Transformation of coccolithophorid Emiliania huxleyi harboring a marine virus(Coccolithoviruses)serine palmitoyltransferase(SPT)gene by electroporation  

作　　者：Weicong CAI Xueting WANG Jinjing SU Jian LI Jun ZENG Guiling LI Jingwen LIU 

机构地区：[1]College of Food and Bioengineering,Jimei University,Xiamen 361021,China [2]Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering,Xiamen 361021,China

出　　处：《Journal of Oceanology and Limnology》2021年第2期693-704,共12页海洋湖沼学报（英文）

基　　金：Supported by the National Natural Science Foundation of China(Nos.41576166,21707042,31771972);the Fujian Province Natural Science Foundation of China(Nos.2019J01696,2017J01447,2017J01636)。

摘　　要：Emiliania huxleyi is the most prominent modern coccolithophore,a group of marine unicellular eukaryotes that play a critical role in ocean biogeochemistry.Coccolithoviruses are large double stranded DNA viruses,which is responsible for the demise of large oceanic blooms formed by E.huxleyi.E.huxleyi virus(EhVs)acquired a series of enzyme-coding genes predicted to be involved in the sphingolipid biosynthesis by horizontal gene transfer between virus-host.Currently,there is limited experimental validation identifying the functions of these genes in EhV.Genetic transformation of eukaryotic cells is a powerful tool to get an insight into gene functions of the studied organisms.Serine palmitoyltransferase(SPT)catalyzes the first committed step in de novo sphingolipid biosynthetic pathway.Here,a novel vector system for the transformation of E.huxleyi was designed.It contained fragments of promoter and terminator sequences of E.huxleyi endogenic fucoxanthin chlorophyll a/c-binding protein gene“fcp”and harbored EhV-99B1 spt gene.The resultant recombinant transformation vectors pEhux-I-spt and pEhux-II were co-transferred into E.huxleyi BOF92 by electroporation.Transformants were obtained upon glufosinate-ammonium selection,and confirmed by Southern hybridization,genome PCR,qRT-PCR and Western blot screening of spt gene,which indicated that spt gene was integrated into the nuclear genome and was expressed at the mRNA and protein levels.The expression of the viral spt gene led to differences in lipid compositions analyzed using thin-layer chromatography(TLC).The results present the genetic transformation system for E.huxleyi,providing additional genetic resource with potential for exploring basic biological questions such as the virus-host interactions.

关 键 词：E miliania huxleyi coccolithovirus genetic transformation serine palmitoyltransferase(SPT) total lipid 

分 类 号：Q943.2[生物学—植物学]