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本文以小新月菱形藻(Nitzschia closterium f.minutissima)B248为研究对象，利用常压室温等离子体(ARTP)诱变育种技术对其进行诱变，并从中筛选出生长、光合活性和岩藻黄素含量均优于出发株的优良突变株。研究表明：小新月菱形藻B248产生有利突变的最佳条件为ARTP输入功率120 W、气流速度10 L/min、处理距离2 mm、处理时间16或20 s,在上述条件下藻的致死率在90%以上。将诱变的突变株经过固体平板第一轮筛选、96孔板第二轮筛选、20 mL试管第三轮筛选、100 mL锥形瓶第四轮筛选，最终获得6株优良突变株，分别编号为Z-A1、Z-A2、Z-A3、Z-A4、Z-A5和Z-A6,6株优良突变株的PSⅡ最大光能转化效率Fv/Fm为0.577～0.686,相对电子传递速率rETR为15.0～19.0,单位体积干质量为0.55～0.71 g/L,岩藻黄素含量为1.01～1.53 mg/L,与出发株相比，分别提高了9.8%～30.4%、15.1%～45.7%、33.4%～71.5%和14.7%～73.1%。遗传稳定性研究表明，编号为Z-A1、Z-A4和Z-A6的突变株遗传稳定性较高。研究结果表明，ARTP诱变育种技术是选育高产岩藻黄素小新月菱形藻藻株的有效手段，最终筛选出的3株突变株在生长、光合活性和岩藻黄素含量方面均优于出发株，且经5次传代后性状稳定。
Abstract：In this study, Nitzschia closterium f. minutissima B248 was mutated with atmospheric and room temperature plasma(ARTP) technology, and a set of mutants with better growth, photosynthetic activity and fucoxanthin content than the original strain were screened out. The results showed that the optimal mutation condition for N. closterium f. minutissima B248 was 120 W(ARTP input power), 10 L/min(airflow speed), 2 mm(treatment distance), either 16 or 20 s(mutation time). Under this condition, the lethal rate was more than 90%. After being mutation, 6 optimum mutant strains were obtained through the first round screening on solid plate, the second round screening in 96-well plates, the third round screening in test tubes(20 mL in volume), the fourth round screening in conical flask(100 mL in volume), which were numbered Z-A1, Z-A2, Z-A3, Z-A4, Z-A5 and Z-A6, respectively. The maximal photochemical efficiency of PSⅡ(Fv/Fm), the relative electron transport rate(rETR), dry weight per unit volume and fucoxanthin content of the 6 mutants varied between 0.577 and 0.686, 15.0 and 19.0, 0.55 and 0.71 g/L, and 1.01 and 1.53 mg/L, respectively. These parameters increased by 9.8%～30.4%, 15.1%～45.7%, 33.4%～71.5% and 14.7%～73.1%, respectively, in comparison with the original strain. Three mutants, Z-A1, Z-A4 and Z-A6, were higher in genetic stability. In conclusion, ARTP mutation technology is effective for breeding high-yield fucoxanthin N. closterium f. minutissima strains. The final three mutants are better than the original strain in growth, photosynthetic activity and fucoxanthin content, and they are genetically stable in at least 5 generations.
[1] 徐润洁，龚一富，陈文婷，等.不同发光二极管单色光质对三角褐指藻中岩藻黄素含量及相关基因表达的影响[J].光学学报，2019,39(9):299-307.Xu R J,Gong Y F,Chen W T,et al.Effects of LED monochromatic light quality of different colors on fucoxanthin content and expression levels of related genes in Phaeodactylum tricornutum[J].Acta Optica Sinica,2019,39(9):299-307.
[2] 臧正蓉，解修俊，赵佩佩，等.温度和光照对三角褐指藻的生长及岩藻黄素含量的影响[J].海洋科学，2015,39(7):1-6.Zang Z R,Xie X J,Zhao P P,et al.Effect of different temperatures and light conditions on the growth and fucoxanthin content of Phaeodactylum tricornutum[J].Marine Sciences,2015,39(7):1-6.
[3] Kong Z L,Sudirman S,Hsu Y C,et al.Fucoxanthin-Rich brown algae extract improves male reproductive function on streptozotocin-nicotinamide-induced diabetic rat model[J].International Journal of Molecular Sciences,2019,20(18):4485.
