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1.
With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based
biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation
procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are
suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media.
Transformation was verified through β-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification
of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes.
The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic
selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials
and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3–4 months, rather than the 6–12 months
required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes
them better suited for large-scale functional genomics studies in poplars. 相似文献
2.
DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent
applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for
isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP)
(Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA
was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A260/230 ratio of 1.836, A260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion. 相似文献
3.
A modified CTAB DNA extraction procedure for plants belonging to the family proteaceae 总被引:3,自引:0,他引:3
Tina L. Maguire Graham G. Collins Margaret Sedgley 《Plant Molecular Biology Reporter》1994,12(2):106-109
This paper describes rapid and efficient DNA extraction methods for mature leaves, resting buds and seedling leaves of genera
in the family Proteaceae. The procedures combine and modify previously published techniques. The DNA can be digested by restriction
endonucleases and is suitable for subsequent PCR amplification. 相似文献
4.
Hai-Linh Tran Seong-Joo Hong Choul-Gyun Lee 《Biotechnology and Bioprocess Engineering》2009,14(2):187-192
Microalgae are a very diverse group of organisms that consist in both prokaryotic and eukaryotic forms. Some species of microalgae
can be induced to overproduce particular fatty acids through simple manipulations of the physical and chemical properties
of the culture medium. In this paper, the effect of different extraction techniques on the recovery of fatty acids from the
freeze-dried biomass from two lipid-producing microalgal strains: Botryococcus braunii LB 572 (green algae) and Synechocystis sp. PCC 6803 (cyanobacteria) was examined. Five procedures were used: after conversion of the lipid material into the corresponding
fatty acid methyl esters (FAMEs) via suitable derivatization reactions (extraction-transesterification) and direct transesterification of biomass to produce FAMEs
(without the initial extraction step) that used differential types of catalysts and processing conditions. This study has
shown that procedure 3, a one step practical procedure for lipid extraction and in situ methyl ester derivation could be used successfully for the determination of the fatty acid compositions of microalgae and
cyanobacteria. 相似文献
5.
José A. Mercado Iman El Mansouri Silvia Jiménez-Bermúdez Fernando Pliego-Alfaro Miguel A. Quesada 《In vitro cellular & developmental biology. Plant》1999,35(2):152-153
Summary In this paper we describe a simple and efficient DNA extraction protocol for Fragaria species, a highly recalcitrant genus due to the large amount of polyphenols and polymeric carbohydrates present in strawberry
tissues. The protocol yields a high quality DNA that can be amplified by polymerase chain reaction and digested with restriction
endonucleases. 相似文献
6.
Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of juncaceae 总被引:2,自引:1,他引:1
Seven DNA extraction protocols were used to obtain DNA from herbarium specimens ofJuncus andLuzula (Juncaceae) of various ages. DNA of historical samples is difficult to extract, and the extracts are seldom of good quality.
The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260/280 absorbance ratio. The total
DNA yield was measured by a fluorometer. The results indicate the success of using both mixer mill grinding and a DNeasy Plant
Kit. Another extraction protocol (grinding with mortar and pestle, using liquid nitrogen) yielded DNA from many samples. Modified
CTAB extraction, with a lengthy precipitation, usually provided good amounts of DNA. Other protocols did not give satisfactory
results. 相似文献
7.
C. El Adlouni M. J. Mukhopadhyay P. Walsh G. G. Poirier D. Nadeau 《Molecular and cellular biochemistry》1995,142(1):19-23
Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from theEisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually refered to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12–18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using32P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such asLumbricus terrestris. 相似文献
8.
Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability. 相似文献
9.
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
10.
High-throughput DNA extraction from forest trees 总被引:2,自引:1,他引:1
Mervyn Shepherd Michael Cross Rhonda L. Stokoe Leon J. Scott Megan E. Jones 《Plant Molecular Biology Reporter》2002,20(4):425-425
It is difficult to extract pure high-quality DNA from trees, which may not be amenable to advances in extraction methods suitable
for other plants. A new commercial high-throughput DNA extraction system, using a silica binding matrix for purification and
a multisample mixer mill for tissue disruption, was evaluated for its suitability withEucalyptus spp.,Pinus spp., andAraucaria cunninghamii (hoop pine). DNA suitable for a range of molecular biology applications was successfully extracted from all genera. The method
was highly reliable when tested in more than 500 preparations and could be adapted to different tree species with relatively
minor modifications. 相似文献
11.
Latha C 《Biotechnology letters》2007,29(2):319-322
A rapid and efficient microwave-assisted extraction (MAE) process for the selective extraction of embelin from Embelia ribes was developed. Solvent selection, microwave energy input and solid loading were optimized. The rate of extraction and purity
of embelin depended upon the solvent used and exposure time to microwaves. Maximum MAE was achieved in acetone with total
yield of 92% (w/w) embelin with 90% (w/w) purity with 1% (w/v) raw material loading at 150 W power level in 80 s. Non-polar
solvents, such as hexane and dichloromethane, were not effective for the selective extraction of embelin. 相似文献
12.
