一株海洋微藻的鉴定及其原生质体制备条件优化
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摘要
从烟台海滨水域分离出一株海洋单细胞微藻。通过形态学鉴定和18S r DNA基因序列分析确定该株真核微藻属于四爿藻属(Tetraselmis sp.),命名为138号藻种。利用Central Composite法的Box-Behnken实验设计优化了此藻株的原生质体备条件。获得了以原生质体制备率为目标函数的三元二次回归方程:Y_1=88. 30-0. 19X_1+2. 44X_2+2. 28X_3-0. 95X_1X_2-3. 13X_1X_3+3. 78X_2X_3-9. 85X_1~2-7. 95X_2~2-7. 22X_3~2。得到最优制备条件:混合酶比例纤维素酶∶果胶酶为5∶2;混合酶浓度为4. 5%(纤维素酶浓度为2. 7%,果胶酶浓度为1. 8%);缓冲液pH为6. 4,最高理论制备率81. 5%。经过3次平行试验,实际得到的原生质体制备率为80. 5%,与模型预测值的误差为1. 24%。绿色荧光蛋白报告基因瞬时表达检测了所制备原生质体的转化能力。
A unicellular marine microalgae was isolated at the seaside of Yantai. Both morphological identification and sequence analysis of 18 S rDNA showed that this eukaryotic marine microalgae strain is Tetraselmis sp.,named 138 algae. A Box-Behnken central composite design was utilized as an optimization of protoplast preparation rate within the experimental range. An equation was obtained to fit the empirical evidence of protoplast formation rate( Y). Y_1= 88. 30-0. 19X_1+ 2. 44X_2+ 2. 28X3-0. 95X_1X_2-3. 13X_1X_3+ 3. 78X_2X_3-9. 85X_1~2-7. 95X_2~2-7. 22X_3~2. The optimum conditions of protoplasts formation was cellulose: macerozyme at5∶ 2,concentration of mixed enzymes at 4. 5%( 2. 7% cellulose and 1. 8% macerozyme) and buffer pH at 6. 4.The theoretical maximum rate is 81. 5%. Three parallel verification test proved actual measured rate is 80. 5%.The error between model prediction and experimental testing is about 1. 24%. Then the transformation efficiency of protoplast was confirmed by the transient expression of GFP gene.
A unicellular marine microalgae was isolated at the seaside of Yantai. Both morphological identification and sequence analysis of 18 S rDNA showed that this eukaryotic marine microalgae strain is Tetraselmis sp.,named 138 algae. A Box-Behnken central composite design was utilized as an optimization of protoplast preparation rate within the experimental range. An equation was obtained to fit the empirical evidence of protoplast formation rate( Y). Y_1= 88. 30-0. 19X_1+ 2. 44X_2+ 2. 28X3-0. 95X_1X_2-3. 13X_1X_3+ 3. 78X_2X_3-9. 85X_1~2-7. 95X_2~2-7. 22X_3~2. The optimum conditions of protoplasts formation was cellulose: macerozyme at5∶ 2,concentration of mixed enzymes at 4. 5%( 2. 7% cellulose and 1. 8% macerozyme) and buffer pH at 6. 4.The theoretical maximum rate is 81. 5%. Three parallel verification test proved actual measured rate is 80. 5%.The error between model prediction and experimental testing is about 1. 24%. Then the transformation efficiency of protoplast was confirmed by the transient expression of GFP gene.
引文
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[14]Berliner M D.Protoplast induction in Chlorella vugaris.Plant Science Letters,1977,9(2):201-204.
[15]Hatano S,Joh T,Miyamoto T,et al.Preparation of protoplasts from Chiorella ellipsoidea C-27.Plant and Cell Physiology,1992,33(5):651-655.
[16]Box G E P,Behnken D W.Simplex-sum designs:a class of second order rotatable designs derivable from those of first order.The Annals of Mathematical Statistics,1960,31(4):838-864.
[17]Ragonese R,Macka M,Hughes J,et al.The use of the BoxBehnken experimental design in the optimisation and robustness testing of a capillary electrophoresis method for the analysis of ethambutol hydrochloride in a pharmaceutical formulation.Journal of Pharmaceutical and Biomedical Analysis,2002,27(6):995-1007.
[18]许良,席海山,藏洪禹,等.荞麦籽壳中芦丁的微波辅助提取工艺的SPSS正交设计优化.计算机与应用化学,2012,29(6):719-722.Xu L,Xi H S,Zang H Y,et al.Optimization of microwaveextraction technology of flavonoids from shells of tarlary buckwheat by SPSS 17.0.Computers and Applied Chemistry,2012,29(6):719-722.
[19]Norris R E,Hori T,Chihara M.Revision of the genus Tetraselmis(Class Prasinophyceae).Journal of Plant Research,1980,93(4):317-339.
