琉璃苣BoD6D载体的构建及转化

doi:10.13560/j.cnki.biotech.bull.1985.2020-0791

摘要/Abstract

摘要：

运用植物基因工程手段构建琉璃苣BoD6D转化载体,为提高油料作物油份中γ-亚麻酸含量奠定基础。以琉璃苣基因组DNA为模板克隆BoD6D,构建酵母表达载体并转化酿酒酵母,对酵母进行诱导表达,提取脂肪酸后进行甲酯化反应,利用气相色谱分析脂肪酸含量;同时构建植物双元表达载体,经农杆菌介导通过蘸花法转化拟南芥,最后对转基因拟南芥通过抗性筛选及PCR进行鉴定。结果表明,BoD6D编码区不含有内含子,可以直接用于后续的功能研究;成功构建了酿酒酵母表达载体pYES2-BoD6D,气相色谱检测结果表明BoD6D在酵母中能够成功的将亚油酸催化生成γ-亚麻酸;成功构建了植物双元表达载体pBI121-BoD6D,卡那霉素抗性筛选和PCR鉴定结果表明已经成功获得BoD6D的拟南芥转化植株。通过以琉璃苣基因组DNA为模板比较方便的获得有功能的BoD6D用于转基因植物研究。

关键词: 琉璃苣, BoD6D, γ-亚麻酸, 酵母表达载体, 植物表达载体

Abstract:

The purpose of this study is to lay a foundation for increasing the content of γ-linolenic acid in oil crops via constructing Borago officinalis BoD6D transforming vector by means of plant genetic engineering. The BoD6D gene was cloned using borage genomic DNA as a template. Then,a yeast expression vector was constructed and transformed into Saccharomyces cerevisiae. The fatty acids were extracted to have methyl esterification reaction,and the fatty acid content was analyzed by gas chromatography. Concurrently,the plant binary expression vector was constructed,and the Arabidopsis thaliana was transformed by the dipping method mediated by Agrobacterium,and finally the transgenic Arabidopsis was identified by resistance screening and PCR. Analysis found that the coding region of BoD6D gene did not contain introns and could be directly used for subsequent functional research. The expression vector pYES2-BoD6D of S. cerevisiae was successfully constructed,and the results of gas chromatography showed that BoD6D gene successfully catalyzed linoleic acid to γ-linolenic acid. The plant binary expression vector pBI121-BoD6D was successfully constructed. The results of kanamycin resistance screening and PCR identification demonstrated that the Arabidopsis transformed plants with the BoD6D gene were ultimately obtained. The above results indicate that the borage genomic DNA can be directly used as a template to easily obtain functional BoD6D gene for transgenic plant research.

Key words: Borago officinalis, BoD6D gene, γ-linolenic acid, yeast expression vector, plant expression vector

巩元勇, 赵丽华, 闫飞, 郭书巧, 束红梅, 倪万潮. 琉璃苣BoD6D载体的构建及转化[J]. 生物技术通报, 2021, 37(3): 227-232.

GONG Yuan-yong, ZHAO Li-hua, YAN Fei, GUO Shu-qiao, SHU Hong-mei, NI Wan-chao. Construction and Transformation of Borago officinalis BoD6D Gene Vector[J]. Biotechnology Bulletin, 2021, 37(3): 227-232.

图/表 6

图1 BoD6D PCR电泳检测图

图2 pEASY-Blunt-BoD6D和pYES2载体BamHI和Xho I双酶切检测 A：pEASY-Blunt-BoD6D载体;B：pYES2载体;M：DL5000 DNA Marker

图3 转基因酿酒酵母总脂肪酸气相色谱分析图

图4 pBI121-BoD6D载体Xba I和BamHI双酶切检测 A：pBI121-BoD6D载体;M：1 kb plus DNA ladder

图5 用含有卡那霉素的MS培养基筛选转化成功的拟南芥植株箭头所示为具有卡那霉素抗性的拟南芥转化植株

图6 拟南芥转化植株BoD6D的PCR电泳结果 M：DL2000 DNA ladder;1-8：不同拟南芥转化植株;+：pBI121-BoD6D质粒;-：水

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