Isolation of Monoterpene Synthase Gene (NsTPS2) and Evaluation of Terpenoid Compounds in Black Cumin Medicinal Plant (Nigella sativa L.)

Black cumin (Nigella sativa) is a medicinal plant of the Ranunculacea family which raised attention due to its pharmaceutical properties. Medical significance of N. sativa mainly attributed to its oxygenated monoterpenes which are biosynthesized via the methyl erythritol phosphate (MEP) pathway located in plastids. In this study, the essential oil components of leaves, flowers, and developmental stages of seed including half black seeds, soft black seeds, and hard black seeds were analyzed in N. sativa. Whereas no terpene was detected in flowers and leaves, seeds were found to be the major site of biosynthesis and accumulation of terpenes, and the amount of terpene compounds changed during seed maturation. The essential oil consists of monoterpenes (more than 99%) and sesquiterpenes (less than 1%). In order to improve our understanding of monoterpene metabolism, the partial sequence of a hypothetical monoterpene synthase (NsTPS2) was isolated from N. sativa plant using RACE-PCR technique. This monoterpene synthase was identified from RNA sequencing data from soft black seeds. Except of the highly conserved DDXXD motif in NsTPS2 which is necessary to validate monoterpene synthases, no other conserved regions of other identified monoterpene synthases were observed. Dendrogram analysis revealed that NsTPS2 had the highest homology with a terpene synthase (72.89%) from Aconitum carmichaelii and these two sequences were grouped in the same group. Nigella sativa and Aconitum carmichaelii both belong to the Ranunculacea family. This indicates that the genetic information of plants of the Ranunculacea family can be used to isolate different monoterpene synthase. The results of this research can be useful in genetic manipulation and metabolic engineering of Nigella sativa.