棉花纤维素合酶复合体蛋白的分离与鉴定  

Characterization of the Cellulose Synthase Complex in Cotton

作　　者：李先良[1,2] 李傲[1] 彭良才[1] 夏涛[1] 

机构地区：[1]华中农业大学作物遗传改良国家重点实验室/生物质与生物能源研究中心/植物科学技术学院/生命科学技术学院,武汉430070 [2]荆楚理工学院生物工程学院,湖北荆门448000

出　　处：《棉花学报》2013年第2期129-134,共6页Cotton Science

基　　金：转基因生物新品种培育重大转项(2009ZX08009-119B);国家重点基础研究发展计划(2010CB134401);高等学校科技创新引智计划(B08032)

摘　　要：选用开花后24 d的棉纤维提取原生质膜,用Triton X-100溶解后通过纤维素合酶1(Gossypium hirsutumcellulose synthase1,GhCESA1)抗体进行免疫共沉淀(Co-Immunoprecipitation,Co-IP),以聚丙烯酰胺凝胶电泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)分离免疫共沉淀的产物,用液相色谱-质谱联用仪(Liquid chromatography-mass spectrometry,LC-MS)进行鉴定。结果表明,棉纤维纤维素合酶复合体可能存在68种蛋白,其中包括8种纤维素合酶(Cellolose synthse,CESA),它们涵盖了棉纤维初生壁和次生壁2种类型纤维素合酶。这说明在次生壁形成的细胞中存在2种类型CESA。本研究也表明复合体中存在非CESA蛋白。To identify the components of CSCs, plasma membrane （PM） was extracted from cotton fibers at 24 days post anthesis （DPA） and solubilized by triton X-100, from which CSCs were isolated by co-immunoprecipitation mediated by Gossypium hit- suture cellulose synthase 1 （GhCESA 1）. The proteins isolated by co-immunoprecipitation mediated by GhCESA 1 were character- ized by western-blotting using the antibodies of GhCESA1 and Gossypium hirsutum sucrose synthase （GhSuSy）. The results show there are GhCESA1 and GhSuSy proteins, which indicates this method can isolate the components of CSCs. Then, CSCs proteins through SDS-PAGE were subjected to nanoflow liquid chromatography-tandem mass spectrometry （LC-MS）. 68 kinds of proteins were identificated, in which there were 8 kinds of CESA proteins. GhCESA3, 5, 6 can take part in synthesizing cellu- lose in promary walls, and the other CESA proteins can be responsible for formation of cellulose in secondary walls. This study suggests that the CSC machinary may be aided by other proteins in addition to cellulose synthase proteins.

关 键 词：棉纤维 纤维素合酶 复合体 次生壁 

分 类 号：S562[农业科学—作物学] Q814[生物学—生物工程]