RESUMEN
Normalization based on inappropriate reference gene may lead to the reduction of the accuracy of RT-qPCR. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in Ulva linza, a ubiquitous green-tide forming alga, are lacking. The expression levels of ten candidate reference genes were analyzed in U. linza across different experimental treatments, and the best-ranked reference genes differed across the treatments. The most suitable reference genes were tubulin2 (TUB2) among different salinity and UV treatments. Histone 2 (H2) was stably expressed in different temperature and desiccation stress treatments. 18S rRNA exhibited better expression stability in different light intensity treatments. While all tested samples were considered, none of single gene was widely applicable as a reference gene. Moreover, using a combination of two genes as reference genes might improve the reliability of gene expression by RT-qPCR, and the combination of TUB1 and TUB2 was selected as ideal for all tested samples. The results suggest that assessing the stability of reference gene expression patterns, determining candidates, and testing their suitability are required for each experimental investigation. The results will guide the selection of reference genes for gene expression studies in U. linza.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Genes de Plantas , Ulva/genética , Perfilación de la Expresión Génica/normas , Regulación de la Expresión Génica de las Plantas , ARN Ribosómico 18S , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tubulina (Proteína)/genética , Rayos Ultravioleta , Ulva/efectos de la radiaciónRESUMEN
Light-harvesting complexes (LHCs) play essential roles in light capture and photoprotection. Although the functional diversity of individual LHCs in many plants has been well described, knowledge regarding the extent of this family in the majority of green algal groups is still limited. In this study, two different LhcSR genes, LhcSR1 and LhcSR2 from Chlamydomonas sp. ICE-L, were cloned from the total cDNA and characterized in response to high light (HL), low light (LL), UV-B radiation and high salinity. The lower F (v)/F (m) as well as the associated induction of non-photochemical quenching (NPQ), observed under those conditions, indicated that Chlamydomonas sp. ICE-L was under stress. Under HL stress, the expression of LhcSR1 and LhcSR2 increased rapidly from 0.5 h HL and reached a maximum after 3 h. In LL, LhcSR2 expression was up-regulated during the first 0.5 h after which it decreased, while the expression of LhcSR1 decreased gradually from the beginning of the experiment. In addition, the transcript levels of LhcSR1 and LhcSR2 increased under UV-B radiation and high salinity. These results showed that both genes were inducible and up-regulated under stress conditions. A higher NPQ was accompanied by the up-regulated LhcSR genes, suggesting that LhcSR plays a role in thermal energy dissipation. Overall, the results presented here suggest that LhcSR1 and LhcSR2 play a primary role in photoprotection in Chlamydomonas sp. ICE-L under stress conditions and provide an important basis for investigation of the adaptation mechanism of LhcSR in Antarctic green algae.
Asunto(s)
Proteínas Algáceas/genética , Chlamydomonas/genética , Chlamydomonas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Complejos de Proteína Captadores de Luz/genética , Proteínas Algáceas/biosíntesis , Secuencia de Aminoácidos , Regiones Antárticas , Clorofila/química , Clonación Molecular , Biología Computacional/métodos , Cartilla de ADN/genética , ADN Complementario/metabolismo , Calor , Luz , Complejos de Proteína Captadores de Luz/biosíntesis , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Fotoquímica/métodos , Filogenia , Homología de Secuencia de Aminoácido , Temperatura , Factores de Tiempo , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
The green-tide-forming macroalga Ulva linza was profiled by transcriptome sequencing to ascertain whether the alga carries both C3 and C4 photosynthesis genes. The key enzymes involved in C4 metabolism including pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK) were found. When measured under normal and different stress conditions, expression of rbcL was higher under normal conditions and lower under the adverse conditions, whereas that of PPDK was higher under some adverse conditions, namely desiccation, high salinity, and low salinity. Both ribulose-1, 5-biphosphate carboxylase (RuBPCase) and PPDK were found to play a role in carbon fixation, with significantly higher PPDK activity across the stress conditions. These results suggest that elevated PPDK activity alters carbon metabolism in U. linza leading to partial operation of the C4 carbon metabolism, a pathway that, under stress conditions, probably contributes to the hardy character of U. linza and thus to its wide distribution.