IC4R009-Proteomic-2007-17385905

Project Title

• Proteome and Phosphoproteome Differential Expression under Salinity Stress in Rice (Oryza sativa) Roots

Plant Culture & Treatment

• Rice seeds (Oryza sativa, cv. Nipponbare) were germinated at 25 °C in Hoagland solution.27 After 20 days, seedlings were treated with 150 mM NaCl in Hoagland solution and the roots were harvested at 0,10, and 24 h of treatment.

Protein Extraction and 2-D PAGE

• Rice root tissues were ground in liquid nitrogen with mortar and pestle into fine powders.Proteins were extracted using a modified phenol extraction protocol28 as follows: Ground tissues were suspended in an extraction buffer (0.9 M sucrose, 0.5 M Tris-HCl, 0.05 M EDTA,0.1 M KCl, and 2% â-mercaptoethanol added freshly, the final pH was 8.7), were mixed with an equal volume of saturated phenol (pH 8.0), and then were homogenized for 10 min. The homogenate was centrifuged at 2500g for 10 min, the phenol

phase was recovered, and the phenol extraction was repeated three times. The final collection of phenol was mixed with five volumes of precipitation buffer (methanol with 0.1 M ammonium acetate and 1% â-mercaptoethanol). Precipitation was carried out at -70 °C overnight. The precipitant was recovered by centrifugation at 13 400g for 10 min, and the pellet was washed three times with cold precipitation buffer and another three times with ice cold 70% ethanol. The protein pellet was lyophilized to powder in a speed vacuum (LABCONCO, model LYPH-LOCK 6) and was stored at -70 °C. At least three biological replicas were extracted for each treatment.

Labs working on this Project

• Department of Biochemistry and Molecular Biology, Mississippi State University,Mail box 9650, Mississippi State, Mississippi 39762

Corresponding Author

• Zhaohua Peng:zp7@ra.msstate.edu.