Acorus calamus terpene

Ibdah, Mwafaq, Hino, Shada, Nawade, Bhagwat, Yahyaa, Mosaab, Bosamia, Tejas C. & Shaltiel-Harpaz, Liora, 2022, Identification and characterization of three nearly identical linalool / nerolidol synthase from Acorus calamus, Phytochemistry (113318) 202, pp. 1-10 : 8

4.6. Isolation and characterization of Acorus calamus terpene synthase genes

To identify putative TPSs genes responsible for the volatile formation, we used the transcriptome datasets of A. calamus available in the NCBI Sequence Read Archive (SRA) under BioProject accession SRR8298303. The SRR accessions were extracted in FASTA/Q format from NCBI (http://www.ncbi.nlm.nih.gov/) using Galaxy tools (htt ps://usegalaxy.org/). Further, de novo assembly of RNA-Seq data was performed using Trinity on Pittsburgh Supercomputing Centres (PSCs) Bridges. Local BLAST was performed against known plant TPSs as queries. The contigs obtained from the BLAST were processed with an Expasy translate tool (https://web.expasy.org/translate/) to identify full-length TPS genes. A total of 6 TPS genes were identified from these transcriptome databases, and gene-specific primer pairs were designed using the IDT primer tool (Supplementary Table S1 ). The A. calamus cDNA was amplified with gene-specific primers using Platinum Taq DNA polymerase (Invitrogen, Israel), yielding a targeted product size for eight AcTPS genes (Supplementary Table S2). Despite numerous attempts, we were not able to amplify intact full-length protein-coding cDNA for only one AcTPS s. The amplified full-length genes, which after digestion with specific restriction enzymes (see Supplementary Table S1 ) was ligated in-frame into the vector pEXP5-CT/TOPO TA expression vector (Invitrogen Corporation, Carlsbad, CA), yielding pEXP-AcTPS3, pEXP-AcTPS4, and pEXP-AcTPS4 genes construct in which the coding sequences of the gene was fused with a His tag-coding extension at the Cterminus. The AcTPS3, AcTPS4, and AcTPS5 gene constructs were introduced into E. coli Top 10 cells. The construct fidelity and orientation of the insert in the vector were verified by DNA sequencing. Subsequently, an empty vector and vectors harboring the AcTPS3 genes were introduced to the E. coli strain BL21 (DE3) for protein expression.