陈小凡,王路路,肖腾飞,朱葆华,李赟,潘克厚

• 1:海水养殖教育部重点实验室(中国海洋大学)
• 2:崂山实验室海洋渔业科学与食物产出过程功能实验室

摘要(Abstract)：

微拟球藻(Nannochloropsis oceanica)生长速度快、油脂含量高，已成为生产生物柴油的理想材料。为获得中性脂产率高的藻株，本研究从染色时间和染色浓度两个方面优化了尼罗红染色测定中性脂含量的方法，对不同育种方法得到的75株微拟球藻藻株的产油能力进行了评价。采用优化后的染色条件(尼罗红浓度1 mg/mL,染色时间10 min),测定了第9天微藻的中性脂相对含量及对应的生物量，获得藻株中性脂的相对产率。研究表明，与原始藻株相比，088号转基因藻株相对产率为252.16%,甲基磺酸乙酯(EMS)诱变藻株101号相对产率为259.02%,亚硝基胍(NTG)诱变藻株231号相对产率为323.84%。本研究获得了3株中性脂产率高的微拟球藻藻株，这3株藻株有望应用于生物柴油的生产，研究结果可为高油脂含量微藻的快速筛选提供参考。

关键词(KeyWords)： 微拟球藻;中性脂;尼罗红;藻种选育

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(41976118)资助~~

作者(Author): 陈小凡,王路路,肖腾飞,朱葆华,李赟,潘克厚

DOI: 10.16441/j.cnki.hdxb.20220372

参考文献(References)：

• [1] 高保燕，张成武，万凌琳，等.真眼点藻纲的系统分类、生物学特性及应用研究[J].水生生物学报，2014,38(5):945-956.Gao B Y,Zhang C W,Wan L L,et al.Systematics,biological characteristics and potential application of Eustigmatophyceae[J].Acta Hydrobiologica Sinica,2014,38(5):945-956.
• [2] Wang T T,Ji Y T,Wang Y,et al.Quantitative dynamics of triacylglycerol accumulation in microalgae populations at single-cell resolution revealed by Raman microspectroscopy[J].Biotechnol Biofuels,2014,7:58.
• [3] Hu Q,Sommerfeld M,Jarvis E,et al.Microalgal triacylglycerols as feedstocks for biofuel production:Perspectives and advances[J].The Plant Journal,2008,54(4):621-639.
• [4] Doan T T,Sivaloganathan B,Obbard J P.Screening of marine microalgae for biodiesel feedstock[J].Biomass and Bioenergy,2011,35(7):2534-2544.
• [5] 梁英，闫译允，赖秋璇，等.微藻诱变育种研究进展[J].中国海洋大学学报(自然科学版),2020,50(6):19-32.Liang Y,Yan Y Y,Lan Q X,et al.Researching advances in microalgal mutation breeding[J].Periodical of Ocean University of China,2020,50(6):19-32.
• [6] Anandarajah K,Mahendraperumal G,Sommerfeld M,et al.Characterization of microalga Nannochloropsis sp.mutants for improved production of biofuels[J].Applied Energy,2012,96:371-377.
• [7] Wong D M,Nguyen T T N,Franz A K,et al.Ethylenediaminetetraacetic acid (EDTA) enhances intracellular lipid staining with Nile red in microalgae Tetraselmis suecica[J].Algal Research,2014,5:158-163.
• [8] Alemán-Nava G S,Cuellar-Bermudez S P,Cuaresma M,et al.How to use Nile Red,a selective fluorescent stain for microalgal neutral lipids[J].Journal of Microbiological Methods,2016,128:74-79.
• [9] 张琳.微拟球藻PUFA和TAG合成的分子调控研究[D].青岛：中国海洋大学，2014.Zhang L.Study on Molecular Regulation of PUFA and TAG Biosynthesis in Nannochloropsis oceanica[D].Qingdao:Ocean university of China,2014.
• [10] 冯磊力.微拟球藻常压室温等离子(ARTP)诱变及富油藻株的高通量筛选研究[D].青岛：中国海洋大学，2016.Feng L L.Atmospheric and Room Temperature Plasma Mutating and High Throughput Screening of Lipid-rich Nannochloropsis oceanica[D].Qingdao:Ocean University of China,2016.
• [11] Liang S S,Guo L,Lin G M,et al.Improvement of nannochloropsis oceanica growth performance through chemical mutation and characterization of fast growth physiology by transcriptome profiling[J].Chinese Journal of Oceanology and Limnology,2017,35(4):792-802.
• [12] 曲晓梅.微拟球藻诱变育种及高脂藻株筛选方法研究[D].青岛：中国海洋大学，2013.Qu X M.The Mutagenesis Breeding of Nannochloropsis oceanica and Screening Methods of High-Lipid Microalgae Strains[D].Qingdao:Ocean University of China,2013.
