DOI: 10.57715/unr/5tsedo
title: Transcription levels of some genes involved in starch metabolism in ChlreSEX4 overexpressing algal lines

description: The presented dataset illustrates the transcriptional levels of select genes implicated in starch metabolism within ChlreSEX4 overexpressing algal strains. The analysis of gene expression levels was conducted with the aim of elucidating the impact of upregulating a glucan phosphatase enzyme within the chloroplast of green algae. The file displays tables with the relative expression for each studied gene in different algal lines (WT, 13, 24, and 48), with each gene shown in different colors. The column "SAMPLE NAME" indicates which algal line is being analyzed. The column "TARGET NAME" indicates which gene is being analyzed, including its name and its ORF name. The column "CT" shows the Ct value obtained after qRT-PCR for each gene and each algal line, using the StepOnePlus Real-Time PCR System under the following conditions: 2 min denaturation at 94 °C; 40 cycles at 94 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s, followed by 10 min extension at 72 °C. Three replicates were performed for each sample and each gene. The column "AVERAGE CT" shows the average Ct obtained for the EEF1A gene in each line, which is used as an internal control. The column "DELTA CT" is the difference between the Ct for the specific gene under study and the average Ct for the EEF1A gene in each algal line. The column "2^-DELTA CT" is the result of calculating 2^-DELTA CT for each gene in each line. The column "average exp" is the average 2^-DELTA CT value obtained for each gene for each algal line. The column "relative expression" is the individual value of 2^-DELTA CT obtained for each gene and each line after considering the average 2^-DELTA CT for that gene in the WT line as 1. Methodology. Total RNA was extracted from Chlamydomonas reinhardtii cells using TRI reagent (Molecular Research Center, Inc, USA) according to the manufacturer's instructions. RNA quality was determined by agarose gel electrophoresis, and its concentration was determined on the Microplate Spectrophotometer. The first strand cDNAs were synthesized by M-MLV reverse transcriptase (Promega, Fitchburg,WI, USA) and an oligo-dT 18 primer. Pair of primers for qRT-PCR were: AGPaseFw (GTGATGGAGAGCAGACGGAGGT) and AGPaseRv (CTCGCTTCAACAGTACCCGAGC) for transcript STA6AGPase (CHLRE_03g188250v5 transcript locus); GBSSIFw (GTGACTGTGTCGCCCAACTACG) and GBSSI Rv (AGGAACTTGTCGGTCTTGGGGT) for transcript GBSSI (locus CHLRE_17g721500v5); SSII Fw (GGACGCACTGTGACATACGCTT) and SSII Rv (CTTCAAGCCCACACGCCCTAAC) for transcript SSII (locus CHLRE_03g185250v5); SBE2 Fw (GTACAAGTGGCTGCTGGCCTTC) and SBE2 Rv (GGGTGGGGTGGAAGTTGAACAC) for transcript SBE2 (locus CHLRE_06g270100v5); GWD2 Fw (CTACGACGAGCCGTTCCTGGAG) and GWD2 Rv (CGTGATATGGTCAATCCCCTCCCC) for transcript GWD2 (locus CHLRE_07g332300v5); PWD Fw (CATCGCCAACTTCTCCACGGC) and PWD Rv (TAGTCCACTTCCCGGTCCACCA) for transcript PWD (locus CHLRE_17g719900v5); AMY2 Fw (CTTGAGCAGTTGGGAGAGGCGA) and AMY2 Rv (CGTTCATGCACCCGAATAGCGG) for transcript AMY2 (locus CHLRE_08g362450v5); and, EEF1 UP (CTAGGATGCTAACCGCGCAAGAG) and EEF1 DW (AACAGCGAAGCTCCACGCAC) for transcript EEF1 (Eukaryotic translation Elongation Factor 1 alpha 2 (CHLRE_12g498600v5)). EEF1 was used as an internal control and the relative expression ratio was calculated as described in Pfaffl, M. W. (2001). The entire process of qRT-PCR was carried out with The StepOnePlus Real-Time PCR System under the following conditions: 2 min denaturation at 94 °C; 40 cycles at 94 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s, followed by 10 min extension at 72 °C. Three replicates were performed for each sample. Melt curve analysis was conducted to confirm the specificity of each assay. Value of the data: The transcription levels of some genes involved in starch metabolism were analyzed and may contribute to gain insight into the intricately regulated mechanism of starch synthesis/degradation and phosphorylation/dephosphorylation in vivo.