共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
Isaacson T Damasceno CM Saravanan RS He Y Catalá C Saladié M Rose JK 《Nature protocols》2006,1(2):769-774
Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e.g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d. 相似文献
3.
Evaluation of protein extraction methods for Vitis vinifera leaf and root proteome analysis by two-dimensional electrophoresis 总被引:1,自引:0,他引:1
An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction. 相似文献
4.
《植物学报(英文版)》2010,52(1)
An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis(2-DE)for recalcitrant plant species, in particular for grapevine(Vitis vinifera L.). Trichloroacetic acid-acetone(TCA-acetone)and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield,a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction. 相似文献
5.
A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues 总被引:26,自引:0,他引:26
Most published proteomics studies of bulk plant tissues use a procedure in which proteins are precipitated with trichloroacetic acid (TCA) and acetone (TCA-A), but few attempts have been made to contrast this approach in a systematic way with alternative methods against a spectrum of tissues. To address this, TCA-A was compared with another acetone-based protocol (TCA-B) or a phenol (Phe)-based method, targeting a range of tomato tissues and three species of fruits that contain high levels of contaminating compounds: banana, avocado and orange. The Phe method gave a higher protein yield and typically greater resolution and spot intensity, particularly with extracts from tissues containing high levels of soluble polysaccharides. The methods also generated remarkably different two-dimensional gel electrophoresis (2-DE) protein spot patterns. Peptide mass fingerprinting was used to identify polypeptides that were common to multiple extracts or uniquely present in one extract type. While no clear pattern emerged to explain the basis for the differential protein extraction, it was noted that the Phe method showed enhanced extraction of glycoproteins. These results suggest that the Phe protocol is highly effective with more recalcitrant tissues and that a combination of TCA-A and Phe methods provides enhanced 2-DE based proteomic analyses of most plant tissues. 相似文献
6.
7.
Kyoungwon Cho Nilka Lineth Torres Subhashree Subramanyam Saligrama A Deepak Nagesh Sardesai Oksoo Han Christie E. Williams Hideo Ishii Hitoshi Iwahashi Randeep Rakwal 《Journal of Plant Biology》2006,49(6):413-420
Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here,
we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly
improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal
crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB)
and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation
of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an
increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than
when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB,
which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy
of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this
is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples
of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT
solubilized proteins. 相似文献
8.
Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis 总被引:23,自引:0,他引:23
This study focuses on the specific problems of protein extraction from recalcitrant plant tissues and evaluates several methods to bypass them. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is absolutely essential for good results. We evaluated four methods: the classical trichloroacetic acid (TCA)/acetone precipitation, TCA/acetone precipitation and fractionation, an alternative based on fractionation and without precipitation, and phenol extraction methanol/ammonium acetate precipitation. We optimized the phenol extraction protocol for small amounts of tissue, which is essential when the study material is limited. The protocol was optimized for banana (Musa spp.) and was subsequently applied to two other plant species: apple (Malus domestica L.) and potato (Solanum tuberosum L.). Banana (Musa spp.) is a good representative of a "difficult" plant species since it contains many interfering metabolites. Only classical TCA/acetone precipitation and phenol extraction methods proved useful as standard methods. Both methods are associated with a minor but reproducible loss of proteins. Every extraction method and the subsequent analytical procedure have their physicochemical limitations; both methods should be investigated before selecting an appropriate protocol. The study, which is presented in this paper, is useful for guiding the experimental setup of many other nonmodel species, containing various interfering elements. 相似文献
9.
Wei Wang Laura Bianchi Monica Scali Liangwei Liu Luca Bini Mauro Cresti 《Acta Physiologiae Plantarum》2009,31(3):597-604
Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of
secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis
separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts
were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels
was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues
were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39)
in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of
β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF
analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins
than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression.
The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening. 相似文献
10.
Using silica particles to isolate total RNA from plant tissues recalcitrant to extraction in guanidine thiocyanate 总被引:1,自引:0,他引:1
The most commonly used protocol of the RNA isolation, the guanidine thiocyanate method, was unsuitable for recalcitrant plant tissues containing a large amount of storage proteins and secondary metabolites. We demonstrated that RNA could bind to the silica particles, which have been used successfully in DNA isolation from various sources, under a high concentration of NaCl in the presence of ethanol and sodium acetate. Based on this observation, an efficient, inexpensive, and highly reproducible technique, the acid phenol-silica method, was developed to isolate high-quality RNAs from various plant tissues recalcitrant to extraction in guanidine thiocyanate. 相似文献
11.
