# Exploring Metalloprotease from _Dunaliella_ sp.: Production, Regulation, and Structural Insight
## 2 Materials and Methods
### RNA Extraction and Quantitative Real-Time Reverse Transcription PCR (qRT-PCR)  
Total RNA was extracted from the algal biomass using an RNA purification kit from MACHEREY-NAGEL according to the manufacturer's recommendations and then treated with DNase (Promega, Madison, WI, USA) to remove any contaminating genomic DNA. cDNA was synthesized from 1 \(\upmu\)g total RNA using an M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative PCR (qRT-PCR) was performed on a CFX 96 Touch Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA, USA) using PowerUp\({}^{\upmu\text{M}}\) SFBR\({}^{\upmu\text{M}}\) Green Master Mix (Thermo Scientific, Waltham, MA, USA). The housekeeping gene (18S rRNA) was used as an endogenous reference for expression data normalization [52]. The relative expression ratio of _DsFtsH2_ was computed in the presence of the following primers: DsFtsH2_Fw-F: 5\({}^{\prime}\)-CTGGGAGATGGCGTCCTG-3\({}^{\prime}\) and DsFtsH2_Rev: 5\({}^{\prime}\)-ACCTGAAGGAGATGGCCTC-3\({}^{\prime}\) qPCR reactions, amplification, and analysis were performed as described by [PERSON] et al. [53].  
In Silico and Functional Analysis of FtsH2 Metalloprotease from Dunaliella salina 2.8.1. Identification of _FtsH_-Encoding Genes in the _Dunaliella salina_ Genome  
Genome sequences of _Dunaliella salina_ were downloaded from the Phytosome 13 database ([[https://phytozome-next.jgi.doe.gov/](https://phytozome-next.jgi.doe.gov/)]([https://phytozome-next.jgi.doe.gov/](https://phytozome-next.jgi.doe.gov/)) accessed on 17 June 2024). FtsH protein sequences of _A. thaliana_ were downloaded from the _Arabidopsis_ Information Resource (TAIR, [[https://www.arabidopsis.org/](https://www.arabidopsis.org/)]([https://www.arabidopsis.org/](https://www.arabidopsis.org/)), accessed on 17 June 2024). These sequences were used as a references to identify the genes encoding FtsH proteins in the _Dunaliella salina_ genome. The BLAST program of TB Tools software (v 1.098722) was used to search for the potential _FtsH_-encoding genes in the whole genome. A Pfam database ([[http://pfam.xfam.org](http://pfam.xfam.org)]([http://pfam.xfam.org](http://pfam.xfam.org)), accessed on 17 June 2024) screening of candidate sequences was conducted to determine the presence of FtsH-conserved domains (PF0004, PF01434, and PF17862). Candidate sequences were further subjected to an online search with the CD-Search ([[https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)]([https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)), accessed on 17 June 2024) to confirm the accuracy of the results. After removing the redundant sequences using the Decrease Redundancy program ([[https://www.expasy.org/resources/decrease-redundancy](https://www.expasy.org/resources/decrease-redundancy)]([https://www.expasy.org/resources/decrease-redundancy](https://www.expasy.org/resources/decrease-redundancy)), accessed on 17 June 2024), seven DsFtsH proteins were obtained and selected for phylogenetic analysis.