Isolation of High Quality RNA from Bilberry (Vaccinium myrtillus L.) Fruit

Jaakola, Laura; Pirttilä, Anna Maria; Halonen, Minna; Hohtola, Anja

doi:10.1385/mb:19:2:201

Cited by 370 publications

(265 citation statements)

References 5 publications

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“…Total RNA was isolated from fruits harvested in 2006 from two separate pools of tissue (stage 5/6 and stage 7/8) using the protocol from Jaakola et al (2001). Poly(A + ) RNA was isolated using Dynabeads Oligo(dT) 25 (Dynal) according to the manufacturer's instructions.…”

Section: Cdna Library Construction and Est Sequencingmentioning

confidence: 99%

“…Subsample(s) of approximately 1 g of tissue were divided into aliquots for RNA extraction. Total RNA was isolated from all tissues using the cetyl-trimethyl-ammonium bromide method originally designed for bilberry (Vaccinium myrtillus) fruit, also high in phenolics and carbohydrates (Jaakola et al, 2001). The extraction buffer contained 2% cetyltrimethyl-ammonium bromide, 0.3 g g 21 polyvinylpolypyrrolidone, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl, and 0.5 g L 21 spermidine.…”

Section: Rna Isolation and Cdna Synthesis For Qrt-pcr Gene Expressionmentioning

confidence: 99%

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Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

Zifkin

Jin²,

Ozga³

et al. 2011

Plant Physiology

256

281

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Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative realtime reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3#-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3#5#-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.Highbush blueberry (Vaccinium corymbosum; Ericaceae) is one of the most economically important fruit crops in North America. Blueberry fruit have been the focus of much recent attention due to numerous reports of their positive effects on human health. These benefits are generally attributed to high levels of polyphenolics, in particular the flavonoids (Rasmussen et al., 2005). Highbush blueberries have one of the highest in vitro antioxidant capacities of any fruit or vegetable (Prior and Gu, 2005;Wu et al., 2006). The major health benefits linked to the consumption of blueberries include a reduced risk for cardiovascular (Basu et al., 2010) and neurodegenerative (Neto, 2007) diseases. Furthermore, experiments on rodents suggest that blueberry extracts may also prevent cancer, slow tumor growth, and reverse cognitive and behavioral deficits related to stroke and aging (Lau et al., 2005;Gordillo et al., 2009).The three common types of flavonoids that accumulate in blueberry fruit are the flavonols, anthocya-

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Section: Cdna Library Construction and Est Sequencingmentioning

confidence: 99%

Section: Rna Isolation and Cdna Synthesis For Qrt-pcr Gene Expressionmentioning

confidence: 99%

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

Zifkin

Jin²,

Ozga³

et al. 2011

Plant Physiology

256

281

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“…Total RNAs were isolated separately from healthy leaves, leaf discs showing the translucent symptoms of blister blight disease, and leaf discs from healthy areas of infected leaves, all from the blister blight-resistant tea 'TRI2043', using the cetyl-trimethyl-ammonium bromide method (Jaakola et al, 2001). Equal amounts of total RNA from the three leaf samples were mixed together, and poly(A + ) RNA was isolated using an Oligotex mRNA Mini Kit (Qiagen).…”

Section: Construction Sequencing and Statistical Analyses Of A Leafmentioning

confidence: 99%

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

et al. 2013

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Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blightresistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (2)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (2)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.

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“…The root, stem and leaf were collected separately and frozen immediately in liquid nitrogen and stored at À80 C freezer until use. Subsequently, total RNA was extracted from all the different tissues using cetyltrimethylammonium bromide (CTAB) method (Jaakola et al 2001). Genomic DNA was extracted from the leaves of G. biloba following the protocol of Jiang and Cai (2000).…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning, characterization and heterologous expression in Saccharomyces cerevisiae of a mevalonate diphosphate decarboxylase cDNA from Ginkgo biloba

Pang

Shen

Bergès

et al. 2006

Physiologia Plantarum

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Mevalonate diphosphate decarboxylase (MVD; EC4.1.1.33) catalyses the conversion of mevalonate diphosphate to isopentenyl diphosphate (IPP), which is a key skeleton for numerous functionally important secondary metabolites. A full‐length cDNA of MVD was isolated from Ginkgo biloba (designated as GbMVD) using rapid amplification of cDNA ends (RACE). GbMVD was 1958 bp with poly(A) tailing and contained a 1290‐bp open reading frame encoding a 430‐amino acid protein. The deduced GbMVD protein had a deduced molecular weight of about 48 kDa and isoelectric point of 5.83, and it showed high similarity to MVDs from other species. The 3D‐structure modelling showed that the 3D model of GbMVD had a similar P loop as that of yeast. Phylogenetic tree analysis revealed that GbMVD shared the same ancestor with MVDs from other species, and it had a closer relationship with those from plant species. Southern blot analysis indicated that GbMVD might belong to a small multigene family. Transcript accumulation analysis revealed that GbMVD was transcribed in root, stem and leaf tissues. Functional complementation of GbMVD in an MVD‐deficient Saccharomyces cerevisiae strain MN19‐34 (erg19ts) confirmed that GbMVD mediated the conversion of mevalonate diphosphate to IPP and that it is a functional gene.

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Isolation of High Quality RNA from Bilberry (Vaccinium myrtillus L.) Fruit

Cited by 370 publications

References 5 publications

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

Molecular cloning, characterization and heterologous expression in Saccharomyces cerevisiae of a mevalonate diphosphate decarboxylase cDNA from Ginkgo biloba

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