文献类型：期刊文献

英文题名：Stable nuclear transformation of the industrial alga Chlorella pyrenoidosa

作者：Run, Conglin[1];Fang, Lei[1];Fan, Jianhua[1];Fan, Chengming[2];Luo, Yuanchan[1];Hu, Zanmin[2];Li, Yuanguang[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Inst Genet & Dev Biol, Beijing 100101, Peoples R China

年份：2016

卷号：17

起止页码：196

外文期刊名：ALGAL RESEARCH-BIOMASS BIOFUELS AND BIOPRODUCTS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000378863700021)】；

基金：This work was supported by National Natural Science Foundation of China 31300295 and 21306222, National Special Fund for State Key Laboratory of Bioreactor Engineering 2060204, National Basic Research Program of China 2011CB200904, the Fundamental Research Funds for the Central Universities WF1314033, and Open Funding Project of the State Key Laboratory of Bioreactor Engineering 2060204, "Chenguang Program" supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission 14CG27.

语种：英文

外文关键词：Genetic engineering; Electroporation; Chlorella pyrenoidosa; Nuclear transformation; eGFP

摘要：Chlorella pyrenoidosa is an extensively investigated species. This species has been used as a commercial microalgal feedstock of proteins, lipids, and chlorophyll for nourishment and aquaculture. As such, reliable and easy genetic modification procedures should be developed to improve this industrial microalga. In this study, a pGreen 0029 vector containing an eGFP gene under the control of a Ubiquitin promoter and NptII, which is a selective marker gene, was constructed to explore and optimize the electroporation method of C. pyrenoidosa. The optimal transformation efficiency was approximately 101 +/- 7 transformants per mu g plasmid and was obtained under the following conditions: 5 x 106/plate cell density in logarithmic phase; 30 mu g/mL plasmid; 660 V pulse voltage; 3.5 ms pulse width; and 30 mu g/mL G418. The NptII and eGFP genes were identified successfully at the DNA level after transformants were screened and purified. High-intensity green fluorescence was observed in the transformants by using fluorescence and laser confocal microscope. Results showed that foreign genes in the transformants were integrated stably and could be expressed. Hence, the efficient transformation system of C. pyrenoidosa can be used to biotechnologically improve this important industrial microalgal species and to facilitate systematic functional genomic studies. (C) 2016 Elsevier B.V. All rights reserved.