棉花纤维伸长期与次生壁增厚期蛋白质组比较

以陆地棉徐州142为材料，比较了3种不同棉花纤维蛋白质提取方法；利用双向电泳技术比较棉花纤维伸长及初生壁形成期(10 DPA)和次生壁增厚期(25 DPA)蛋白质组的变化；利用PDQuest软件分析各个差异蛋白在10 DPA和25 DPA棉花纤维中的相对表达量，选取质量好、实验重复性高的蛋白质点15个进行MALDI-TOF MS鉴定；根据目的蛋白核苷酸序列设计特异引物，对5种差异蛋白进行半定量RT-PCR分析。结果表明，利用饱和酚-甲醇醋酸铵法提取的棉花纤维蛋白，其蛋白含量较高，且SDS-PAGE电泳条带清晰；进行MALDI-TOF MS鉴定的15个差异蛋白，于NCBI上进行数据查询，分别属于F-box家族蛋白、肌动蛋白、β-微管蛋白、F₁-ATP合成酶、ATP酶β亚基、膜联蛋白、磷酸甘油酸酯激酶I、胞质苹果酸脱氢酶、S-腺苷-L-高半胱氨酸水解酶、谷氨酰胺合成酶、Cu-Zn超氧化物歧化酶、profilin、4-香豆酸辅酶A连接酶等。查询结果表明，上述蛋白参与能量代谢、碳代谢、细胞周期调控和发育等。

Comparison of Proteome in Cotton Fiber Cell between Elongation and Secondary Wall Thickening Stages

Three cotton fiber protein extraction methods were compared using Gossypium hirsutum, cv. Xuzhou 142. Two-dimensional polyacrylamide gel electrophoresis was used to exhibit the protein profile differences between two stages of cotton fiber cells developments. The two stages are cell elongating and synthesizing primary cell walls at 10 days postanthesis (DPA), and the secondary cell wall thickening at 25 DPA. PDQuest8.0 software was used to analysis the relative abundance of different proteins between 10 and 25 DPA cotton fibers. Fifteen of the proteins which had good quality and reproducibility were identified by MALDI-TOF-MS. Gene specific PCR primers were designed according to the interest proteins nucleotide sequences. Semiquantitative RT-PCR was used to detect the five different proteins. The results showed that, the phenol-methanol-ammonium acetate extraction of cotton fiber proteins was the best method among three methods with a extract higher amount of protein extracted and clean SDS-PAGE loading pattern. According to the NCBI database searching results, the differentially expressed proteins were F-box family proteins, actin, β-tubulin, F1-ATP synthase subunit beta, annexin, cytosolic phosphoglycerate kinase 1, cytosolic malate dehydrogenase, adenosylhomocysteinase, putative glutamine synthetase, ATPase subunit, profilin, copper/zinc superoxide dismutase and putative 4-coumarate coA ligase. These proteins are involved in a wide range of physiological processes, such as energy metabolism, carbohydrate metabolism, cell cycle control and development.