An Improved CTAB–Ammonium Acetate Method for Total RNA Isolation from Cotton

BackgroundDNA methylation, with a cryptic role in genome stability, gene transcription and expression, is involved in the drought response process in plants, but the complex regulatory mechanism is still largely unknown.ResultsHere, we performed whole-genome bisulfite sequencing (WGBS) and identified long non-coding RNAs on cotton leaves under drought stress and re-watering treatments. We obtained 31,223 and 30,997 differentially methylated regions (representing 2.48% of the genome) after drought stress and re-watering treatments, respectively. Our data also showed that three sequence contexts, including mCpG, mCHG, mCHH, all presented a hyper-methylation pattern under drought stress and were nearly restored to normal levels after the re-watering treatment. Among all the methylation variations, asymmetric CHH methylation was the most consistent with external environments, suggesting that methylation/demethylation in a CHH context may constitute a novel epigenetic modification in response to drought stress. Combined with the targets of long non-coding RNAs, we found that long non-coding RNAs may mediate variations in methylation patterns by splicing into microRNAs. Furthermore, the many hormone-related genes with methylation variations suggested that plant hormones might be a potential mechanism in the drought response.ConclusionsFuture crop-improvement strategies may benefit by taking into account not only the DNA genetic variations in cotton varieties but also the epigenetic modifications of the genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3681-y) contains supplementary material, which is available to authorized users.

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“…Genomic DNA and RNA were extracted with CTAB method [52]. Genomic DNA and RNA degradation and contamination were monitored on 1% agarose gels.…”

Section: Methodsmentioning

confidence: 99%

Single-base resolution methylomes of upland cotton (Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress

Wang

Chen

et al. 2017

BMC Genomics

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“…), high NaCl concentrations induce leaf senescence . Nitric oxide (NO) has been found to delay salt‐induced leaf senescence in cotton through regulation of SOS1 , NHX1 , NCED2 , NCED9 , and IPT1 expression . Changes in expression of these genes decrease intracellular Na + levels and abscisic acid (ABA) contents and increase intracellular K + levels and cytokinin expression .…”

Section: Salt Stressmentioning

confidence: 99%

Molecular mechanisms underlying stress response and adaptation

Sun

Zhou

2017

Thoracic Cancer

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Environmental stresses are ubiquitous and unavoidable to all living things. Organisms respond and adapt to stresses through defined regulatory mechanisms that drive changes in gene expression, organismal morphology, or physiology. Immune responses illustrate adaptation to bacterial and viral biotic stresses in animals. Dysregulation of the genotoxic stress response system is frequently associated with various types of human cancer. With respect to plants, especially halophytes, complicated systems have been developed to allow for plant growth in high salt environments. In addition, drought, waterlogging, and low temperatures represent other common plant stresses. In this review, we summarize representative examples of organismal response and adaptation to various stresses. We also discuss the molecular mechanisms underlying the above phenomena with a focus on the improvement of organismal tolerance to unfavorable environments.

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“…The CTAB method has been widely used for RNA isolation from polyphenols and polysaccharides-rich plants, including pine needles (Chang et al, 1993), a variety of tissues and organs of Chinese fir (Wang et al, 2007(Wang et al, , 2011, cotton (Zhao et al, 2012), Spurge (Xu et al, 2010), and tea (Muoki et al, 2012). Unexpectedly, it failed to extract high quality RNA from floral buds of tree peony.…”

Section: Resultsmentioning

confidence: 99%

Comparison of Different Methods for RNA Extraction from Floral Buds of Tree Peony (<i>Paeonia suffruticosa</i> Andr.)

Gao

Zhao

Jiang

et al. 2016

Not Bot Horti Agrobo

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Tree peony (Paeonia suffruticosa Andr.), a species native to China, is one of the most important ornamental and medicinal plants. Like other tree species in temperate and boreal zones, the dormancy-activity transition of floral buds is critical for blooming time and fruit production. However, floral buds contain high levels of secondary metabolites, making the isolation of high quality RNA difficult. To obtain a method suitable for extracting RNA from floral buds of tree peony, we evaluated five different methods, including the cetyltrimethylammonium bromide (CTAB) and Sodium dodecyl sulfate (SDS)-based methods, a modified SDS-TRNzol protocol, and two commercial kits (TRNzol and Qiagen RNeasy Plant Mini Kit). The modified SDS-TRNzol method was capable of efficiently removing polyphenols and other metabolites in floral buds. The isolated RNA was of high purity and integrity, as demonstrated by the the A 260/280 ratio of approximately 2.0, and RIN values of more than 9.0. Gel electrophoresis analysis indicated that the extracted RNA had clear 28S and 18S ribosomal RNA bands without DNA contamination. The RNA isolated by this protocol was successfully used for downstream manipulations, such as RT-PCR, RACE, and real-time PCR. Together, the modified SDS-TRNzol protocol is an easy, efficient, and highly reproducible method for RNA isolation from floral buds rich in secondary metabolites.

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An Improved CTAB–Ammonium Acetate Method for Total RNA Isolation from Cotton

Abstract: The CTAB-ammonium acetate method is easy, rapid, low-cost and effective for high-quality RNA isolation from polysaccharide- and polyphenol-rich cotton tissues.

Cited by 61 publications

References 16 publications

Single-base resolution methylomes of upland cotton (Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress

Single-base resolution methylomes of upland cotton (Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress

Molecular mechanisms underlying stress response and adaptation

Comparison of Different Methods for RNA Extraction from Floral Buds of Tree Peony (<i>Paeonia suffruticosa</i> Andr.)

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