[2]林玉艳.一株海洋微藻的分离鉴定及环境因素对其生长的影响.青岛:中国海洋大学,2015.Lin Y Y.The isolation and identification of a marine microalga and the effect of environmental factors on its growth pattern.Qingdao:Ocean University of China,2015.
[3]郭立亮.海洋微藻DNA条形码基因的评估及环境样本多样性分析和定量基因的开发.青岛:中国海洋大学,2015.Guo L L.Assessment of marine microalgae DNA barcode genes and development of new gene for the diversity analysis and quantitation of environmental samples.Qingdao:Ocean University of China,2015.
[4]李芳芳,隋正红,龚春霞,等.5种沙漠微藻的分离鉴定及其18S r DNA保守区片段差异分析.石河子大学学报(自然科学版),2012,30(3):265-270.Li F F,Sui Z H,Gong C X,et al.Isolation and identification of five desert microalgae and analysis of 18S r DNA conserved fragment.Journal of Shihezi University(Natural Science),2012,30(3):265-270.
[5]Pau L S,Malcolm S Y T,Dillirani N,et al.A holistic approach to managing microalgae for biofuel applications.International Journal of Molecular Sciences,2017,18(1):215.
[6]崔玉林.亚心型扁藻细胞核与叶绿体稳定转化系统的构建.青岛:中国科学院海洋研究所,2011.Cui Y L.Establishment of the nuclear and chloroplast transformation system of Platymonas subcordiformis.Qingdao:Institute of Oceanology,Chines Academy of Sciences,2011.
[7]Umemoto Y,Araki T S.Cell wall regeneration in Bangia atropurpurea(Rhodophyta)protoplasts observed using a mannanspecific carbohydrate-binding module.Marine Biotechnology,2010,12(1):24-31.
[8]Millner P A,Callow M E,Evans L V.Preparation of protoplasts from the green alga Enteromorpha intestinalis(L.)Link.Planta,1979,147(2):174-177.
[9]叶涛,王明兹,黄建忠,等.雨生红球藻原生质体制备与再生育种.福建师范大学学报(自然科学版),2011,27(4):122-126.Ye T,Wang M Z,Huang J Z,et al.Preparation and regeneration breeding of protoplasts from Haematococcus pluvialis.Journal of Fujian Normal University(Natural Science Edition),2011,27(4):122-126.
[10]林学政,沈继红,李光友.复合酶对卡德藻溶壁效果的研究.海洋科学,2000,24(7):1-4.Lin X Z,Shen J H,Li G Y.Study on effects of complex enzymes on the cell wall degradation of Tetraselmis sp..Marine Sciences,2000,24(7):1-4.
[11]马晓磊,张琳,杨官品,等.微拟球藻原生质体制备与再生.海洋科学,2012,36(2):19-23.Ma X L,Zhang L,Yang G P,et al.Preparation and regeneration of protoplasts from Nannochloropsis oculata.Marine Sciences,2012,36(2):19-23.
[12]王明兹,王世锋,施巧琴,等.紫球藻原生质体再生育种.福建师范大学学报(自然科学科版),2007,23(1):65-69.Wang M Z,Wang S F,Shi Q Q,et al.Regeneration of protoplasts from porphyridium cruentum and its variation for breeding.Journal of Fujian Normal University(Natural Science Edition),2007,23(1):65-69.
[13]Suzuki T,Yamaguchi T,Ishida M.Effects of some factors on protoplast formation of a microalga,Prototheca zopfii.World Journal of Microbiology and Biotechnology,1997,13(3):355-356.
[14]Berliner M D.Protoplast induction in Chlorella vugaris.Plant Science Letters,1977,9(2):201-204.
[15]Hatano S,Joh T,Miyamoto T,et al.Preparation of protoplasts from Chiorella ellipsoidea C-27.Plant and Cell Physiology,1992,33(5):651-655.
[16]Box G E P,Behnken D W.Simplex-sum designs:a class of second order rotatable designs derivable from those of first order.The Annals of Mathematical Statistics,1960,31(4):838-864.
[17]Ragonese R,Macka M,Hughes J,et al.The use of the BoxBehnken experimental design in the optimisation and robustness testing of a capillary electrophoresis method for the analysis of ethambutol hydrochloride in a pharmaceutical formulation.Journal of Pharmaceutical and Biomedical Analysis,2002,27(6):995-1007.
[18]许良,席海山,藏洪禹,等.荞麦籽壳中芦丁的微波辅助提取工艺的SPSS正交设计优化.计算机与应用化学,2012,29(6):719-722.Xu L,Xi H S,Zang H Y,et al.Optimization of microwaveextraction technology of flavonoids from shells of tarlary buckwheat by SPSS 17.0.Computers and Applied Chemistry,2012,29(6):719-722.
[19]Norris R E,Hori T,Chihara M.Revision of the genus Tetraselmis(Class Prasinophyceae).Journal of Plant Research,1980,93(4):317-339.