25-06-2013, 12:49 PM
CHARACTERIZATION OF Triticum Dicoccoides GERMPLASM FOR HIGH GRAIN PROTEIN GENE GpcB1
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INTRODUCTION
Wheat is the largest staple food crop of India. India stands second in the production of wheat after China. Wheat is self-pollinated, C3 and hexaploid plant. The common bread wheat Triticum aestivum is the most important species, occupying more than 90% of the wheat area and 87% of the total wheat production in the country.
Wild emmer (Triticum dicoccoides) grows wild in the fertile crescent of the Near East. It is a tetraploid wheat formed by the hybridization of two diploid wild grasses, Triticum urartu (closely related to wild einkorn), and as yet unidentified Aegilops species related to A. speltoides. Wild emmer wheat is an important genetic resource that could be exploited in breeding for resistance to a broad range of diseases, pests, and for tolerance to poor soil and climatic factors (Nevo 1983, 1989, 1995). Among many agriculturally important characteristics already found in T. dicoccoides is its resistance to stripe rust (Gerechter-Amitai and Stubbs 1970; Nevo et al. 1986; Fahima et al. 1998), stem rust (Nevo et al. 1991), and powdery mildew (Nevo et al. 1985). Wild emmer wheat contains rich and variable genetic resources that will play a major role in future wheat improvement (Nevo 1983,1989, 1995).
OBJECTIVE
The objective of this study was to detect the gene for high grain protein content(GpcB1) in different accessions of wild tetraploid wheat T. turgidum L. var. dicoccoides using gene based markers.
To study the high molecular weight glutenin profile of selected T. dicoccoides accessions.
MATERIAL AND METHOD
The experiment was carried out in Molecular Biology Laboratories of the School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana.
Leaf samples were collected from forty eight different accessions of Triticum dicoccoides during the crop season 2011-12.
Genomic DNA extraction
Genomic DNA was isolated using the CTAB (Cetyltrimethyl ammonium bromide) method as modified by Saghai-Maroof et al (1984). Briefly, young leaves from each accession were collected, placed in the butter paper bags and stored in -80ºC deep freezer. The leaves were grounded into fine powder in liquid nitrogen by constant crushing using sterilized and chilled pestle and mortar, so as to make the leaves brittle and to reduce the DNase activity. Approximately 2g of fine powder tissue were transferred into microcentrifuge tube. The ground tissue was mixed thoroughly with CTAB buffer comprising of 2% CTAB, 100mM Tris, 1.4M NaCl, 20mM EDTA, 0.2% mercaptoethanol and pH adjusted to 8.0. The homogenate was incubated at 65ºC for 45-50 minutes with occasional mixing by inverting the tubes. After incubation of the homogenate, 800 µl of chloroform: isoamylalcohal (24:1) was added and mixed well for 15-20 minutes by placing the microcentrifuge tube on shaker for 45 minutes. After constant shaking on shaker, the tubes were then centrifuged for 15 minutes at 10,000 rpm. After centrifugation the supernatant was transferred to another microcentrifuge tube. Then RNase was added and incubation done at 37ºC for 1 hour. Chilled isopropanol (800µl) was then added and tubes were gently inverted. After this supernatant was kept at -20ºc for 30 mins. Centrifugation was done for 10 mins at 10,00 rpm. Supernatant was removed. DNA was then washed with 70% ethanol and air-dried by simply inverting the tubes for 1-1.5 hours on a blotting paper. DNA was finally dissolved in appropriate quantity of 1X TE buffer.
Assessment of quantity and quality of DNA
Quantity and quality of DNA samples were determined by gel electrophoresis. For this, 2 µl of each DNA sample was loaded in 0.8% agarose gel. For this 0.8 g of agarose was dissolved in 100 ml of 1X TBE electrophoresis buffer. The mixture was heated till the agarose was completely dissolved, i.e. when the solution became transparent and clear. It was cooled down to 60ºC with constant stirring and 5 µl of ethidium bromide (10mg/ml) was added to a final concentration of 0.5 µg/ml buffer. Then the agarose solution was poured in an already prepared gel mould with combs and was left for 40-50 minutes for solidification. DNA samples were prepared by adding loading dye (6X loading dye consisting of 0.4% bromophenol blue, 0.4% xylene cyanol, and 50% glycerol in sterile water) to the DNA such that the final concentration of the loading dye was 1X. When the gel solidified, the DNA samples were loaded into the wells with the help of micropipette. After loading, the gel was run for about 2-3 hours at a constant voltage of 5V/cm in an electrophoresis tank. The gel was then visualised under UV transilluminator. The quality of the DNA samples was judged based on whether DNA formed a single high molecular weight band (good quality) or a smear (poor quality).
Methodology for SDS PAGE
Extraction of proteins- For this 40mg of ground grains were taken and 400µl of extraction buffer was added. After adding extraction buffer to each tube, the mixture was vortexed for 2-3 mins and then allowed to stand for about 1 hour at room temperature. After this samples were centrifuged for 5mins at 10,000 rpm. The supernatant was transferred into separate tube and stored in refrigerator.
Preparation of separating gel– Prepared separating gel of 1.5mm thickness by mixing components as listed in Table-3. The solution was poured into the gel mould below the position of sample wells. The gel was left to polymerise for 1 hour.