Construction of expression vector and transformation via electroporation in coccolithophore <i>Emiliania huxleyi</i>

Wang Xueting; Guo Qiangqiang; Cai Yiqin; Chen Zhifu; Li Jian; Liu Jingwen

doi:10.3969/j.issn.0253-4193.2016.08.011

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Wang Xueting, Guo Qiangqiang, Cai Yiqin, Chen Zhifu, Li Jian, Liu Jingwen. Construction of expression vector and transformation via electroporation in coccolithophore Emiliania huxleyi[J]. Haiyang Xuebao, 2016, 38(8): 103-114. doi: 10.3969/j.issn.0253-4193.2016.08.011

Citation:

Wang Xueting, Guo Qiangqiang, Cai Yiqin, Chen Zhifu, Li Jian, Liu Jingwen. Construction of expression vector and transformation via electroporation in coccolithophore Emiliania huxleyi[J]. Haiyang Xuebao, 2016, 38(8): 103-114. doi: 10.3969/j.issn.0253-4193.2016.08.011

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Construction of expression vector and transformation via electroporation in coccolithophore Emiliania huxleyi

doi: 10.3969/j.issn.0253-4193.2016.08.011

College of Food and Bioengineering, JiMei University, Xiamen 361021, China

Received Date: 2015-12-11
Rev Recd Date: 2016-03-03

Abstract

Abstract

The marine coccolithophore Emiliania huxleyi is a eukaryotic microalga species crucial to the study of global biogeochemical cycles and climate modeling and also much of interest to those in biotechnology due to the capable of abundant bioactive metabolites production. Here, seven different kinds of antibiotics including ampicillin, kanamycin, G418, chloramphenicol, streptomycin, novobiocin and puromycin were used for the screening of antibiotic resistance. G418 was chosen most suitable selective antibiotics and the corresponding resistance gene "neo" as the marker for E. huxleyi genetic system. The promoter of the endogenic fucoxanthin chlorophyll a/c-binding protein gene "fcp" was cloned from E. huxleyi BOF92 strain. A construct was made containing the green fluorescent protein reporter gene "gfp" and screened G418 resistance gene "neo". The resultant recombinant transformation vectors pUC18-fcp-gfp and pUC18-fcp-neo were co-transferred into E. huxleyi by electroporation. Transformants were obtained upon G418 selection. The results presented the new genetic transformation system for E. huxleyi, providing additional genetic resource with potential for exploring basic biological questions and biotechnological applications.
- Emiliania huxleyi,
- vector construction,
- neo marker gene,
- electroporation

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References(42)

References

Bellou S, Baeshen M N, Elazzazy A M, et al. Microalgal lipids biochemistry and biotechnological perspectives[J]. Biotechnol Adv, 2014, 32(8):1476-1493.

De Morais M G, Vaz Bda S, de Morais E G, et al. Biologically active metabolites synthesized by microalgae[J]. Biomed Res Int, 2015, 2015:835761.

Qin S, Jiang P, Tseng C K. Molecular biotechnology of marine algae in China[J]. Hydrobiologia, 2004, 512(1/3):21-26.

Niu Y F, Yang Z K, Zhang M H, et al. Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker[J]. BioTech Rap Dis, 2012, doi: 10.2144/000113881.

Radakovits R, Eduafo P M, Posewitz M C. Genetic engineering of fatty acid chain length in Phaeodactylum tricornutum[J]. Metab Eng, 2011, 13(1):89-95.

Qin S, Lin H Z, Jiang P. Advances in genetic engineering of marine algae[J]. Biotechnol Adv, 2012, 30(6):1602-1613.

Hlavova M, Turoczy Z, Bisova K. Improving microalgae for biotechnology-from genetics to synthetic biology[J]. Biotechnol Adv, 2015, 33(6):1194-1203.

Xue J, Niu Y F, Huang T, et al. Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation[J]. Metab Eng, 2015, 27:1-9.

Joassin P, Delille B, Soetaert K, et al. Carbon and nitrogen flows during a bloom of the coccolithophore Emiliania huxleyi:modelling a mesocosm experiment[J]. J Marine Syst, 2011, 85(3/4):71-85.

