IC4R011-Proteomic-1999-10217180

Project Title

• Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.):Towards a rice proteome

The Background of This Project

• Rice (Oryza sativa L.) is an important crop in Eastern Asia. A vast number of rice cultivars as well as wild species of rice are widely grown and their genetic and molecular makeup is under active investigation. The rice genome conceivably consists of about 450 million basepairs and, at most, ten thousand genes can be expressed in the rice plant tissue [1].
• High-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is useful for separating complex protein mixtures [2]. Due to its high-resolving power, the technique has been employed to study alterations in cellular protein expression in response to various stimuli or as a result of differentiation and development [3].
• Proteins can be recognized by their amino acid composition, their exact molecular weight as determined by mass spectrometry or by partial amino acid sequencing. The latter approach further allows cDNA cloning from the resultant sequence(s). Sequencing of protein separated by 2-D PAGE became possible with the introduction of protein electroblotting methods, which allow efficient transfer of sample from the gel matrix onto a support suitable for gas-phase sequencing or related techniques [4].

Plant Culture & Treatment

• Green and etiolated shoots of rice (Oryza sativa L. cultivar Nipponbare) were used for this study. Rice seeds were sown on vermiculite and incubated at 25oC under light for 12 h and in the dark for 12 h (for green shoots) or in the dark for 24 h (for etiolated shoots)

Protein Extraction and 2-D PAGE

• A portion (500 mg) of the shoots was removed,homogenized with 1 mL of lysis buffer [2] and centrifuged at 15 000 ” g for 5 min. The supernatant (50 mL) was subjected to 2-D PAGE [2]. IEF was carried out in a glass capillary tube of 13 cm length and 3 mm diameter. Briefly,the gel solution consisted of 10% NP-40, 30% w/v acrylamide, 9.5 M urea, 10% ammonium persulfate, and an equal mixture of 2% carrier ampholytes (pH 3.5±10and 5±8). The sample overlay buffer consisted of 20 mL of 1/2 lysis buffer. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 800 V for 1 h.Sodium dodecyl sulfate (SDS)-PAGE in the second dimension was performed with 15% separation gels and 5% stacking gels at a constant current of 35 mA. The isoelectric point and relative molecular weight of each protein were determined using the Isoelectric Focusing Calibration Kit (pH 3.5±10) from Pharmacia Biotech(Uppsala, Sweden). The localization sites of individual proteins on the gels were evaluated automatically with Image Master 2D Elite software (Pharmacia Biotech).
• Following separation by 2-D PAGE, the proteins were electroblotted onto a polyvinylidene difluoride (PVDF)membrane (ProBlott; Applied Biosystems, Foster City,CA, USA) and detected by Ponceau 3R staining [11]. The spots were excised from the PVDF membrane and applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer (477A; Applied Biosystems). Edman degradation was performed according to the standard program supplied by Applied Biosystems.The released phenylthiohydantoin amino acid derivatives were identified by the on-line systems of high performance liquid chromatography (120A; Applied Biosystems).

Research Findings

• Proteins extracted from green and etiolated shoots at two weeks after germination of rice were separated by 2-D PAGE and detected by CBB staining. More than 300 major protein spots were identified by CBB staining in 2-D PAGE patterns of the green and etiolated shoots (Fig. 1).

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• Protein patterns on the gels were standardized by an image analyzer with reference to molecular weights and isoelectric points of the marker proteins (Fig. 2).

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• Fifty major protein spots were selected from green shoots, and 35 from etiolated shoots, for recent investigations (Table 1, 2 and 3). Following separation by 2-D PAGE, the proteins were electroblotted onto a PVDF membrane and detected by Ponceau 3R staining. The spots were cut from the PVDF membrane and applied to the upper glass block of the reaction chamber in a gasphase protein sequencer; Edman degradation was performed according to the standard program supplied by Applied Biosystems.

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Labs working on this Project

• Department of Molecular Genetics, National Institute of Agrobiological Resources,Tsukuba, Japan

Corresponding Author

• Dr. Setsuko Komatsu:skomatsu@abr.affrc.go.jp