[4] Bae M,Kim M B,Park Y K,et al.Health benefits of fucoxanthin in the prevention of chronic diseases[J].Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids,2020,1865(11):158618.
[5] Lin H V,Tsou Y C,Chen Y T,et al.Effects of low-molecular-weight fucoidan and high stability fucoxanthin on glucose homeostasis,lipid metabolism,and liver fucoxanthin in a mouse model of type Ⅱ diabetes[J].Marine Drugs,2017,15(4):113.
[6] 隋吉槺，汪辉，刘天中.硅藻岩藻黄素特性与其生物合成的研究进展[J].海洋科学，2019,43(12):130-138.Sui J K,Wang H,Liu T Z.Research progress of the characteristics and biosynthesis of diatom fucoxanthin[J].Marine Sciences,2019,43(12):130-138.
[7] 诸德斐，杨润青，宋培钦，等.光及补料条件对室内管道光反应器中三角褐指藻生长和岩藻黄素积累的影响[J].广东海洋大学学报，2021,41(2):18-26.Zhu D F,Yang R Q,Song P Q,et al.Effect of light and fed-batch operation on growth of Phaeodactylum tricornutum and its fucoxa-nthin accumulation in indoor tubular photobioreactor[J].Journal of Guangdong Ocean University,2021,41(2):18-26.
[8] Kim S M,Kang S W,Kwon O,et al.Fucoxanthin as a major carotenoid in Isochrysis aff.galbana:Characterization of extraction for commercial application[J].Journal of the Korean Society for Applied Biological Chemistry,2012,55(4):477-483.
[9] Xia S,Wang K,Wan L,et al.Production,characterization,and antioxidant activity of fucoxanthin from the marine diatom Odontella aurita[J].Marine Drugs,2013,11(7):2667-2681.
[10] Wang L J,Fan Y,Parsons R L,et al.A rapid method for the determination of fucoxanthin in diatom[J].Marine Drugs,2018,16(1):33.
[11] 宋培钦，刘鹭，魏东.三角褐指藻跑道池规模化培养及岩藻黄素积累条件的优化[J].现代食品科技，2018,34(4):150-158;241.Song P Q,Liu L,Wei D.Scaling-up cultivation of Phaeodactylum tricornutum in open raceway pond and optimization of the culture conditions for fucoxanthin accumulation[J].Modern Food Science and Technology,2018,34(4):150-158;241.
[12] 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,Lai Q X,et al.Researching advances in microalgal mutation breeding[J].Periodical of Ocean University of China,2020,50(6):19-32.
[13] Ottenheim C,Nawrath M,Wu J C.Microbial mutagenesis by atmospheric and room-temperature plasma (ARTP):The latest development[J].Bioresources and Bioprocessing,2018,5(1):1-14.
[14] Zhang C,Qin J,Dai Y,et al.Atmospheric and room temperature plasma(ARTP)mutagenesis enables xylitol over-production with yeast Candida tropicalis[J].Journal of Biotechnology,2019,296:7-13.
[15] Zhang C Z,Li Y,Zhang T S,et al.Increasing chitosanase production in Bacillus cereus by a novel mutagenesis and screen method[J].Bioengineered,2021,12(1):266-277.
[16] Liu B,Sun Z,Ma X,et al.Mutation breeding of extracellular polysaccharide-producing microalga Crypthecodinium cohnii by a novel mutagenesis with atmospheric and room temperature plasma[J].International Journal of Molecular Sciences,2015,16(4):8201-8212.
[17] 吴晓英，柳泽深，姜悦.雨生红球藻等离子诱变及高产藻株的筛选[J].食品安全质量检测学报，2016,7(9):3781-3787.Wu X Y,Liu Z S,Jiang Y,et al.Mutation breeding of Haematococcus pluvialis by atmospheric and room temperature plasma and isolation of high-producing mutants[J].Journal of Food Safety and Quality,2016,7(9):3781-3787.
[18] An J,Gao F L,Ma Q Y,et al.Screening for enhanced astaxanthin accumulation among Spirulina platensis mutants generated by atmospheric and room temperature plasmas[J].Algal Research,2017,25:464-472.
[19] Cao S,Zhou X,Jin W,et al.Improving of lipid productivity of the oleaginous microalgae Chlorella pyrenoidosa via atmospheric and room temperature plasma (ARTP)[J].Bioresource Technology,2017,244:1400-1406.