Meinhard Grubert 《Plant Systematics and Evolution》1982,141(1):7-21
The mucilage content of structurally differing myxospermatic diaspores from 49 species belonging to 19 families ofAngiospermae has been determined by applying various extraction procedures. The results demonstrate no obvious relationship between the size, mucilage quantity, and the swelling factor of the diaspores studied. Furthermore, mucilage producing structures and structural peculiarities of the mucilages themselves are elaborated. 相似文献
13.
Lara-Reyna J. Olalde-Portugal V. Olmedo-Alvarez G. 《World journal of microbiology & biotechnology》2000,16(4):345-351
DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples. 相似文献
14.
Maria J. Sanchez James M. Bradeen 《Molecular breeding : new strategies in plant improvement》2006,17(2):137-148
Resistance (R) genes and the proteins they encode are key components of the defense system of plants. The exploration of R
gene diversity enables the study of R gene evolution and may facilitate the isolation of new and functional alleles. Most
cloned R genes occur in clusters of related sequences. Thus, the development of a tool for reliable recovery of orthologous
R gene sequences to the exclusion of paralogous sequences will facilitate R gene diversity analysis. The late blight resistance
gene RB is a single functional locus embedded within a cluster of related sequences. Previously, the functional RB allele was cloned from wild potato using a Long Range-PCR (LR-PCR) technique, suggesting this method may be a promising tool
for recovery of R gene orthologs in other genotypes. Using the RB gene as a model, we explored the limitations and improved three technical aspects of LR-PCR for multi-genotype applications.
We present improved primers for the recovery of the RB locus and have identified efficient DNA extraction procedures and reliable amplification systems. We document that consensus
sequences built from three independently generated LR-PCR clones can be up to 100% accurate. Our results show encouraging
advances toward successful application of LR-PCR for isolating alleles from orthologous R gene loci. 相似文献
15.
Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols,
polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol
for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified
hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a
washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of
polyvinylpyrrolidone (2% [w/v]), CTAB (3% [w/v]), and β-mercaptoethanol (3% [v/v]) in the high-salt-concentration extraction
buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins
and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This
protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream
results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction
(PCR) analyses. 相似文献
16.
A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genusAbelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves. This problem is resolved
using cotyledons of dark-grown seedlings. 相似文献
17.
Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR 总被引:2,自引:0,他引:2
Louela A. Castrillo Lene Thomsen Punita Juneja Ann E. Hajek 《Mycological Research》2007,111(3):324-331
Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores. 相似文献
18.
为寻找适用于中药材莪术基原植物鉴定的DNA条形码序列,探索快速高效的莪术基原植物鉴定的新方法,该文首先利用扩增成功率和测序成功率对中药材莪术三种基原植物,9个样本的7种DNA条形码序列(ITS、ITS2、matK、psbA-trnH、trnL-trnF、rpoB和atpB-rbcL)进行评估,然后利用MEGA6.0软件对获得的高质量的序列通过变异位点分析、遗传距离计算和系统树分析等进一步进行评估,最后将筛选到的DNA条形码序列对未知基原的待测样品进行基原鉴定。结果表明:(1) ITS、ITS2和matK等条形码序列在莪术基原植物中的扩增或测序成功率较低,难以应用于实际鉴定;而psbA-trnH、trnL-trnF和rpoB条形码序列变异位点信息过少,不足于区分莪术的三种不同基原植物;只有atpB-rbcL条形码序列的扩增和测序成功率较高,容易获得高质量的序列,同时序列长度(642~645 bp)理想,变异位点多(11个),可实现莪术的三种不同基原的区分鉴别。(2)待测样品经基于atpB-rbcL序列构建的系统发育树鉴别为温郁金。综上所述,叶绿体atpB-rbcL序列能够准确鉴定莪术不同基原植物,可以作为中药材莪术基原植物鉴定的条形码序列。 相似文献
19.
Summary Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: 1) measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; 2) monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmuno assay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers.UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency. 相似文献
20.
Both the rbcL and rbcS genes, encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase, have been found to be encoded by chloroplast DNA in the marine diatom Cylindrotheca sp. N1. The rbcS gene in this diatom was found to be adjacent to the rbcL gene by a combination of: (i) Southern-blotting analyses, using heterologous probes; (ii) examination of recombinant proteins synthesized in Escherichia coli, directed by cloned rbcL/rbcS genes; and (iii) synthesis of enzymatically active heterologous Rubisco protein in vivo by recombinant DNA procedures using large subunits of Anacystis nidulans and small subunits of Cylindrotheca sp. N1. It appears that two copies of rbcL and rbcS genes are encoded by the chloroplast DNA of this diatom. 相似文献