Gene Sequence mining and primer designing
The full-length gene was mined from transcriptome previously sequenced from leaf samples of C. asiatica [26]. The Hydroxymethyl glutaryl-CoA-synthase (HMGS) gene sequence of C. asiatica was isolated followed by primer designing, RNA isolation, cDNA (complementary DNA) synthesis and further cloning processes. Genes specific primers (listed in Table 4) were synthesized by using OligoAnalyzer™ Tool (www.idtdna.com/pages/tools/oligoanalyzer) [27].
Total RNA isolation
Total RNA extraction was done from different tissues of C. asiatica by using TRI Reagent®(Sigma) using 100 mg of tissue samples. Isolated RNA was dissolved in Milli Q water and checked by Nanodrop spectrophotometer ND1000 and 0.8% agarose gel electrophoresis and further RNA was kept at -80 ̊ C [28] for applications in further applications.
cDNA synthesis
Revert Aid kit from Thermo Scientific (U.S.A.) was used to make cDNA from the isolated total RNA. Synthesized cDNA were confirmed by Semi quantative PCR with Actin primers.
Amplification of the pathway gene CaHMGS by PCR
Amplification of the gene was done with gene specific primers (Table 4) taking synthesized cDNA as a template. For this the reagents like PCR master mix (Thermo Scientific), nuclease-free water, forward primers and reverse primers were added with 35X PCR cycles and temperatures as: Initial denaturation: 94°c:5 minutes; final denaturation: 94°c:35 seconds; primer annealing: 53°c:30 seconds, initial extension: 72°c:2:30 minutes; final extension: 72°c:7:00 minutes used for the successful amplification of HMGS gene. To visualize the bands of the gene, 0.8% agarose gel was casted (0.8g /100 ml 1x TAE) using 1x TAE (Tris-acetate-EDTA) as tank buffer. The amplified band of DNA fragments of interest from the gel was sliced/excised out with a scalpel or razor blade and extracted using the Gen EluteTM gene elution kit from Qiagen.
Cloning and selection of positive clones
The product eluted was further cloned in cloning vector pGEM®-T Easy to obtain a ligated product. The ligated product was then transformed into DH5α strain of E. coli and plated on Luria Agar medium with IPTG (Isopropyl β-d-1-thiogalactopyranoside) and X-gal (5-Bromo-4-Chloro-3-Indolyl-beta-D-Galactoside) as inducer and ampicillin as selection drug. The white colonies were taken for screening through PCR with the gene-specific primers to screen the putative transformants.
Colony PCR for screening the recombinant colonies and isolation of plasmid DNA from E. coli DH5α
Confirmation of the positive clones was done through colony PCR [29]. The conditions for the PCR used were 94 ̊C:7 minutes; 94 ̊C: 30 seconds; 53 ̊C:40 seconds; 72 ̊C: 2 minutes 30 seconds and final extension 72 ̊ C:7 minutes. The resulting product of colony PCR was checked on 0.8 % agarose gel. The plasmid was isolated through the alkaline lysis method and treated with RNaseA to remove RNA contamination. Positive clones were sequenced in an automated sequencer Genetic analyzer Model 3130xl using M13 forward and reverse universal primers [30].
