山西农业大学学报（自然科学版）2018，Vol.38Issue(3)：36-42,7.DOI:10.13842/j.cnki.issn1671-8151.201706012
杜氏盐藻电击转化体系的优化
Optimization of an electroporation-mediated transform system of Dunaliella salina
摘要
Abstract
[Objective]The present study was conducted to construct a highly-efficient genetic transformation system of Dunaliella salina,which would greatly facilitate the assembly of target metabolic pathways in cells,and further pro-mote the large-scale production of high-valued products such as carotenoids and biofuels using Dunaliella salina as the bioreactor.[Methods]In this paper,Dunaliella salina was used as the target for genetic transformation.The foreign target gene was V gDGA T1a which encode a DGAT protein,and a key enzyme responsible for TAG biosynthesis from high-oil plant Vernonia galamensis.The pCAMBIA3301 was selected as expression vector in microalgae.An electro-porationmethod was selected for transformation and the related parameters was further optimized.Molecular testing tools were used to analyze the integration and expression of the target gene in transformants.[Result]Dunaliella salina was sensitive to herbicide glufosinate.Concentration of glufosinate was tested as 20 mg·L -1 and 40 mg·L -1 for effectively selecting the positive tranformant in solid and liquid culture media,respectively.The optimized electropora-tion conditions for Dunaliella salina transformation included 7 d cultured microalgal cells as receptors,6 mg·L -1 plas-mid per transformation,and setting of 0.4 kV and 4 ms for electroperation.The transformation rate of Dunaliella sa-lina was 2.63‰,which was 1.14 times higher than previously reported value.Molecular identification showed that the target gene V gDGA T1a was successfully transferred into Dunaliella salina and also highly expressed.[Conclusion] The optimized electroporationmethod developed here significantly increased the transformation rate of Dunaliella sali-na.The herbicide glufosinate resistant Bar gene and pCAMBIA3301 expression vector widely used in higher plant transformation also can be successfully used in Dunaliella salina genetic transformation.关键词
杜氏盐藻/遗传转化/电击法/VgDGAT1a基因Key words
Dunaliella salina/Genetic transformation/Electroporation/V gDGA T1a gene分类
农业科技引用本文复制引用
宋程飞,郝敬云,程蔚兰,史飞飞,季春丽,李润植..杜氏盐藻电击转化体系的优化[J].山西农业大学学报（自然科学版）,2018,38(3):36-42,7.基金项目
国家自然科学基金(30971806、31201266和31401430) （30971806、31201266和31401430）
国家"948"项目(2014-Z39) （2014-Z39）
山西省煤基重点科技攻关项目(FT-2014-01) （FT-2014-01）
山西省重点研发计划重点项目(201603D312005) （201603D312005）
