大麻蛋白质组的双向电泳技术建立  

Establishment and optimization of two-dimensional electrophoresis( 2-DE) technology for proteomic analysis of hemp

作　　者：程霞[1] 苏源[1] 刘开庆[1] 窦玉敏[1] 刘飞虎[2] 邓纲[2] 

机构地区：[1]昆明学院生命科学与技术系云南省高校特色生物资源开发与利用重点实验室,云南昆明650214 [2]云南大学农学院,云南昆明650500

出　　处：《云南大学学报（自然科学版）》2016年第4期661-668,共8页Journal of Yunnan University(Natural Sciences Edition)

基　　金：云南省教育厅科研基金(2014Y391;2015Z007);云南省高校特色生物资源开发与利用重点实验室开放基金和主任基金(GXKM201502);国家自然科学基金(31501350)

摘　　要：蛋白组学研究中最关键步骤是样品制备,为得到高质量的双向电泳蛋白图谱,作者通过对TCA/丙酮法对大麻茎、叶进行蛋白样品制备、双向电泳技术流程进行优化,建立了适于大麻茎、叶的双向电泳技术体系.优化双向电泳体系为：裂解液在传统配方中加入Tris-HCl（p H=8.8）,以Triton X-100代替CHAPS,蛋白裂解后加入4倍体积100%预冷丙酮等均可有效去除各种非蛋白杂质;IPG胶条p H=4~7,24 cm,考染上样量800μg/胶条可得到背景清晰、分辨率高的双向电泳图谱.大麻茎中可分辨的蛋白点为1 027±12,叶中为905±8.该体系能同时适用于大麻茎、叶,并能满足下一步蛋白质谱测序分析的要求,可为大麻蛋白组学的发展提供技术支持.Sample preparation is the key to the success of proteomics studies.To obtain a high-quality electrophorectogram, a two-dimensional electrophoresist was established which was suitable for the proteomic analysis of ramie＇s various parts（ stems ,leave） by optimizing the traditional TCA/acetone protein extraction method.An im- proved 2-DE technology system was obtained ：Tris and Triton X-100 were added together instead of CHAPS to the lysis buffer, protein purification （4 times volume of acetone） after protein cleavage, IPG strips （ pH = 4-7, 24 cm）,loading amount was 800 μg/strip （Coomassie Brilliant Blue stain）.The method effectively eliminated nonprotein impurities and obtained a clear two-dimensional gel electrophoresis array. The protein of hemp was found mainly distributed at the range of pH= 5-6,the protein spots of stem and leaves were 1 027± 12,905-±8 respectively.This technology was also applied to the stems and leaves of hemp and provided reliable technical support to the proteomic study of hemp.

关 键 词：大麻 蛋白质组 双向电泳 TCA/丙酮 

分 类 号：Q946.1[生物学—植物学]