Genes induced by high concentration of salicylic acid in Mitragyna speciosa

Mitragyna speciosa is rich in secondary metabolites which are similar in effects as opium. Treatment with plant growth regulator such as salicylic acid (SA) is able to increase plant defense mechanism which later induces the expression of genes that encode secondary metabolite production. To identify genes that respond to elicitation of high concentration of SA, suppression subtractive hybridization (SSH) library was constructed using mRNA from SA-treated leaves and mRNA from non-SA-treated leaves. A total of 292 EST clones were randomly sequenced and all cleaned clone sequences (111) were analyzed using BLASTX against nonredundant NCBI databases. Results showed that most genes responding to acute SA treatment are related to stress and signaling pathways which eventually led to cell death. This include genes encoding chaperone, heatshock proteins (HSPs), antioxidants and genes involved in secondary metabolite biosynthesis, such as sinapyl alcohol dehydrogenase (SAD), cinnamyl alcohol dehydrogenase (CAD) and Cytochrome P450 (CYP 450). Further analysis was carried out using 8 out of 60 differentially expressed unique sequences through semi-quantitative RT-PCR on samples before and after treatment with 5 mM SA in 4 consecutive days. The result revealed that their expression changed over time when the plant was treated with 5 mM SA.