МОЛЕКУЛЯРНАЯ БИОЛОГИЯ, 2012, том 46, № 4, с. 598-604
ГЕНОМИКА. ТРАНСКРИПТОМИКА
УДК 577.2.08
POLYSACCHARIDE-FREE NUCLEIC ACIDS AND PROTEINS OF Abelmoschus esculentus FOR VERSATILE MOLECULAR STUDIES © 2012 A. Manoj-Kumar1, K. N. Reddy1*, M. Manjulatha2, L. Blanco1
functional Genomics of Eukaryotes, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico 2Department of Biotechnology, Sri Krishnadevaya University, Ananthapur, Andhra Pradesh, India
Received August 08, 2011 Accepted for publication September 19, 2011
Abelmoschus esculentus (okra) is one of the polysaccharide rich crop plants. The polysaccharides interfere with nucleic acids and protein isolation thereby affecting the downstream molecular analysis. So, to understand the molecular systematics of okra, high quality DNA, RNA and proteins are essential. In this study we present a method for extracting genomic DNA, RNA and proteins from polysaccharide rich okra tissues. The conventional extraction procedures were integrated with purification treatments with pectinase, RNase and proteinase K, which improved the quality and quantity of DNA as well. Using SDS, additional washes with CIA and NaCl precipitation improved the RNA isolation both quantitatively and qualitatively. Finally, ammonium acetate mediated protein precipitation and re-solubilization increased the quality of total protein extracts from the okra leaves. All of the methods above not only eliminated the impurities but also improved the quality and quantity of nucleic acids and proteins. Further, we subjected these samples to versatile downstream molecular analyses such as restriction endonuclease digestion, RAPD, Southern, reverse transcription-PCR and Western analysis and were proved to be successful.
Keywords: Abelmoschus esculentus, nucleic acid isolation, total proteins, superior quality, molecular analysis.
INTRODUCTION
High quality nucleic acids and protein samples are essential for most of molecular analysis techniques. Though large numbers of DNA and RNA isolation methods were developed in the recent years for a variety of plant species, they are indeed not always simple and moreover cannot be reproduced in other species [1]. In plants, several factors are known to limit the isolation of pure DNA or RNA due to co-extraction of impurities such as polysaccharides, terpenes, polyphenols, melicera colloidal hyalosome etc. Of these impurities polysaccharide contamination is the most common problem while isolating DNA and RNA of higher plants. Viscous or glue-like texture of polysaccharides makes the nucleic acids unmanagable during pipetting and unsuitable for PCR, since they inhibit the activity of Taq polymerase, ligases and restriction enzymes [15]. The samples contaminated with melicera colloidal hyalosome, are often more difficult to dissolve in water or the TE buffer [6]. Sometimes, phenolic compounds also interfere with extrac-
Abbreviations: PC — phenol (pH 4.5): chloroform; CIA — chloro-form:isoamyl alcohol; PCIA — phenol:chloroform:isoamyl alcohol; CTAB — hexadecyltrimethylammonium bromide; DTT — dithiotreitol; PMSF— phenylmethanesulfonyl fluoride; PVPP — polyvinyl polypyrrolidone; DEPC — diethyl pyrocarbonate; RH — relative humidity; RT — room temperature.
* E-mail: kalpana_reddyn@rediffmail.com
tion procedures, bringing down the purity ofDNA and RNA, making them unfit for most of molecular analyses [7, 8]. Many perennials are rich with these contaminants which greatly affect the quality and quantity of the extracted nucleic acids [9—11], as a result, rehy-drated ethanol-precipitated DNA will be a viscous slurry. The most common CTAB method [12] and its modifications [13, 14] are widely used in various laboratories, but they are not suitable for all the plant species. Extraction protocols using diatomaceous earth and spin filters for DNA extraction and benzyl chloride for RNA extraction, though capable of isolating good quality samples, are tedious and expensive [15— 17] for small scale laboratories. The conventional DNA extraction protocols, which can remove some contaminants [18], require large amounts of tissue samples.
Similarly, protein extraction from plant tissues has been of a great deal for an array for proteomic analysis [19]. Plant tissues usually contain relatively low amounts of proteins and high quantities of secondary metabolites and polysaccharides which are indeed responsible for tissue disintegration and interfere with protein resolution on SDS-PAGE [20]. Hence, an effective protein extraction protocol is essential to eliminate or minimize the secondary metabolite variations across the
various tissues such as leaves, roots, fruit, seeds and stems, and also between different species [21].
Abelmoschus esculentus (L.) Moench, (okra) is a potential multiple-purpose crop [22] reported to have high polysaccharide content in leafs, buds, flowers, and pods, resulting in a highly viscous solution with a slimy appearance when okra is extracted with water or standard buffer solutions [23]. Isolation of nucleic acids (DNA and RNA) and proteins is much more difficult with this crop because of the impurities mentioned above. Such samples with high contamination levels have higher risks of interference during further molecular manipulations with the nucleic acids associated with enzymes and subsequent electrophoresis and with proteins during SDS-PAGE and Western analysis. In this article, we suggest isolation methods giving high quality nucleic acids and proteins from okra suitable for a variety ofversatile molecular studies such as RAPD, Southern, Reverse Transcription-PCR and Western analyses.