• [13] 王松.微拟球藻化学诱变及富油藻株的高通量筛选研究[D].青岛：中国海洋大学，2015.Wang S.Chemical Mutagenesis of Nannochloropsis oceanica and High-throughput Screening of Lipid-Rich Microalgae Strains[D].Qingdao:Ocean University of China,2015.
• [14] 何文栋，朱葆华，冯磊力，等.微拟球藻富油藻株筛选及柱状光生物反应器培养评价研究[J].水生生物学报，2017,41(5):1112-1117.He W D,Zhu B H,Feng L L,et al.Screening of lipid-rich Nannochloropsis oceanica mutants and evaluation using a bubble column photobioreactor[J].Acta Hydrobiologica Sinica,2017,41(5):1112-1117.
• [15] 曲晓梅，宓文义，朱葆华，等.精喹禾灵筛选高脂微藻的有效性研究[J].中国海洋大学学报，2013,43(6):25-28.Qu X M,Mi W Y,Zhu B H,et al.Applicability of herbicide quizalofop-p-ethyl to the screening of lipid rich Nannochloropsis oceanica[J].Periodical of Ocean University ofChina,2013,43(6):25-28.
• [16] 李玉芹，苏裴，马怡然，等.尼罗红染色荧光定量海洋微拟球藻胞内油脂含量[J].中国油脂，2021,46(11):121-127.Li Y Q,Su P,Ma Y R,et al.Fluorescent quantitation of oil content in marine Nannochloropsis oceanica by Nile Red staining[J].China Oils and Fats,2021,46(11):121-127.
• [17] Chen W,Zhang C,Song L,et al.A high throughput Nile Red Method for quantitative measurement of neutral lipids in microalgae[J].Journal of Microbiological Methods,2009,77(1):41-47.
• [18] Griffiths M J,Garcin C,Hille R P,et al.Interference by pigment in the estimation of microalgal biomass concentration by optical density[J].Journal of Microbiological Methods,2011,85(2):119-123.
• [19] 耿升英，韩吉昌，朱葆华，等.高固碳、高油脂产率微拟球藻筛选[J].中国海洋大学学报，2020,50(S1):64-70.Geng S Y,Han J C,Zhu B H,et al.Screening of high carbon dioxide fixing and lipid yielding Nannochloropsis oceanica strains[J].Periodical of Ocean University of China,2020,50(S1):64-70.
• [20] Katayama T,Kishi M,Takahashi K,et al.Isolation of lipid-rich marine microalgae by flow cytometric screening with Nile Red staining[J].Aquaculture International,2019,27:509-518.
• [21] Doan T T Y,Obbard J P.Improved Nile Red staining of Nannochloropsis sp.[J].Journal of Applied Phycology,2011,23:895-901.
• [22] 潘克厚，韩吉昌，朱葆华，等.基因工程在提高微藻生产生物柴油能力中的应用前景[J].海洋湖沼通报，2012(2):33-43.Pan K H,Han J C,Zhu B H,et al.The application of genetic engineering in improving the ability of microalgae for biodiesel production[J].Transaction of Oceanology and Limnology,2012(2):33-43.
• [23] Cao S,Zhou X,Jin W B,et al.Improving of lipid productivity of the oleaginous microalgae Chlorella pyrenoidosa via atmospheric and room temperature plasma (ARTP)[J].Bioresource Technology,2017,244(2):1400-1406.
• [24] Sun X,Meng L S,Li P F,et al.Increasing lipid production of Desmodesmus sp.through atmospheric and room temperature plasma orientated with malonic acid:Performance and biochemical mechanism[J].Journal of Cleaner Production,2022,342:130911.
• [25] Chou H H,Su H Y,Song X D,et al.Isolation and characterization of Chlorella sp.mutants with enhanced thermo- and CO2 tolerances for CO2 sequestration and utilization of flue gases[J].Biotechnol Biofuels,2019,12:251.
• [26] 张琳，马晓磊，朱葆华，等.微拟球藻抗生素抗性分析和选择标记筛选[J].渔业科学进展，2011,32(4):72-76.Zhang L,Ma X L,Zhu B H,et al.Antibiotic resistance of Nannochloropsis oculata and determination of selection markers for its genetic transformation[J].Progress in Fishery Sciences,2011,32(4):72-76.

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