不同品系球等鞭金藻在生长及脂肪酸组成上的种内差异
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摘要
球等鞭金藻作为一种优良的单细胞生物饵料具有选育价值。目前我国水产养殖上使用的大多数球等鞭金藻品系都来源于同一株藻株Isochrysis galbana OA-3011,经过多年的地理隔离形成了不同的品系,为了揭示地理隔离对球等鞭金藻表型性状的影响,从而为球等鞭金藻的选育工作提供依据,我们对5个品系的球等鞭金藻(I.galbana FACHB-861,OA3011-QD,OA3011-LK,OA3011-AY和OA3011-LZ)的种属关系、生长和脂肪酸组成进行了比较分析。实验在相同条件下对5个品系球等鞭金藻培养6 d,提取基因组DNA并PCR扩增测定18S rDNA基因序列,每天定时记录各品系的OD_(660),绘制生长曲线,在进入稳定期后收获并进行生物量的测定及脂肪酸的气相色谱分析。18S rDNA基因序列的进化分析表明,OA3011-LK、OA3011-AY和OA3011-LZ具有一致的序列,而OA3011-QD因1个碱基的差异与其他OA3011出现了分离,FACHB-861与OA3011品系亲缘关系较远;对生长和脂肪酸进行对比分析后,结果显示,5个品系的球等鞭金藻在不同的时间达到稳定期,其中FACHB-861表现出了与其他品系明显不同的生长模式。FACHB-861和OA-3011品系的生物量具有显著差异,OA3011-LK、OA3011-AY和OA3011-LZ之间也有显著差异(P<0.05),但特异生长率各品系间无显著差异(P<0.05)。FACHB-861和OA3011-LZ的总脂肪酸(TFA)含量显著低于OA3011-LK、OA3011-QD和OA3011-AY(P<0.05)。FACHB-861的脂肪酸各成分与OA-3011各品系明显不同。然而,在OA-3011各品系间脂肪酸组成也有明显差异,这表明球等鞭金藻存在广泛和频繁的数量性状表型差异。总之,地理隔离造成的这些差异为球等鞭金藻的选育提供了可行性。根据生长速度或DHA、EPA、多不饱和脂肪酸含量等筛选标准初步分析显示,可对OA3011-LK,OA3011-QD进行进一步的选育。
Isochrysis galbana is an excellent unicellular bait for the larvae of many aquaculture animals. Thus, screening for I. galbana strains with rapid growth rate and high content of docosahexaenoic acid(DHA) and eicosapentaenoic acid(EPA) is very important for the breeding of aquaculture animals. However, most of the I. galbana strains in China have been derived from a common ancestor called I. galbana OA-3011, and different strains of I. galbana have been developed after a long duration of geographical isolation. Whether the traits of these strains have changed or not remains unknown. In order to evaluate the status of the phenotypic traits of different geographic isolates for future selective breeding, comparative analysis of five strains of I. galbana(FACHB-861, OA3011-QD, OA3011-LK, OA3011-AY, and OA3011-LZ) with regard to their taxonomy, growth, and fatty acid composition was conducted. Different strains were cultured under identical conditions for six days until they reached a stationary phase. Genomic DNA was extracted for polymerase chain reaction sequencing of the 18 S rDNA gene. The OD_(660) was recorded daily for plotting a growth curve. When the cultures reached stationary phase, they were harvested by high-speed centrifugation for biomass determination and fatty acid analysis by using gas chromatography. Phylogenetic analysis of the 18 S rDNA gene suggested that FACHB-861 was distantly related with the OA-3011 strains. OA3011-LK, OA3011-AY, and OA3011-LZ had identical 18 S rDNA sequences, but the 18 S rDNA sequence of OA3011-QD differed from that of the other OA-3011 strains at one position, resulting in a single base substitution. The growth curve showed that the five strains reached stationary phase at different times. FACHB-861 showed a distinct pattern of growth, unlike the other four OA-3011 strains. The final biomass concentrations were significantly different between I. galbana FACHB-861 and OA-3011 strains as well as among OA3011-LK, OA3011-AY, and OA3011-LZ(P<0.05), whereas the growth rates were not significantly different among the strains(P>0.05). The total fatty acid content of FACHB-861 and OA3011-LZ were significantly lower than that of OA3011-LK, OA3011-QD, and OA3011-AY(P<0.05). The fatty acid profile of FACHB-861 was remarkably different from that of the OA-3011 strains. However, a conspicuous variation in fatty acid compositions was also observed among OA-3011 strains, indicating a wide and frequent quantitative phenotypic variation among the strains of I. galbana. These variations resulting from geographic isolation might provide a feasible method for the selective breeding of I. galbana strains. According to the screening criteria such as growth rate or DHA, EPA, and polyunsaturated fatty acid content, our preliminary analysis showed that OA3011-LK, which has the highest growth rate, and OA3011-QD, which contains the highest amount of DHA, are promising candidates for further screening.
