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1.
Mustapha Aitchitt Charles C. Ainsworth Madan Thangavelu 《Plant Molecular Biology Reporter》1993,11(4):317-319
A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable
for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for
at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may
be applicable to other species of palms. 相似文献
2.
Abolghasem Abbasi Kejani Sayed Ali Hosseini Tafreshi Sayed Mojtaba Khayyam Nekouei Mohammad Reza Mofid 《Molecular biology reports》2010,37(2):797-800
Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew
(Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation
were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform
extraction steps from the isolation procedure. Both spectrophotometric (A260/A280 and A260/A230 ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the
average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g
leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively. 相似文献
3.
We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality
DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity,
clear color, A260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability. 相似文献
4.
5.
RNA isolation from high-phenolic tea leaves and apical buds 总被引:6,自引:0,他引:6
Lakhvir Lal Rashmita Sahoo Rajesh Kumar Gupta Priti Sharma Sanjay Kumar 《Plant Molecular Biology Reporter》2001,19(2):181-181
6.
Varietal resistance of tea towardsBipolaris carbonum was tested following detached leaf inoculation technique. Among the fourteen varieties tested, three were found to be highly
susceptible, while other three were resistant. Leaf exudates and diffusates collected from the resistant varieties were more
fungitoxic than those from the susceptible ones. Two antifungal compounds isolated from healthy andB. carbonum-infected tea leaves exhibited clear inhibition zones atR
F 0.8 and 0.65, respectively, in a chromatographic bioassay. On the basis of their color reaction on TLC and UV-spectra these
were identified to be catechin and pyrocatechol. Resistant and susceptible varieties accumulated 439–510 and 187–212 μg/g
fresh mass tissue of pyrocatechol, respectively, 2 d after inoculation withB. carbonum, while a low concentration (45–58 μg/g) of this compound was detected in healthy leaf tissue. 相似文献
7.
RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the
authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with
PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along
with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric
analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g)
and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at
126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream
molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely
to be widely applicable. 相似文献
8.
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
9.
High-quality RNA is important in studying gene expression. This report describes an improved method for isolating intact purified
RNA from dehydrated organs of chili pepper plants. Common RNA extraction protocols have produced poor yields because dehydrated
leaves accumulate polysaccharides and RNases. Our protocol is based on a guanidine thiocyanate extraction combined with additional
purification steps using butanol and the ionic detergent CTAB (cetyltrimethylammonium bromide). Using this protocol, RNA yields
ranged from 40–70 μg of total RNA per 200 mg of fresh tissue. This method can be adapted to large-scale isolations, allowing
the recovery of larger amounts of intact RNA (up to 250 μg per gram of fresh tissue). 相似文献
10.
Salix gracilistyla is one of the dominant plants in the riparian vegetation of the upper-middle reaches of rivers in western Japan. This species
colonizes mainly sandy habitats, where soil nutrient levels are low, but shows high potential for production. We hypothesized
that S.␣gracilistyla uses nutrients conservatively within stands, showing a high resorption efficiency during leaf senescence. To test this hypothesis,
we examined seasonal changes in nitrogen (N) and phosphorus (P) concentrations in aboveground organs of S. gracilistyla stands on a fluvial bar in the Ohtagawa River, western Japan. The concentrations in leaves decreased from April to May as
leaves expanded. Thereafter, the concentrations showed little fluctuation until September. They declined considerably in autumn,
possibly owing to nutrient resorption. We converted the nutrient concentrations in each organ to nutrient amounts per stand
area on the basis of the biomass of each organ. The resorption efficiency of N and P in leaves during senescence were estimated
to be 44 and 46%, respectively. Annual net increments of N and P in aboveground organs, calculated by adding the amounts in
inflorescences and leaf litter to the annual increments in perennial organs, were estimated to be 9.9 g and 0.83 g m−2 year−1, respectively. The amounts released in leaf litter were 6.7 g N and 0.44 g P m−2. These values are comparable to or larger than those of other deciduous trees. We conclude that S. gracilistyla stands acquire large amounts of nutrients and release a large proportion in leaf litter. 相似文献
11.
Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.) 总被引:1,自引:1,他引:0
A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy
for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons
and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing.
Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast
to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the
latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications
as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning. 相似文献
12.
Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components 总被引:57,自引:1,他引:57
A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large
quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of
these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt
concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase
treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and
cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5
ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue
that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR
amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae. 相似文献
13.
Selenium-enriched tea was suggested as a possible source of supplemental Se. The result of this study indicates that it is
not practicable to make selenium-enriched tea as a beverage like traditional green tea or black tea for the supplementation
of selenium in human diet. The selenium dispersibilities of fresh tea leaves, green tea, and black tea highly correlated with
those of protein (r
2 = 0.998). The high protein dispersibility (85.0%) of fresh tea leaves in water solution was accompanied by that of selenium
(93.8%). Decreases in protein dispersibility of green tea and black tea to 2.5% and 4.2 % coincided with those of selenium
to only 8.3% and 10.1%, respectively. The amount (14.90 μg) of selenium in saturated ammonium sulphate (a protein precipitating
reagent) precipitate was 83.8% of that (17.79 μg) in fresh tea leaf extract, and after the saturated ammonium sulphate precipitate
was dialyzed against distilled water overnight, the amount (14.37 μg) of selenium remaining in the dialyzed precipitate (protein)
was still 80.8% of that in the fresh tea leaf extract. However, there were no significant differences (p > 0.05) between the amount of selenium in the saturated ammonium sulphate precipitate and that in the saturated ammonium
sulphate precipitate that was dialyzed. 相似文献
14.
