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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Reference genes for transcript quantification in Gracilaria tenuistipitata under drought stress

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Alves-Lima, Cicero ; Cavacana, Natale ; Teixeira Chaves, Gustavo Antonio ; de Lima, Natalia Oliveira ; Stefanello, Eliezer ; Colepicolo, Pio ; Hotta, Carlos Takeshi
Total Authors: 7
Document type: Journal article
Source: JOURNAL OF APPLIED PHYCOLOGY; v. 29, n. 2, p. 731-740, APR 2017.
Web of Science Citations: 4

Gracilaria tenuistipitata is a red macroalga often found in intertidal environments, where it is frequently under drought stress due to tidal water scarcity. In order to be able to study the molecular mechanisms involved in G. tenuistipitata resistance to dehydration, we have done a dehydration assay, tested different RNA extraction protocols, and selected new reference genes for reverse transcription quantitative PCR (RT-qPCR). After testing four different methods, we found that an adapted TRIzol method resulted in the highest quantities of pure RNA from less biomass. This method yielded an average of 783.6 ng mg(-1) of clean RNA from 25 mg of fresh tissue in only 1.5 h. Additionally, DNAse I treatment and silica spin column purification were done prior to RT-qPCR. Of the nine putative reference genes tested for expression stability under control and drought-like conditions, a combination of four genes: beta-GALACTOSIDASE B (GLB), TRANSCRIPTION INITIATION FACTOR IIB (GTF2B), EUKARYOTIC ELONGATION FACTOR 2 (EEF2), and beta-TUBULIN (TUB) was selected. This allowed us to measure the transcription level of THIOREDOXIN (TRX), OXYGEN-EVOLVING ENHANCER 1 (OEE), and NITRATE REDUCTASE 2 (NIA2). TRX was upregulated after 1-h dehydration and kept higher transcriptional levels even 4 h later. In contrast, OEE1 transcriptional levels were slowly downregulated, and NIA2 transcriptional levels were downregulated 1 h after dehydration but returned to regular levels 4 h after. This suggests that 40 % dehydration of G. tenuistipitata induced transcriptional responses without changing the relative growth rate or affecting the thalli mortality. (AU)

FAPESP's process: 11/00818-8 - Development of alternative biological models for the study of sugarcane regulatory networks
Grantee:Carlos Takeshi Hotta
Support type: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants
FAPESP's process: 13/05301-9 - Study on the interactions between the biological clock and desiccation in photosynthetic eukaryotes
Grantee:Cícero Alves Lima Júnior
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 10/14893-9 - Study of metabolic effects of anthracene on microalgae Chlamydomonas reinhardtii
Grantee:Eliezer Stefanello
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 13/08574-6 - Auxin signaling pathways and their interactions with the circadian clock in sugarcane
Grantee:Gustavo Antônio Teixeira Chaves
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 10/50193-1 - Marine algae as source of metabolites of economical impact
Grantee:Pio Colepicolo Neto
Support type: BIOTA-FAPESP Program - Thematic Grants