Isochrysis galbana is an excellent unicellular bait for the larvae of many aquaculture animals. Thus, screening for I. galbana strains with rapid growth rate and high content of docosahexaenoic acid(DHA) and eicosapentaenoic acid(EPA) is very important for the breeding of aquaculture animals. However, most of the I. galbana strains in China have been derived from a common ancestor called I. galbana OA-3011, and different strains of I. galbana have been developed after a long duration of geographical isolation. Whether the traits of these strains have changed or not remains unknown. In order to evaluate the status of the phenotypic traits of different geographic isolates for future selective breeding, comparative analysis of five strains of I. galbana(FACHB-861, OA3011-QD, OA3011-LK, OA3011-AY, and OA3011-LZ) with regard to their taxonomy, growth, and fatty acid composition was conducted. Different strains were cultured under identical conditions for six days until they reached a stationary phase. Genomic DNA was extracted for polymerase chain reaction sequencing of the 18 S rDNA gene. The OD_(660) was recorded daily for plotting a growth curve. When the cultures reached stationary phase, they were harvested by high-speed centrifugation for biomass determination and fatty acid analysis by using gas chromatography. Phylogenetic analysis of the 18 S rDNA gene suggested that FACHB-861 was distantly related with the OA-3011 strains. OA3011-LK, OA3011-AY, and OA3011-LZ had identical 18 S rDNA sequences, but the 18 S rDNA sequence of OA3011-QD differed from that of the other OA-3011 strains at one position, resulting in a single base substitution. The growth curve showed that the five strains reached stationary phase at different times. FACHB-861 showed a distinct pattern of growth, unlike the other four OA-3011 strains. The final biomass concentrations were significantly different between I. galbana FACHB-861 and OA-3011 strains as well as among OA3011-LK, OA3011-AY, and OA3011-LZ(P<0.05), whereas the growth rates were not significantly different among the strains(P>0.05). The total fatty acid content of FACHB-861 and OA3011-LZ were significantly lower than that of OA3011-LK, OA3011-QD, and OA3011-AY(P<0.05). The fatty acid profile of FACHB-861 was remarkably different from that of the OA-3011 strains. However, a conspicuous variation in fatty acid compositions was also observed among OA-3011 strains, indicating a wide and frequent quantitative phenotypic variation among the strains of I. galbana. These variations resulting from geographic isolation might provide a feasible method for the selective breeding of I. galbana strains. According to the screening criteria such as growth rate or DHA, EPA, and polyunsaturated fatty acid content, our preliminary analysis showed that OA3011-LK, which has the highest growth rate, and OA3011-QD, which contains the highest amount of DHA, are promising candidates for further screening.
引文
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[12]Jamali H,Ahmadifard N,Abdollahi D.Evaluation of growth,survival and body composition of larval white shrimp(Litopenaeus vannamei)fed the combination of three types of algae[J].Internat Aquat Res,2015,7(2):115-122.
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[15]Zhang L,Li L,Liu J.Comparison of the photosynthetic characteristics of two Isochrysis galbana strains under high light[J].Botanica Mar,2014,57(6):477-481.
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[19]Guillard R R L,Ryther J H.Studies of marine planktonic diatoms:I.Cyclotella nana Hustedt,and Detonula confervacea(CLEVE)Gran[J].Can J Microbiol,1962,8(2):229-239.
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[22]Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molec Biol Evol,2011,28(10):2731-2739.
[23]Qiao H,Wang J,Zhang L,et al.An improved direct transesterification method for fatty acid determination of Phaeodactylum tricornutum[J].J Appl Phycol,2015,27(2):697-701.
[24]Duncan D B.Multiple range and multiple F tests[J].Biometrics,1955,11(1):1-42.
[25]Kain J M,Fogg G E.Studies on the growth of marine phytoplankton I.Asterionella japonica Gran[J].J Mar Biolog Assoc UK,1958,37(2):397-413.
[26]Liu J,Sommerfeld M,Hu Q.Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production[J].Appl Microbiol Biotechnol,2013,97(11):4785-4798.
[27]Burgess J G,Iwamoto K,Miura Y,et al.An optical fibre photobioreactor for enhanced production of the marine unicellular alga Isochrysis aff.galbana T-Iso(UTEX LB 2307)rich in docosahexaenoic acid[J].Appl Microbiol Biotechnol,1993,39(4-5):456-459.
