Dataset Information

Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses.

ABSTRACT: Synechococcus elongatus PCC 7942 (S. elongatus PCC 7942) is a model cyanobacteria species for circadian clock mechanism studies. It has also been widely used as a bioreactor to produce biofuels and other metabolic products. Quantitative real-time PCR (qPCR) technology is the most commonly used method for studying the expression of specific genes, in which the relative expression level of target genes is calibrated by stably expressed internal reference genes. In this work, we examined the expression of nine candidate reference genes in time-course samples of S. elongatus PCC 7942 under no treatment (control), NaCl-stress conditions, H₂O₂-stress conditions, and high light-stress conditions. Based on the qPCR amplification parameters, the stability ranking of these candidate reference genes was established by three statistical software programs, geNorm, NormFinder, and BestKeeper. Considering all the stress conditions or high light stress alone, the results showed that the combination of prs and secA was the best choice for the double reference gene calibration method by qPCR. The combination of secA and ppc, rimM and rnpA, rnpA, and ilvD was most stable under no treatment, NaCl-stress conditions, and H₂O₂-stress conditions, respectively. rimM was stable under only special conditions and should be carefully chosen. 16S and rnpB were not suitable as internal reference genes for S. elongatus PCC 7942 qPCR experiments under all experimental conditions. To validate the above results, a cyanobacterial core clock gene, kaiC, was used to evaluate the actual performance of the optimized reference genes by qPCR, as well as the worst reference genes under different stress conditions. The results indicated that the best reference gene yielded more accurate calibration results for qPCR experiments carried out in S. elongatus PCC 7942 time-course samples.

SUBMITTER: Luo X

PROVIDER: S-EPMC6500708 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses.

Luo Xiao X Li Jiaxing J Chang Tianliang T He Hongyan H Zhao Yi Y Yang Xue X Zhao Yuwei Y Xu Yao Y

BioMed research international 20190421

Synechococcus elongatus PCC 7942 (S. elongatus PCC 7942) is a model cyanobacteria species for circadian clock mechanism studies. It has also been widely used as a bioreactor to produce biofuels and other metabolic products. Quantitative real-time PCR (qPCR) technology is the most commonly used method for studying the expression of specific genes, in which the relative expression level of target genes is calibrated by stably expressed internal reference genes. In this work, we exami ...[more]

PMID: 31139651

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Project description:The direct conversion of carbon dioxide into biofuels by photosynthetic microorganisms is a promising alternative energy solution. In this study, a model cyanobacterium, Synechococcus elongatus PCC 7942, is engineered to produce free fatty acids (FFA), potential biodiesel precursors, via gene knockout of the FFA-recycling acyl-ACP synthetase and expression of a thioesterase for release of the FFA. Similar to previous efforts, the engineered strains produce and excrete FFA, but the yields are too low for large-scale production. While other efforts have applied additional metabolic engineering strategies in an attempt to boost FFA production, we focus on characterizing the engineered strains to identify the physiological effects that limit cell growth and FFA synthesis. The strains engineered for FFA-production show reduced photosynthetic yields, chlorophyll-a degradation, and changes in the cellular localization of the light-harvesting pigments, phycocyanin and allophycocyanin. Possible causes of these physiological effects are also identified. The addition of exogenous linolenic acid, a polyunsaturated FFA, to cultures of S. elongatus 7942 yielded a physiological response similar to that observed in the FFA-producing strains with only one notable difference. In addition, the lipid constituents of the cell and thylakoid membranes in the FFA-producing strains show changes in both the relative amounts of lipid components and the degree of saturation of the fatty acid side chains. These changes in lipid composition may affect membrane integrity and structure, the binding and diffusion of phycobilisomes, and the activity of membrane-bound enzymes including those involved in photosynthesis. Thus, the toxicity of unsaturated FFA and changes in membrane composition may be responsible for the physiological effects observed in FFA-producing S. elongatus 7942. These issues must be addressed to enable the high yields of FFA synthesis necessary for large-scale biofuel production.

Dataset Information

Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses.

Publications

Stable Reference Gene Selection for RT-qPCR Analysis in <i>Synechococcus elongatus</i> PCC 7942 under Abiotic Stresses.

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