棉花学报  Cotton Science 2005，17(3)：146-150

An Improved Method for Extraction and SDS-PAGE of Total Protein in Cotton Leaves
SHA Yue-xia^1，2，JIAN Gui-liang²，XIAO Chong-gang¹，LU Mei-guang²，CAO Cheng-zhong²
( l. Plant Protection College，South-West Agricultural University，Chongqing 400716，China；2. Insti tute of Plant Protection，Chinese Academy of Agricultural Sciences，The State Key Lab for Biology of Plant Diseases and Insect Pest，Beijing 100094，China)

Abstract：The methods of total protein extraction in cotton leaves were compared and SDS-PAGE were improved. Ameliorated steps included protein extraction, modified pyruvic subside and destaining.The experimental cultivars were resisitance cultivars: Wenwu，9456D, X033(Gossypium hirsutum)，sea-island cotton Hai7124(G. barbadense) and susceptive cultivar, Junmian l. which were induced by Verticillium dahilae. Same varieties were planted without pathogeny. All of the experimental breeds were planted in cotton disease garden in Institute of Plant Protection，Chinese Academy of Agricultural Sciences. Four methods could be used to extract the total protein in cotton leaves. It was citrate，1，200 and 25 mmol•L^-1，pH6.8 Tris-HCl. The results showed that it could make better to utilize 25mmol•L^-1, pH 8.0 Tris-HCI on total protein extraction. Using this method, the protein was got completely and efficiently. Moreover, satisfied results were achieved in SDS-PAGE. We used three ways in SDS-PAGE. It was modified pyruvic subside, (NH₄)₂SO₄ subside and direct extract. The method of modified pyruvic subside in SDS-PAGE in cotton leaves can be used to test toothful. We can get clear electrophoresis result: the cingula were clear，numerous and with high resolution. But the results of the other two methods were not satisfied. Direct extract to SDS-PAGE in cotton, the consistency was low, it was difficult to detect the protein. the method of (NH₄)₂SO₄ subside in SDS-PAGE, the cingula was not recognizable. In addition, both of them were complex in the experiment. When we used modified pyruvic subside in SDS-PAGE, confecting different consistent separate gel, the consisitence of 10%-12% fit for SDS-PAGE of total protein in cotton leaves. Through SDS-PAGE, wefound a conclusion that the temperature related to destaining: the higher temperature (lower than 45 ℃ )，the shorter time of destaining. Depending on experiment, we got an appropriate method on total protein extraction, fix quantify, gel preparation, electrophoresis, destaining and wafer preparation in cotton leaves.
Key words：cotton leaves；total protein extraction；SDS-PAGE    [Full Text, 2139]