油葵转化体系建立和重组人胰岛素的初步表达

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-...

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Bibliographic Details
Published in西南农业学报 Vol. 29; no. 11; pp. 2682 - 2687
Main Author 焦展 安胜军 邵铁梅 仵陶 李雪 刘培 高维娟 柴锡庆
Format Journal Article
LanguageChinese
Published 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%河北中医学院,河北石家庄,050200 2016
Subjects
花粉管通道
融合表达
转基因油葵
重组人胰岛素
Recombinant human insulin
Fusion expression
Transgenic Oil Sunflower
转基因油葵
重组人胰岛素
Pollen tube pathway
融合表达
花粉管通道
Online AccessGet full text
ISSN1001-4829
DOI10.16213/j.cnki.scjas.2016.11.031

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Summary: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。
Bibliography:51-1213/S
Transgenic Oil Sunflower; Pollen tube pathway; Recombinant human insulin; Fusion expression
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
ISSN:1001-4829
DOI:10.16213/j.cnki.scjas.2016.11.031