[20] Sun X,Li P,Liu X,et al.Strategies for enhanced lipid production of Desmodesmus sp.mutated by atmospheric and room temperature plasma with a new efficient screening method[J].Journal of Cleaner Production,2020,250:119509.
[21] 王青岩，陈书秀.小新月菱形藻培养基优化研究[J].科学养鱼，2018(9):27-28.Wang Q Y,Chen S X.Optimization of culture medium for Nitzschia parvum[J].Scientific Fish Farming,2018(9):27-28.
[22] 朱帅旗，龚一富，刘浩，等.硫酸铈铵对三角褐指藻岩藻黄素含量的影响及转录差异研究[J].中国稀土学报，2014,32(6):750-757.Zhu S Q,Gong Y F,Liu H,et al.Effects of ammonium cerous sulfate on fucoxanthin content in Phaeodactylum tricornutum and research of transcriptional differences[J].Journal of the Chinese Society of Rare Earths,2014,32(6):750-757.
[23] Guillard R R L,Ryther J H.Studies of marine planktonic diatoms:I.Cyclotella nana (Hustedt) and Detonula confervacea (Cleve) Gran[J].Canadian Journal of Microbiology,1962,8(2):229-239.
[24] Liang Y,Sun M,Tian C,et al.Effects of salinity stress on the growth and chlorophyll fluorescence of Phaeodactylum tricornutum and Chaetoceros gracilis (Bacillariophyceae)[J].Botanica Marina,2014,57(6):469-476.
[25] 张雪，张晓菲，王立言，等.常压室温等离子体生物诱变育种及其应用研究进展[J].化工学报，2014,65(7):2676-2684.Zhang X,Zhang X F,Wang L Y,et al.Recent progress on atmospheric and room temperature plasma mutation breeding technology and its applications[J].CIESC,2014,65(7):2676-2684.
[26] 艾江宁，姚长洪，孟迎迎，等.生长速度快且油脂产率高的湛江等鞭金藻诱变株的筛选[J].微生物学通报，2015,42(1):142-147.Ai J N,Yao C H,Meng Y Y,et al.Screening of fast growth and high oil productivity mutants of Isochrysis zhangjiangensis[J].Microbiology China,2015,42(1):142-147.
[27] Fang M,Jin L,Zhang C,et al.Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes[J].PLoS One,2013,8(10):e77046.
[28] 李青，吴洪，蔡忠贞，等.利用流式细胞仪筛选紫外诱变高含油小球藻[J].中国油脂，2018,43(5):110-112;122.Li Q,Wu H,Cai Z Z,et al.Screening of UV-mutated Chlorella zofingensis with high lipid content using flow cytometry[J].China Oils and Fats,2018,43(5):110-112;122.
[29] 魏婷婷.高产油蛋白核小球藻60Co-γ射线诱变育种的初步研究[D].上海：华东理工大学，2018.Wei T T.Preliminary Studies on Mutation Breeding of Chlorella pyrenoidosa for High Lipid Productivity by 60Co-γ Irradiation[D].Shanghai:East China University of Science and Technology,2018.
[30] 陈建楠，陈由强，薛婷.利用UV和ARTP诱变筛选优良性状的球等鞭金藻[J].福建农业科技，2020(2):9-16.Chen J N,Chen Y Q,Xue T.Mutation screening of Isochrysis galbana with excellent characters by using UV and ARTP treatments[J].Fujian Agricultural Science and Technology,2020(2):9-16.
[31] Wang X,Lu M,Wang S,et al.The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure[J].International Journal of Biological Macromolecules,2014,70:284-291.
[32] 江胜滔.虾青素产生菌的筛选鉴定及其转化子的研究[D].福州：福建师范大学，2005.Jiang S T.Screening and Identification of the Epiphyte Producing Astaxanthin and Study on Its Transformant[D].Fuzhou:Fujian Normal University,2005.
基本信息:
DOI：10.16441/j.cnki.hdxb.20210358
中图分类号:S968.4
引用信息:
[1]梁英,辛红翠,闫译允,等.小新月菱形藻的常压室温等离子体诱变及高产岩藻黄素藻株的筛选[J],2022,52(07):39-48.DOI:10.16441/j.cnki.hdxb.20210358.
基金信息:
山东省科技攻关项目(2017GHY15104)资助~~