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Purification of Urea Transporter in Paddy (oryza Sativa) Induced by Applied Urea in the Soil During Growing Stage

M. S. Haque
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

M.K. Hasan
Department of Biochemistry and Molecular Biology, Laboratory of Protein and Enzyme Research, University of Rajshahi, Rajshahi-6205, Bangladesh

M.A. Haque
Department of Biotechnology, BSMRAU, Salna, Gazipur, Bangladesh

Mohammad Golam Mostofa
Department of Biotechnology, BSMRAU, Salna, Gazipur, Bangladesh

S.M.N. Islam
Department of Biotechnology, BSMRAU, Salna, Gazipur, Bangladesh

Abstract/ Executive Summary:

In plant urea is believed to be transported through urea transporter. To purify urea transporter, we prepared extract of paddy plant induced by urea applied in the soil. Total protein in the extract for urea-induced paddy was 37.3 mg/100 g of tissue while for the control, the value was 27.27 mg. Ammonium sulphate precipitation produced the higher protein content in urea-treated plant. G-50 chromatography of total protein for both groups produced major three peaks. The absorbance at 280 nm was higher than the control when plotted against the elution volume. The first major fraction F-1 was considered to have the urea-transporter activity because of the higher molecules. For molecular weight determination, a mixture of marker proteins was applied to G-50 column. Depending on the elution profile, urea transporter has the molecular weight of 50.6 kDa compared to the molecular weight of BSA. The efficiency of purification of the protein increased up to the G-50 chromatography and was higher than the control. Our investigations suggest that the urea-transporter in paddy will give a new insight for the clarification of mechanism of urea transport in plants.

Keywords: Urea transporter, Purification, Gel filtration, Soil urea, Paddy extract

Location: Rajshahi University Campus

Start Date: 00-00-2008

End Date: 00-00-2008

Program Area: Crop-Soil-Water Management

Commodity: Rice

Objectives:

1. To clarify the mechanism of the transport of urea.

Methodology:

The rice variety BRRI-27 seedlings were raised in the Rajshahi University Campus. The seeds were collected from the near by Bangladesh Rice Research Institute (BRRI), Sympur, Rajshahi, Bangladesh. Plants in the control seedling were not treated with urea while the other seedling was treated with urea (80 kg/ha). Stem and leaf were collected from the cultivation field during the summer season and were kept in refrigerator. 165 g of tissue from control plant crushed in a mortar and pestle were suspended in 50 ml of ice-cool distilled water. After occasional gentle stirring for 3 hours at 4⁰C, the suspension was filtered through double layer of muslin cloth. The filtrate was collected and centrifuged at 8000 rpm for 15 minutes at 40C. The supernatant was used as crude extract. For urea treated plant, crude extract was obtained from 185 g of paddy tissue. The homogenized tissues from control and urea treated paddy were dissolved with another 500 ml ice cooled distilled water separately. 300 ml crude extracts from each group were treated with 80% ammonium sulfate and the precipitated protein after centrifugation was placed in a bag of semi permeable membrane for dialysis. After 48 hours dialysis against ice cool distilled water, the suspension was centrifuged at 8000 rpm for 15 min at 40C and the supernatant was collected, the precipitate was dissolved with distilled water and centrifuged again. The supernatant was collected and used as a pure protein solution. 5-10 ml of protein sample was loaded on to Sephadex G-50 column equilibrated with 10 mM phosphate buffer, pH 7.2 at 4⁰C and the samples were eluted with the same buffer at a flow rate of about 27 ml/hour and 3 ml fractions were collected by an automatic fraction collector. Absorbance of each fraction was measured at 280 nm. The molecular weight of urea transporter was determined. Briefly, a column (3.0 x 100 cm) was packed with activated Sephadex G-50 gel suspension equilibrated with 10 mM phosphate buffer, pH 7.2. The standard proteins and the sample containing urea transporter were applied to the column. During gel filtration, identical conditions were maintained in each time. The buffer was finally allowed to flow continuously through the column at flow rate of 27 ml/hour and 3 ml fractions of the eluate were collected by an automatic fraction collector. Absorbance of each fraction was measured at 280 nm. The molecular weight of the urea transporter was determined from a standard curve, which was constructed by plotting the eluent volume against log of molecular weight of standard proteins. Amount of total protein was estimated according to the Folin-Lowry method. Determination of molecular weight by gel filtration method: The molecular weight of the purified urea transporter from paddy plant extract was determined by gel filtration on Sephadex G-50 column. β-galactosidase (MW-116 kDa), Bovine Serum Albumin (MW-67 kDa), Egg white albumin (MW-45kDa), Pepsin (MW-36 kDa), Trypsin inhibitor (MW-20 kDa) and Lysozyme (MW-14 kDa) were used as molecular weight markers. Marker proteins and purified protein were applied separately on to Sephadex G-50 column under identical conditions. The molecular weight was calculated from the standard curve of reference proteins constructed by plotting against elution volume on gel filtration and the molecular weight of urea transporter was 50.6 kDa.

Source of Information: BANGLADESH RESEARCH PUBLICATIONS JOURNAL; ISSN: 1998-2003, Volume: 2, Issue: 3, Page: 597-604, May - June, 2009

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Total protein in the extract for urea-induced paddy was 37.3 mg/100 g of tissue while for the control, the value was 27.27 mg. Ammonium sulphate precipitation produced the higher protein content in urea-treated plant. G-50 chromatography of total protein for both groups produced major three peaks. The absorbance at 280 nm was higher than the control when plotted against the elution volume. The first major fraction F-1 was considered to have the urea-transporter activity because of the higher molecules. For molecular weight determination, a mixture of marker proteins was applied to G-50 column. Depending on the elution profile, urea transporter has the molecular weight of 50.6 kDa compared to the molecular weight of BSA. The efficiency of purification of the protein increased up to the G-50 chromatography and was higher than the control. Our investigations suggest that the urea-transporter in paddy will give a new insight for the clarification of mechanism of urea transport in plants.

Knowledge Management: Journal