficient transformation by A. tumefaciens harboring pBI121 plasmid and GUS gene as reporter gene. Plant regeneration of transformed tissue was performed through organogenesis. Transformed tissue was selected on MS Medium; 0,5 µM 2.4-D; 0,1 µM BAP; with addidition of 20 mg/l kanamycin as selectable agent and 400 mg/l cefotaxime  as antibiotic to eliminate A.tumefaciens. Histochemical gus assay showed that frequency of gus gene expression was 61,6%; obtained from transformed calli.  The PCR analysis followed by visualisation on 1% agarose gel showed the presence of 338 bp on the line of transform callus and postive control of pBI121. This results indicated that gus gene has been inserted into plant tissue.  Transform tissue regeneration marked by emerged shoot on transformed callus. The frequency of gus gene expression on this bud was 85%. Based on the results, it can be concluded that gus gene from pBI121 have been succesfully inserted into plant genome and stably integrated on regenerated A.paniculata tissue.

LIST OF RESEARCH OUTPUT

1. Prototype ‘green house’ (rumah kaca) untuk pemeliharaan tanaman A. paniculata hasil transformasi

2. Transformasi genetik pada Sambiloto (Andrographis paniculata (Burm.F.) dengan perantara Agrobacterium tumefaciens  strain GV3101 pembawa vektor pBI121 dan regenerasi jaringan tertransformasi membawa gen pelapor GUS.

Gambar 1. Bangunan  ‘green house’

HEAD OF RESEARCH TEAM : Dr. Erly Marwani
TEAM MEMBERS : Dr. Fenny Matrha Dwivany
OFFICIAL ADDRESS : Labtek  XI -ITB, Jalan Ganesha 10 Bandung 40132
EMAIL : erly@sith.itb.ac.id


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