Transcriptome analysis and identification of genes associated with bryonolic acid biosynthesis in Trichosanthes cucumerina L.

Abstract

Trichosanthes cucumerina L. (TC) is a Thai medicinal plant belonging to Cucurbitaceae family. This plant has been used in Thai traditional medicine for purgative, emetics, treating dandruff and relieving the sinusitis. Additionally, it has been reported about its activities, especially cytotoxicity and many studies showed that these activities related with a compound named bryonolic acid. This compound is one of major triterpenoid compounds that can be generally found in TC root. This study aimed to improve bryonolic acid content using plant tissue culture techniques for the study of genes in bryonolic acid biosynthesis. The callus was induced using Murashige and Skoog (MS) plant media supplemented with 0.5 mg/ml of indole- 3-butyric acid (IBA) and 1 mg/ml of benzylaminopurine (BA). For the cell suspensions, they were cultured in MS media with 1 mg/ml of IBA and 1 mg/ml of BA. Three elicitors, methyl jasmonate (50 - 200 μM), yeast extract (0.1 - 2% w/v) and chitosan (1 - 100 mg/ml), were added into the cell suspension. The results from HPLC analysis showed that callus and cell suspension presented the higher bryonolic acid levels by just over twenty and seven times compared with TC root (approximately 2 mg/g dry weight), respectively. In the presence of elicitors, the best treated condition was to treat cell suspensions with 1 mg/ml of chitosan for 8 days (approximately 24 mg/g dry weight). To carry out the candidate genes in bryonolic acid biosynthesis pathway, RNA was extracted from TC calli for the de novo transcriptome analysis compared with TC leaf and fruit to carry out the candidate genes associated with bryonolic acid biosynthetic pathway.