Sayanova O, Haslam R P, Calerón M V, et al. Identification and functional characterisation of genes encoding the omega-3 polyunsaturated fatty acid biosynthetic pathway from the coccolithophore Emiliania huxleyi[J]. Phytochemistry, 2011, 72(7):594-600.

Read B A, Kegel J, Klute M J, et al. Pan genome of the phytoplankton Emiliania underpins its global distribution[J]. Nature, 2013, 499(7457):209-213.

Evans C, Pond D W, Wilson W H. Changes in Emiliania huxleyi fatty acid profiles during infection with E. huxleyi virus 86:physiological and ecological implications[J]. Aquat Microb Ecol, 2009, 55(3):219-228.

Michaelson L V, Dunn T M, Napier J A. Viral trans-dominant manipulation of algal sphingolipids[J]. Trends Plant Sci, 2010, 15(12):651-655.

Wilson W H, Schroeder D C, Allen M J, et al. Complete genome sequence and lytic phase transcription profile of a Coccolithovirus[J]. Science, 2005, 309(5737):1090-1092.

Liu X H, Zheng T L, Cai Y X, et al. Cloning, expression and characterization of serine palmitoyltransferase (SPT)-like gene subunit (LCB2) from marine Emiliania huxleyi virus (Coccolithovirus)[J]. Acta Oceanologica Sinica, 2012, 31(6):127-138.

Vardi A, Van Mooy B A S, Fredricks H F, et al. Viral glycosphingolipids induce lytic infection and cell death in marine phytoplankton[J]. Science, 2009, 326(5954):861-865.

Laguna R, Romo J, Read B A, et al. Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi[J]. Appl Environ Microbiol, 2001, 67(9):3824-3831.

Sambrook J, Russell D W. 分子克隆实验指南[M]. 3版. 黄培堂, 译. 北京:科学出版社, 2002. Sambrook J, Russell D W. Molecular Cloning:A Laboratory Manual[M]. 3rd ed. Huang Peitang,Trans. Beijing:Science Press, 2002.

Muto M, Fukuda Y, Nemoto M, et al. Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580-a high triglyceride producer[J]. Mar Biotechnol, 2013, 15(1):48-55.

余爱丽, 赵晋锋, 王高鸿, 等. 两个谷子CIPK基因在非生物逆境胁迫下的表达分析[J]. 作物学报, 2016, 42(2):295-302. Yu Aili, Zhao Jinfeng, Wang Gaohong, et al. Expression analysis of two CIPK genes under abiotic stress in foxtail millet[J]. Acta Agronomica Sinica, 2016, 42(2):295-302.

郑晓瑜, 郭晋艳, 张毅, 等. 植物非生物胁迫诱导启动子顺式作用元件的研究方法[J]. 植物生理学报, 2011, 47(2):129-135. Zheng Xiaoyu, Guo Jinyan, Zhang Yi, et al. Research methods of cis-acting elements in plant abiotic stress inducible promoters[J]. Plant Physiology Journal, 2011, 47(2):129-135.

Cheng S J, Wang Z Y, Hong M M. Rice bZIP protein, REB, interacts with GCN4 motif in promoter of Waxy gene[J]. Science in China Series C:Life Sciences, 2002, 45(4):352-360.

张积森, 林清凡, 方静平, 等. 甘蔗SPS Ⅲ启动子区ATCT-motif和CAT-box元件的酵母单杂交报告载体构建[J]. 福建师范大学学报(自然科学版), 2013, 29(1):86-89. Zhang Jisen, Lin Qingfan, Fang Jingping, et al. Construction of yeast one-hybrid reporter vector for screening the binding proteins of ATCT-motif and CAT-box in SPS Ⅲ promoter[J]. Journal of Fujian Normal University (Natural Science Edition), 2013, 29(1):86-89.

许家辉, 朱娜, 温超, 等. 龙眼LEAFY同源基因启动子的克隆与序列分析[J]. 果树学报, 2011, 28(4):689-693. Xu Jiahui, Zhu Na, Wen Chao, et al. Cloning and sequence analysis of LEAFY gene promoter from longan (Dimocarpus longan)[J]. Journal of Fruit Science, 2011, 28(4):689-693.