EXPERIMENTAL
Plant material and sampling. Seeds of A. esculentus cv Arka Anamika were surface sterilized with 96% ethyl alcohol for 1 min followed by 10% sodium hypochlorite for 15 min. The seeds were washed 4 times for 5 min with sterile distilled water after treatment with each of the above and were soaked overnight in sterile distilled water with low agitation (30 rpm) on a shaker. They were germinated on sterile moistened filter paper in Petri dishes (10 cm diameter) for 2 days in the dark at 37°C. Healthy germinated seeds were transferred into sterile soil moistened with a nutrient solution in polypropylene pots. The pots were kept in a green house at 28 ± 1°C, ~55% RH and irrigated daily. Young, fully opened, healthy leaves from two week old plants were collected and frozen in liquid nitrogen and were stored at —80°C till use.
Nucleic acids isolation. Genomic DNA: About 0.4 g of tissue plus ~40 mg of polyvinyl pyrrolidone (PVP) were homogenized with a pestle in a pre-cooled sterile mortar containing liquid nitrogen until a fine powder was obtained, then the frozen powder was transferred to a 15 mL falcon tube. 10 folds (w/v) of sterilized C-TEN buffer (3% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0 and 1.4 M NaCl: preheated at 55°C) amended with 0.006% P-mercaptoethanol (add just prior to use) was added to the homogenized powder. The tubes were incubated for 30 min at 55°C and mixed at every 10 min interval. The samples were brought to RT and an equal volume of 24 : 1 CIA was added and mixed thoroughly. The tubes were centrifuged twice at 2500 g for 20 min at RT. 1/10th volume of 5 M NaCl and 2.5 volumes of absolute ethanol were added to the supernatant and incubated at 4°C overnight. The DNA was pelleted at 2500 g for 20 min at
RT and the pellet was washed with 70% ethanol. The pellet was dried for 15 min at RT and re-suspended with 300 |L of 0.1 x TE at 4°C. To purify the DNA, RNase (10 |g/mL) and pectinase (30—80 U) were added and incubated at 37°C for 45—60 min, followed by proteinase K (20 |g/mL) treatment for 15 min. Later an equal volume (v/v) 25 : 23 : 2 of PCIA was added, mixed thoroughly and centrifuged at maximum speed for 5 min. Then 1/10th volume of 3 M C2H3NaO2 and 2.5 volumes of chilled absolute ethanol were added to the supernatant and incubated at 4°C overnight. The DNA was pelleted at maximum speed for 10 min at RT and washed once with 70% ethanol. The pellet was dried for 30 min at RT and then dissolved in 100 |L of 0.1 x TE at 4°C.
Total RNA: (All of the reagents should be prepared with DEPC water and centrifuged at 14000 g at 4°C). Approximately 100—150 mg of leaf sample were ground with a pestle in a pre-cooled sterile mortar containing liquid nitrogen until a fine powder was obtained, then 500 |L of buffer-1 (38% phenol, 0.8 M ammonium thiocyanate, 0.4 M guanidium thiocyan-ate, 10 mM C2H3NaO2, 5% glycerol) were added and homogenized (may take 10—15 min). The homoge-nate was transferred to a 1.5 mL sterile tube and half the volume of PC (1 : 1 v/v) was added, mixed by inverting and centrifuged. 500 |L of freshly prepared buffer-2 (1.5% SDS, 100 mM Tris pH 8, 50 mM EDTA, 150 mM LiCl, 1.5% P-mercaptoethanol) were added to the supernatant, the tubes were inverted for 1 min and incubated for 10 min at RT. 1/6th volume of 24 : 1 CIA was added, mixed thoroughly and centri-fuged for 15 min. The supernatant was transferred to a fresh tube and 300 |L isopropanol plus 250 |L NaCl (1.2 M) were added and mixed gently for 1 min. The tubes were incubated at —80°C for 20 min and centrifuged for 20 min. The samples were washed twice with 70% chilled ethanol, dried and finally re-suspended in 75-100 |L of sterile DEPC water at 4°C.
The total DNA or RNA was spectrophotometrical-ly quantified by using NanoDrop1000. The samples were resolved on appropriate gels according to Sam-brook and Russell [24].
Southern and RAPD analysis. In order to analyze the digestibility of the total genomic DNA and to ensure its quality for Southern analysis, 10 ^g of genomic DNA were digested using 3U/^g of DNA for 16 h with EcoRI ("Fermentas®", USA), which does not cut the target gene sequence of the elongation factor (elf). The digested DNA samples were electrophoresed on a 0.8% agarose gel. The separated fragments were trans
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