大豆油体与EGF融合基因的克隆及其在红花种子中的表达

doi:10.13523/j.cb.20140412

中国生物工程杂志

2014, Vol. 34

Issue (4): 71-77 DOI: 10.13523/j.cb.20140412

技术与方法

大豆油体与EGF融合基因的克隆及其在红花种子中的表达

庞实锋¹, 姜潮², 李文荣^1,2, 冯治国², 刘敏², 楚生辉², 李校堃², 郑克勤¹

1. 广东医学院基础医学院东莞 523808;
2. 温州医科大学药学院温州 325035

Cloning of Soybean Oleosin and EGF Fusion Gene and Expression in Safflower Seeds

PANG Shi-feng¹, JIANG Chao², LI Wen-rong^1,2, FENG Zhi-guo², LIU Min², CHU Sheng-hui², LI Xiao-kun², ZHENG Ke-qin¹

1. Guangdong Medical College, Dongguang 523808, China;
2. College of Pharmacy, Wenzhou Medical University, Wenzhou 325035, China

全文: PDF(942 KB) HTML

摘要：

目的：旨在建立一种在红花油体中表达EGF的转基因植物的方法。方法：通过PCR技术把大豆油体基因（DDoil）与EGF构建成融合基因，克隆至植物表达载体pCAMBIA1390R中，构建成植物表达载体p1390Do-EGF，然后转化进农杆菌 LBA4404 中用于侵染红花外植体，通过甘露糖筛选培养基培养可获得红花转化苗。通过PCR、实时荧光相对定量RT-PCR、SDS-PAGE和Western blot分析目的基因的表达情况，通过MTT法检测EGF的促细胞增殖活性。结果：PCR结果显示，红花叶片中能检测到EGF基因；实时荧光相对定量RT-PCR结果显示，在红花种子中EGF能成功实现转录；SDS-PAGE和Western blot检测证明，在转基因红花种子中能有效表达出EGF，并具有其原有的免疫原性，MTT法实验结果表明EGF具有促进balb/c 3T3细胞增殖的生物活性。结论：大豆油体和EGF融合基因已经成功转化进红花细胞的基因组中，并实现了EGF外源蛋白在红花种子油体中的表达，为EGF蛋白的产业化生产探讨了一种新的生产途径。

关键词： 油体; 表皮生长因子; 转基因红花; 融合基因

Abstract:

Aim: The purpose is expressing EGF in oil-body of transgenic safflower. Method: The Ddoil-EGF fusion gene was got with polymerase chain reaction (PCR) amplification, and the Ddoil-EGF fusion gene was cloned into plant expression vector pCAMBIA1390R to gain p1390Do-EGF. The recombinant plasmid p1390Do-EGF was transferred into Agrobacterium tumefaciens LBA4404. By using co-cultivated method,the fusion gene Ddoil-EGF was transferred into safflower cells. Transferred shoots were selected on solid medium containing mannose.EGF was detected by PCR and real-time RT-PCR in safflower. The expression of EGF in safflower was detected by SDS-PAGE and Western blot. Effect of EGF on the proliferation of Bab/c 3T3 cells assessed by MTT. Result: PCR result showed that EGF gene was transformed into safflower. Relative quantitative real-time RT-PCR showed EGF mRNA was transcripted in safflower. The transgenic safflower seeds expressing Ddoil-EGF fusion protein through SDS-PAGE were confirmed. Western blot result showed that recombinant protein expressed by transgenic safflower could eliciting immunoresponse to EGF antibody. MTT result showed that EGF can stimulate the proliferation of Bab/c 3T3 cells. Conclusion:The Ddoil-EGF fusion gene was transformed into safflower and was expressed in safflower seeds.It is a new ways to express EGF in plant.

Key words: Oil body Epidermal Growth Factor Transgenic safflower Fusion gene

收稿日期: 2014-01-20 出版日期: 2014-04-25

ZTFLH:

Q786

基金资助:

东莞市科技局科技计划资助项目（201010815209）

通讯作者: 李校堃, 郑克勤 E-mail: zkqgdmc@126.com;xiaokunL@163.net

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引用本文:

庞实锋, 姜潮, 李文荣, 冯治国, 刘敏, 楚生辉, 李校堃, 郑克勤. 大豆油体与EGF融合基因的克隆及其在红花种子中的表达[J]. 中国生物工程杂志, 2014, 34(4): 71-77.

PANG Shi-feng, JIANG Chao, LI Wen-rong, FENG Zhi-guo, LIU Min, CHU Sheng-hui, LI Xiao-kun, ZHENG Ke-qin. Cloning of Soybean Oleosin and EGF Fusion Gene and Expression in Safflower Seeds. China Biotechnology, 2014, 34(4): 71-77.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140412 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I4/71

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