同源重组敲除三角褐指藻基因的研究  

The research of gene knockout via homologous recombination in Phaeodactylum tricornutum

作　　者：郭婷[1] 代易颖 陈孔翔 高莉[1] 张克亚 曹志远[1] 王冠儒[1] 兰利琼[1] 卿人伟[1] 

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《四川大学学报（自然科学版）》2017年第1期173-177,共5页Journal of Sichuan University(Natural Science Edition)

基　　金：国家自然科学基金(40976092);四川省科技厅项目(2014JY0171);四川省电科院项目(13H1138)

摘　　要：为探索应用基因敲除技术研究三角褐指藻基因功能的研究体系,本文以三角褐指藻甘油激酶基因作为靶基因,构建了同源重组基因敲除载体,利用微弹轰击法将该载体成功转化至三角褐指藻中,经100μg/mL Zeocin筛选和PCR验证获得了34个阳性转基因藻株;并进一步对三角褐指藻甘油激酶基因敲除转基因藻株的甘油激酶表达量和生长两方面进行分析,结果显示胞外甘油兼养不影响转基因藻细胞生长,甘油激酶不表达或表达量降低.本文通过构建敲除载体,完成了遗传转化,筛选获得阳性转基因藻,并进一步研究了转基因藻的性状,最终建立了应用同源重组基因敲除技术靶向研究三角褐指藻基因功能的研究体系.This paper aimed at exploring a knockout system for further gene function research in P. tri- cornutum. The gene encoding glycerol kinase was selected as a target gene in P. tricornutum, constructed the homologous recombination vector for P. tricornutum, and using particle bombardment success-fully converted the homologous recombination vector to wild type P. tricornutum by particle bombardment. After screened on the f/2 solid medium which contain Zeocin （final concentration 100μg/mL） and identified the strains by PCR, 34 transformations were obtained. The analysis of cell growth showed that extracellular glycerin had no effect on the cell growth of the transformats. Detected by western blot, the results presented that the expression of glycerol kinase were repressed or reduced in the trans- formants. This research explored a system for application of gene knockout technology in researching gene function in P. tricornutum, via constructed a gene knockout vector, transformed the vector to the algae cell, screened and identified the knockout algae strains, and studied the characteristics of knockout algae strains.

关 键 词：三角褐指藻 同源重组 基因敲除 甘油激酶 

分 类 号：Q786[生物学—分子生物学]