In silico and in vitro analysis of THCA synthase gene in Moroccan Cannabis sativa, L

Abstract

Background: Cannabis ( Cannabis sativa, L.) is an archaic, most infamous plant with unique therapeutic characteristics, and great economic interest. The identification of its THCAS synthesizing enzyme was a great leap forward in Cannabis investigation. However, basic molecular biology for THCAS gene expression remains largely unstudied. In this paper, we explored the ability of bioinformatics resources in the design of molecular cloning protocol and prediction of three-dimensional structure of recombinant THCAS in Moroccan Cannabis sativa variety. Methods: We described conventional experiments performed in vitro. We used specific primers to isolate the nucleotide sequence of THCAS from the leaves of Cannabis, without including the native signal sequence.we prepared the gene by amors containing specific adapters to integrate the gene into the expression vector PpinkαHC, transforming E.coli bacteria, in order to express the gene in Pichia pasteri yeast. Results: As a result, we found high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported. In addition, we predicted the structure of the recombinant THCAS using the organic crystal structure prediction method, the amino acid sequence reference, and the artificial intelligence technology. Conclusions: In this study, we have successfuly cloned recombinant THCAS enzyme in E.Coli for further expression in host Pichia pastori.