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Isolation of functional RNA from cactus fruit 总被引:1,自引:0,他引:1
María Leonor Valderrama-Cháirez Andrés Cruz-Hernández Octavio Paredes-López 《Plant Molecular Biology Reporter》2002,20(3):279-286
Isolating RNA from cactus fruit is notoriously difficult because the fruit contains high amounts of secondary metabolites
and polysaccharides. These form insoluble complexes with nucleic acids during extraction and can inhibit enzyme action. Our
procedure allows for the extraction of RNA from finely ground tissue. The RNA we isolated was of high quality and undegraded,
as gauged by spectrophotometry and electrophoresis in agarose gels. Quality was further assessed through use of the RNA in
RT-PCR and northern blot analysis, indicating that it could be used to construct cDNA libraries. Using this modified protocol,
90μg of RNA was routinely obtained from 1 g of dried cactus fruit. Isolating RNA from other polysaccharide-rich fruits was
also possible. 相似文献
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A simple procedure for the isolation of high quality RNA from ripening banana fruit 总被引:18,自引:0,他引:18
Isolation of high quality RNA from ripening banana fruit tissue is difficult due to high levels of polysaccharides and other
substances that interfere when using conventional procedures for RNA isolation. These substances not only decrease the yield
but the quality of RNA is almost unusable. We describe here a simple RNA procedure that effectively removes these contaminating
substances without affecting the yield. Following this procedure, we routinely obtained 80–150 μg of total RNA per g fresh
tissue. The RNA is of good quality and suitable for northern analysis, RT-PCR and cDNA library construction.
NBRI publication No. 488(NS). 相似文献
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An ethylene-related cDNA from ripening apples 总被引:17,自引:0,他引:17
We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)+ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit. 相似文献
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A modified procedure for PCR-based differential display and demonstration of use in plants for isolation of genes related to fruit ripening 总被引:1,自引:1,他引:0
We have modified and optimized PCR-based differential display for efficient identification and isolation of genes whose expression
patterns are correlated with changes in growth, development, physiology, and/or environmental response. This protocol is general
in nature and can be applied for analysis of virtually any plant tissues from which several μg of total RNA can be extracted.
We report here the use of tomato fruit ripening as a model system in which to test and optimize differential display in plants.
Specifically, mRNA from ripe, early breaker, mature green, and ethylene-treated mature green tomato fruit were examined to
identify and distinguish non-ethylene-inducible from ethylene-inducible genes related to ripening. DNA-free total RNA was
utilized as template for synthesis of first-strand complementary DNA using each of 12 possible 5′-T11 XY-3′ anchor primers (X=A, C, or G; Y=A, C, G, or T). PCR amplification products of the resulting cDNA populations were generated
via use of random primers in combination with the corresponding anchor primer employed for cDNA synthesis. We demonstrate
that degenerative anchor primers are useful for making representative cDNA populations, but are not effective for representative
display-PCR. cDNA, resulting from degenerative anchor primer synthesis, yielded substantially fewer ripening-related display-PCR
products when amplified with the same degenerative anchor primer employed in cDNA synthesis, versus the corresponding set
of specific anchor primers. Amplification products specific to ripe fruit cDNA were isolated directly from display gels, reamplified,
cloned, and confirmed for ripening-related gene expression via RNA gel-blot analysis. 相似文献
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Laura Jaakola Anna Maria Pirttilä Minna Halonen Anja Hohtola 《Molecular biotechnology》2001,19(2):201-203
A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the
use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and β-mercaptoethanol in an extraction buffer
in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of
the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable
for a variety of plant tissues. 相似文献
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Up-regulation of two cDNA clones encoding metallothionein-like proteins in apple fruit during cool storage 总被引:3,自引:0,他引:3
We are investigating the molecular basis of low temperature responses in apples, by identifying and characterising fruit genes which show altered expression in response to cool-storage, Two independent cold-regulated clones (pAMTI and pAMT2) were isolated from a cDNA library derived from cool-stored apple ( Malus domestics Borkh. cv. Granny Smith) fruit. These clones share only 27% amino acid identity with each other, but both show high similarity to plant metallothionein (MT)-like proteins. The polypeptide encoded by pAMTI shares similarity with type 2 MT-like sequences, while that encoded by pAMT2 is similar to others which share a different distribution of cysteine residues. We suggest, these form a 'type 3' group of MT-like clones. Genomic Southern analysis confirmed that there is a family of MT-like genes in apple. There are differing patterns of pAMTI and pAMT2 expression during apple fruit development, amt 1 RNA was abundant in flowers and during the early stages of development, and decreased as the fruit approached maturity, while amt 2 RNA was barely detectable in flowers and young fruit and accumulated with fruit development. In ripe fruit. amt 1. expression was up-regulated, while amt 2 expression was down-regulated. In leaves, both genes showed increased expression with leaf age. In Granny Smith, Cox's Orange Pippin and Braeburn apple cultivars. both genes were up-regulated in cool-stored fruit. In Granny Smith contical tissue, amt RNA levels were elevated within the first 45 min at both 0.5°C and 4°C, but not at 12.5°C. The different patterns of amt 1 and amt 2 expression during fruit development and in different tissues suggest that the respective genes have distinct controlling elements and may be functionally different. The in vivo roles of the encoded polypeptides, particularly in relation to chilling tolerance or acclimation, are as yet unknown. 相似文献
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A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites 总被引:31,自引:0,他引:31
A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides.
The method is based on the Nucleon PhytoPure™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same
time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried
out with the RiboGreen™ reagent and of PCR products with the PicoGreen™ reagent. 相似文献
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A Rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants 总被引:1,自引:0,他引:1
Introduction – RNA quality and integrity are critical for many studies in plant molecular biology. High‐quality RNA extraction from grapevine and other woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co‐precipitate with the RNA. Objective – To develop an optimised cetyltrimethylammonium bromide (CTAB)‐based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from polysaccharide‐rich tissues of several plants. Methodology – Several changes were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C, the sample weight was decreased and the concentrations of PVP‐40 and LiCl were increased reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with six different RNA extraction methods from three grapevine tissues, namely, in vitro plantlets, and leaves and mature canes from actively growing field vines. Results – The rapid CTAB method gave high‐quality RNA in only 3 h at low cost with efficiency equal to or higher than that obtained with other time‐consuming and expensive protocols. The procedure was applied to RNA extraction from other grapevine tissues and other woody species including olive, lemon, poplar, chestnut, apple, pear, peach, cherry, apricot, plum and kiwi fruit. RNA of high quality could be isolated from all tissues and from all species. Conclusion – The study has shown that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from grapevine and many woody species. Copyright © 2008 John Wiley & Sons, Ltd. 相似文献
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RNA isolation from loquat and other recalcitrant woody plants with high quality and yield 总被引:1,自引:0,他引:1
Jaime Morante-Carriel Susana Sellés-Marchart Ascensión Martínez-Márquez María José Martínez-Esteso Ignacio Luque Roque Bru-Martínez 《Analytical biochemistry》2014
RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function. 相似文献
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Fan Yang Haidong Tan Yongjin Zhou Xinping Lin Sufang Zhang 《Molecular biotechnology》2011,47(2):144-151
Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the
basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal
protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method
was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the
addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total
RNA per gram of cells was obtained with an average purity measured as A260/A280 of 2.09. A titer of 105 cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel
analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed
the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for
screening the genes related to lipid accumulation. 相似文献