油葵转化体系建立和重组人胰岛素的初步表达  

Establishment of Genetic Transformation System and Primary Expression of Recombinant Human Insulin in Oil Sunflower

作　　者：焦展[1] 安胜军[1] 邵铁梅[1] 仵陶[1] 李雪[1] 刘培[1] 高维娟[2] 柴锡庆[1] 

机构地区：[1]河北化工医药职业技术学院,河北省高校生物反应器与蛋白类药物开发应用技术研发中心,河北石家庄050026 [2]河北中医学院,河北石家庄050200

出　　处：《西南农业学报》2016年第11期2682-2687,共6页Southwest China Journal of Agricultural Sciences

基　　金：河北省高等学校科学技术研究重点项目“利用转基因亚麻悬浮细胞表达阿朴脂蛋白米兰突变体”(ZD2014087);河北省高等学校科学技术研究重点项目“植物生物反应器的方法生产Reteplase的研究与开发”(ZD2010216)

摘　　要：为建立并研究利用油葵油体表达系统表达重组人胰岛素蛋白的方法,采用PCR技术构建植物种子特异性表达载体,经花粉管通道用重组人胰岛素基因转化油葵。利用PCR法检测目的基因的转化情况,用SDS-PAGE和Western blot对胰岛素蛋白的表达进行鉴定。结果表明,合成了重组人胰岛素与花生油体蛋白融合基因,并获得植物种子特异性表达载体p BINOI。花粉管通道转化油葵的适宜条件为,花柱剪切1/2,质粒浓度为50-100 ng/μl转化效率最高。PCR结果显示,油葵花粉管通道法转胰岛素基因总转化率为3.2%,温室转化率（5.20%）高于大田转化率（1.12%）。获得T1-T5代转基因种子,播种为T1-T5代植株。获得的1号转基因株系T5代植株阳性/阴性植株为11∶1。经SDS-PAGE电泳分析和Western blot检测结果显示,融合蛋白约为23 k Da,重组人胰岛素基因在油葵种子中得到了表达。本实验建立了转基因油葵油体表达系统,且该系统能够成功表达重组人胰岛素。In order to establish and study the expression of recombinant human insulin in Oil Sunflower oil body expression system, plant seed-specific expression vector was constructed by PCR technique, and recombinant human insulin gene was introduced into Oil Sunflower via pollen tube pathway. Insulin gene was detected by PCR, and the expression of insulin in Oil Sunflower was detected by SDS-PAGE and Western blot. The result showed that recombinant human insulin fusion gene was synthesized, and seed-specific expressed vector pBINOI was obtained. 1/2 shearing of the style and 50 - 100 ng/μl of plasmid concentration were the suitable conditions of genetic transformation via pollen tube pathway. The result of PCR indicated that the total transgene rate of the insulin genetic transformation in Oil Sunflower via pollen tube pathway was 3.2 % and the transgene rate in glassroom （5.20 % ） was higher than that in field（ 1.12 % ）. The transgenic seeds of T1 - T5 generation were obtained, which grew into plants of T1 - T5 generations. The approximate proportion of positive plants/negative plants of T5 generation of No. 1 plant line was 11 ： 1. The results of SDS-PAGE and Western blot showed that fusion protein was estimated to be 23kDa, and recombinant human insulin gene was successfully expressed in Oil Sunflower seeds. It was concluded that the transgenic Oil Sunflower oil body system was established in this experiment. It could be used effectively for expressing recombinant human insulin.

关 键 词：转基因油葵 花粉管通道 重组人胰岛素 融合表达 

分 类 号：S512.1[农业科学—作物学]