A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae) 总被引:1,自引:0,他引:1
Jia Xu Mahender Aileni Sadanandam Abbagani Peng Zhang 《Phytochemical analysis : PCA》2010,21(5):395-398
Introduction – It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation. Objective – To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)‐based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta). Methodology – Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites. Results – The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT‐PCR analysis. Conclusion – The study has shown that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from various recalcitrant Euphorbiaceae members. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
12.
High-quality total RNA was extracted using a cethyltrimethylammonium bromide-containing buffer followed by an acid guanidium thiocyanate-phenol-chloroform treatment from recalcitrant plant tissues such as tree leaves (pine, Norway spruce, ginkgo, Japanese cedar, rose), flowers (rose, Lotus japonicus) and storage tissues (seeds of Lotus japonicus and rice, sweet potato tuber, banana fruit). This protocol greatly reduced the time required for RNA extraction. 相似文献
13.
14.
Echevarría-Machado I Sánchez-Cach LA Hernández-Zepeda C Rivera-Madrid R Moreno-Valenzuela OA 《Molecular biotechnology》2005,31(2):129-135
A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain
large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification
of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation
is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure
can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used
for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was
used with healthy Bixa orellana and virus-infected Malvaceae plants. 相似文献
15.
Abolfazl Barzegari Sepideh Zununi Vahed Sina Atashpaz Sajjad Khani Yadollah Omidi 《Molecular biology reports》2010,37(2):833-837
Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted
intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant
tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions
associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid,
easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly
efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA
contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from
leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP.
Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues. 相似文献
16.
Cheong-Xin Chan Swee-Sen Teo Chai-Ling Ho Rofina Yasmin Othman Siew-Moi Phang 《Journal of applied phycology》2004,16(4):297-301
RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g–1 fresh weight) with high quality (A260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies. 相似文献
17.
A DNA extraction procedure that does not require hazardous materials, such as CTAB, phenols, or liquid nitrogen, was optimized
forAnthurium andreanum, a plant rich in polysaccharides and polyphenolics. Three DNA isolation techniques were tested. The modified Rowhani protocol
(1983), with slight modifications, was found to yield up to milligrams of DNA suitable for RAPD from spathe and leaf tissues.
High-quality DNA was obtained readily from spathe tissues, while a spermine precipitation step was found to be essential when
DNA was extracted from the leaf tissues. 相似文献
18.
Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the
biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous
plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported.
In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique. 相似文献
19.
Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues 总被引:1,自引:0,他引:1
Isolation of RNA from recalcitrant tree tissues has been problematic due to large amounts of secondary metabolites and interfering
compounds in their cells. We have developed an efficient RNA extraction method, which yielded high-quality RNA preparations
from tissues of the lychee tree. The method reported here utilized EDTA, LSS, and CTAB to successfully inhibit RNase activities.
It was found that a high ionic strength brought about by 2 M NaCl was necessary. In addition, secondary metabolites and other
interfering compounds were effectively removed using sodium borate and PVPP under a deoxidized condition. The quality of purified
RNA was tested by both RACE and Northern blotting analysis, ensuring that the RNA could be used for subsequent gene expression
analysis. This method has been successfully applied to purify RNA from 15 other plant species. In conclusion, the protocol
reported here is expected to have excellent applications for RNA isolation from recalcitrant plant tissues. 相似文献
20.
The most widely used technique for the introduction of new genetic information into plant cells is based on the natural gene
transfer capacity ofAgrobacterium tumefaciens. Currently, this technique is routinely applicable in just a few model species, like tobacco and petunia. Thus far, the numerous
efforts to apply the technique to crop species have had limited success. In this review, an attempt is made to survey all
the research experience onAgrobacterium tumefaciens-mediated transformation of recalcitrant crops and to highlight the problems generally encountered. The main difficulty appears
to be directing the gene transfer towards those plant cells that are amenable to regeneration. The various ways to reduce
stress during the transformation and regeneration process are often beneficial. The influence of the developmental stage of
the plant material and the host range of theAgrobacterium strain depends largely on the plant species used, which hampers the formulation of common procedures. However, some general
guidelines for the development of a transformation protocol are discussed. 相似文献