Silla Fernando Fleury Marina Mediavilla Sonia Escudero Alfonso 《Trees - Structure and Function》2008,22(6):785-793
We examined the effects of simulated folivory by caterpillars on photosynthetic parameters and nitrogen (N) resorption efficiency
in Quercus pyrenaica saplings. We analyzed the differences between intact leaves in control plants, punched leaves in damaged plants, and intact
leaves in damaged plants. We then established two levels of simulated folivory: low (≈13% of the leaf area of one main branch
removed per plant) and high (≈26% of the leaf area of one main branch removed per plant) treatments. No differences were found
in net assimilation rate and conductance between either leaf type or treatment during the most favourable period for photosynthesis.
However, the N content was lower in punched than in intact leaves, and as a result PNUE was higher in damaged leaves from
treated trees. In leaf-litter samples, N mass was significantly higher in punched than in intact leaves in treated plants,
and LMA was significantly higher in damaged than in intact leaves of both the treated and control plants. Consequently, N
resorption efficiency was around 15% lower in damaged leaves as compared with intact leaves from treated and control plants.
Mechanical injury to leaves not only triggered no compensatory photosynthetic response to compensate a lower carbon uptake
due to leaf area loss, but also affected the resorption process that characterizes leaf senescence. 相似文献
15.
16.
An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different
DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and
Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by
Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved
procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g
of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated. 相似文献
17.
1. A marked decrease in the total RNA content during the withering process of tea leaves was found. During the fermentation process, there was a small but significant decrease in the total RNA content. 2. During isolation of RNA from tea leaf tissues, the action of leaf ribonuclease was minimized by the addition of sodium dodecyl sulphate during extraction; 1% (w/v) sodium dodecyl sulphate in 0.2m-tris-hydrochloric acid buffer, pH8.0, containing 0.005% EDTA was found to be most efficient for the extraction and gave about 93% yield. 3. The total RNA preparations isolated from fresh, withered and fermented tea leaves were compared with regard to nucleotide composition and spectral characteristics. The total RNA preparations from all three sources contained more purines than pyrimidines (purine/pyrimidine ratio 1.47-1.52). 相似文献
18.
Many plant RNA isolation techniques aim to prevent contamination by means of secondary phenolics, carbohydrates, RNase, and
other chemicals. However, when applied in our laboratory to the isolation of RNA fromRumex obtusifolius, these protocols failed to produce good quality RNA. A major problem was contamination of the RNA samples with the secondary
metabolite oxalate. The relative quantities of guanidine isothiocyanate extraction buffer to plant tissue used in the protocol
had significant effects on oxalate contamination. An increase in extraction buffer, from 1.5 mL in the original method to
15 mL per 200–300 mg of tissue in our protocol, removed the oxalate from the RNA. This RNA was of a good quality and was suitable
for molecular biology applications. 相似文献
19.
Small RNAs play important roles in plant development, metabolism, signal transduction and responses to biotic and abiotic
stresses by affecting gene expression. Tea (Camellia sinensis L.) is an important commercial crop in the world. To understand the regulatory mechanisms involving small RNAs in tea metabolism,
we constructed a small RNA (sRNA) library from its tea drink manufacturing tissue part i.e. topmost two leaves and a bud.
For the first time, we isolated and cloned six novel small RNAs candidates from tea. These were predicted to target 67 genes
responsible for various important plant functions. Isolated small RNAs were validated through expression analysis in young
leaf and old leaf during non-dormant and dormant growth phases of tea. Results suggest the probable role of isolated small
RNAs in development and seasonal variations of tea. 相似文献
20.
A Modified Protocol for RNA Extraction from Different Peach Tissues Suitable for Gene Isolation and Real-Time PCR Analysis 总被引:1,自引:0,他引:1
RNA extraction is the first step in the study of gene isolation and expression. However, it is difficult to extract high quantity
and quality RNA from tissues containing large quantities of polysaccharides and polyphenols. Peach (Prunus persica), in addition to containing high levels of polysaccharides and polyphenols, is a challenging starting material for RNA isolation
using a single method because of different amounts of those substances in diverse tissues. Based on three reported methods,
we developed a modified RNA isolation protocol to solve this problem, leading to high quality and quantity of total RNA from
peach mesocarp tissues of fruits which were sampled from all developmental stages and different storage periods, as well as
from other tissues including flowers, leaves, stems, and roots. With our modified method, 28–650 μg of total RNA was routinely
obtained from per gram of fresh material, gave at least a 1.16-fold improvement by compared with those isolated by other seven
methods. The RNA extracts were successfully used in downstream applications such as RT-PCR, RACE, and real-time PCR. 相似文献