Primary sequence analysis and phylogenetic study
Gene sequences were translated using EMBOSS Transeq tool (https://www.ebi.ac.uk/Tools/st/emboss_transeq). Obtained translated sequences were further undergoes in silico proteomics studies. First of all, HMGS (GeneBank ID:MZ997833) primary sequence of the protein was subjected to BLASTp against the non-redundant (NR) database of NCBI for searching the closest homologs of CaHMGS. For multiple sequence alignment, all sequences with significant alignment were aligned by ClustalW tool [24] (https://www.genome.jp/tools-bin/clustalw) [25]. Identity cut off >90, E value –0.0, query coverage −100% with the CaHMGS. For the study of Evolutionary relationship of the CaHMGS gene with HMGS of other species, we constructed phylogenetic tree by Neighbour-Joining method having bootstrap value of 1000 replicates [25,28] by the software Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6. [31]. Various bioinformatics tools were used for protein sequence analysis like protein family and domain arrangement within the gene was explored by Pfam database (http://pfam.sanger.ac.uk/) [32], domain and motif was deduced through InterProScan and Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de) Conserved Domain Database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/ cdd/cdd.shtml), was used to deduce the Conserved Domains of the protein. Besides, various parameters of physiochemical properties of the amino acid sequence of CaHMGS protein was also observed with the ExPaSy Protparam (http://web.expasy.org/protparam) tool of the proteomics server [32].
Theoretical modelling and its validation
The CaHMGS primary protein sequence was subjugated to BLAST against the Protein Data Bank database. The closest homolog found was Brassica juncea HMGS (7CQT.1) with >81% identity and >95% coverage was observed with CaHMGS. The sequences were deposited in the Swiss model (swissmodel.expasy.org) for homology modelling. Amongst different models, the best model having maximum favoured score was selected according to Ramachandran Plot. The model selected was then undergoes for refinement by Galaxy Refine server. Also, the obtained refined model was again verified through quantification of the amino acid residues shown in the Ramachandran plot. The stereochemical quality of the CaHMGS model was analyzed through PROCHECK [33,34] in the Structural Analysis and Verification Server (SAVES) (http://nihserver.mbi.ucla.edu/SAVES/) [25]. Also, the model has further visualized using UCSF Chimera-Molecular Modeling System Chimera [35]. Modelled protein was exposed to Protein Structure Analysis (ProSA) [36] and ERRAT (SAVES) tools for quality determination and validation of the modelled protein [37].
Tissue-Specific Expression Analysis
Differential tissue specific Expression of CaHMGS gene was studied in various tissues (node, petiole, leaves, and root) of Centella asiatica using quantitative real-time PCR. For the study, Total RNA from different tissues like leaves, node, petiole and root were isolated [16] using TRI Reagent® from Sigma. 5µg of total RNA was used to prepare cDNA using Gene Sure First Strand cDNA Synthesis Kit (Puregene) [38]. The PRIMER 3 was used to primers designing for real time PCR using having Tm > 50 °C, GC content of 50–60%, primer length of nucleotides approx.18-24 and amplicon size (expected) of 100–120 bp. RT-PCR specific primer sequences of CaHMGS (listed in Table 4). Concentration of cDNA synthesized was optimized to 100-150 ng/μl. Relative expression of genes were also checked by semi-quantitative PCR. Actin gene was used as endogenous control for normalizing the expression of the target gene. Calculation of Relative expression was done by 2−ΔΔCt method [25]. All the reactions have been done in triplicates to ensure the credibility of the data.
Functional complementation
Yeast (Saccharomyces cerevisiae) required MVA pathway for its survival [15] and interrupting MVA pathway genes in yeast could be lethal. pYES2 are autonomously replicating expression vectors that contain a yeast galactose-dependent promoter. Hence to characterized the function of CaHMGS, the vector pYES2-CaHMGS construct was prepared. For this, the cDNA prepared in previous experiment was amplified using primers (listed in Table 4) having restriction sites SacI and XbaI, eluted and ligated in pYES vector underneath to the yeast galactose-dependent GAL1 promoter [39]. pYES2-CaHMGS construct was then transformed into diploid Saccharomyces cerevisiae strain YSC1021 and haploid strain YSC1021 (that lack one HMGS allele). Also, Wild type pYES2 plasmid construct was transformed as a positive control. Positive clones were then sequenced. Both yeast strains were streaked on YEPG (Himedia) and YEPD (Yeast Extract Peptone Dextrose) agar media plates either with or without G418 (Geneticin) and then incubated at 30ﹾC for yeast growth.