耿德贵, 王义琴, 李文彬, 等. 杜氏盐藻基因工程选择标记的研究[J]. 生物技术, 2001, 11(5):1-3. Geng Degui, Wang Yiqin, Li Wenbin, et al. Study on selective marker of Dunaliella salina genetic engineering[J]. Biotechnology, 2001, 11(5):1-3.

陈颖, 李文彬, 张利明, 等. 小球藻对5种常用基因工程抗生素的敏感性研究[J]. 海洋与湖沼, 1999, 30(5):500-505. Chen Ying, Li Wenbing, Zhang Liming, et al. Study on sensitivities of Chlorella Ellipsoidea to 5 antibiotics in genetic engineering[J]. Oceanologia et Limnologia Sinica, 1999, 30(5):500-505.

曹军平, 费志清, 刘必谦, 等. 金藻基因工程选择标记的研究[J]. 海洋科学, 2001, 25(7):6-8. Cao Junping, Fei Zhiqing, Liu Biqian, et al. Study on the selectable marker for Dicrateria inornata gene engineering[J]. Marine Sciences, 2001, 25(7):6-8.

Cerutti H, Johnson A M, Gillham N W, et al. A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii:integration into the nuclear genome and gene expression[J]. Genetics, 1997, 145(1):97-110.

Kovar J L, Zhang J, Funke R P, et al. Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation[J]. Plant J, 2002, 29(1):109-117.

Nelson J A, Savereide P B, Lefebvre P A. The CRY1 gene in Chlamydomonas reinhardtii:structure and use as a dominant selectable marker for nuclear transformation[J]. Mol Cell Biol, 1994, 14(6):4011-4019.

Sizova I, Fuhrmann M, Hegemann P. A streptomyces rimosus aph VⅢ gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii[J]. Gene, 2001, 277(1/2):221-229.

Hallmann A, Rappel A. Genetic engineering of the multicellular green alga Volvox:a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker[J]. Plant J, 1999, 17(1):99-109.

Slavskaia L A, Lippmerier J C, Kroth P G, et al. Transformation of the diatom Phaeodactylum tricornutum(bacillariophyceae) with a variety of selectable marker and reporter genes[J]. J Phycol, 2000, 36(2):379-386.

Apt K E, Kroth-Pancic P G, Grossman A R. Stable nuclear transformation of the diatom Phaeodactylum triconutum[J]. Mol Gen Genet, 1996, 252(5):572-579.

郑国庭, 姜鹏, 秦松, 等. 三角褐指藻(Phaeodactylum tricornutum)通用转化载体的构建[J]. 生物学杂志, 2012, 29(4):8-11. Zheng Guoting, Jiang Peng, Qin Song, et al. Construction of a transformation vector for diatom Phaeodactylum tricornutum[J]. Journal of Biology, 2012, 29(4):8-11.

Paludan K, Duch M, Jørgensen P, et al. Graduated resistance to G418 leads to differential selection of cultured mammalian cells expressing the neo gene[J]. Gene, 1989, 85(2):421-426.

Miyagawa A, Okami T, Kira N, et al. Research note:high efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis[J]. Phycol Res, 2009, 57(2):142-146.

Watanabe S, Ohnuma M, Sato J, et al. Utility of a GFP reporter system in the red alga Cyanidioschyzon merolae[J]. J Gen Appl Microbiol, 2011, 57(1):69-72.

Li F C, Qin S, Jiang P, et al. The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica(Phaeophyta)[J]. J Appl Phycol, 2009, 21(3):287-293.

Miyagawa-Yamaguchi A, Okami T, Kira N, et al. Stable nuclear transformation of the diatom Chaetoceros sp.[J]. Phycol Res, 2011, 59(2):113-119.

Ladygin V G. Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation[J]. Process Biochem, 2004, 39(11):1685-1691.

Falciatore A, Casotti R, Leblanc C, et al. Transformation of nonselectable reporter genes in marine diatoms[J]. Mar Biotechnol, 1999, 1(3):239-251.

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