[28]Grima E M,Pérez J A S,Camacho F G,et al.Effect of growth rate on the eicosapentaenoic acid and docosahexaenoic acid content of Isochrysis galbana in chemostat culture[J].Appl Microbiol Biotechnol,1994,41(1):23-27.
[29]Grima E M,Camacho F G,Pérez J A S,et al.Biochemical productivity and fatty acid profiles of Isochrysis galbana Parke and Tetraselmis sp.as a function of incident light intensity[J].Proc Biochem,1994,29(2):119-126.
[30]Vazhappilly R,Chen F.Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth[J].J Am Oil Chem Soc,1998,75(3):393-397.
[31]Lin Y H,Chang F L,Tsao C Y,et al.Influence of growth phase and nutrient source on fatty acid composition of Isochrysis galbana CCMP 1324 in a batch photoreactor[J].Biochem Engin J,2007,37(2):166-176.
[32]Sukenik A,Wahnon R.Biochemical quality of marine unicellular algae with special emphasis on lipid composition.I.Isochrysis galbana[J].Aquaculture,1991,97(1):61-72.
[33]Zhu C J,Lee Y K,Chao T M.Effects of temperature and growth phase on lipid and biochemical composition of Isochrysis galbana TK1[J].J Appl Phycol,1997,9(5):451-457.
[34]Pernet F,Tremblay R,Demers E,et al.Variation of lipid class and fatty acid composition of Chaetoceros muelleri and Isochrysis sp.grown in a semicontinuous system[J].Aquaculture,2003,221(1):393-406.
[35]Patil V,K?llqvist T,Olsen E,et al.Fatty acid composition of12 microalgae for possible use in aquaculture feed[J].Aquaculture International,2007,15(1):1-9.
[36]Molina Grima E,Sánchez Pérez J A,García Sánchez J L,et al.EPA from Isochrysis galbana.Growth conditions and productivity[J].Proc Biochem,1992,27(5):299-305.
[37]Fidalgo J P,Cid A,Torres E,et al.Effects of nitrogen source and growth phase on proximate biochemical composition,lipid classes and fatty acid profile of the marine microalga Isochrysis galbana[J].Aquaculture,1998,166(1):105-116.
[38]Alonso D L,del Castillo C I S,Sánchez J L G,et al.Quantitative genetics of fatty acid vaiation in Isochrysis galbana(Prymnesiophyceae)and Phaeodactylum tricorntum(Bacillariophyceae)[J].J Phycol,1994,30(3):553-558.
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[2]Gladyshev M I,Sushchik N N,Makhutova O N.Production of EPA and DHA in aquatic ecosystems and their transfer to the land[J].Prostagl Lip Mediat,2013,107:117-126.
[3]Reitan K I,Rainuzzo J R,?ie G,et al.A review of the nutritional effects of algae in marine fish larvae[J].Aquaculture,1997,155(1):207-221.
[4]Muller-Feuga A.The role of microalgae in aquaculture:situation and trends[J].J Appl Phycol,2000,12(3-5):527-534.
[5]Servel M O,Claire C,Derrien A,et al.Fatty acid composition of some marine microalgae[J].Phytochemistry,1994,36(3):691-693.
[6]Volkman J K,Smith D J,Eglinton G,et al.Sterol and fatty acid composition of four marine Haptophycean algae[J].JMar Biolog Associat UK,1981,61(2):509-527.
[7]Volkman J K,Jeffrey S W,Nichols P D,et al.Fatty acid and lipid composition of 10 species of microalgae used in mariculture[J].J Experim Mar Biol Ecol,1989,128(3):219-240.
[8]Alonso L,Grima E M,Pérez J A S,et al.Fatty acid variation among different isolates of a single strain of Isochrysis galbana[J].Phytochemistry,1992,31(11):3901-3904.
[9]Alonso D L,Grima E M,Pérez J A S,et al.Isolation of clones of Isochrysis galbana rich in eicosapentaenoic acid[J].Aquaculture,1992,102(4):363-371.
[10]Fradique M,Batista A P,Nunes M C,et al.Isochrysis galbana and Diacronema vlkianum biomass incorporation in pasta products as PUFA’s source[J].LWT-Food Sci Technol,2013,50(1):312-319.
[11]Freites L,Lodeiros C,Guevara M,et al.Fatty acids profiles as selecting criteria of microalgal diets used for hatchery-rearing of the tropical scallop Euvola ziczac[J].Aquac Res,2016,47(8):2670-1676.
[12]Jamali H,Ahmadifard N,Abdollahi D.Evaluation of growth,survival and body composition of larval white shrimp(Litopenaeus vannamei)fed the combination of three types of algae[J].Internat Aquat Res,2015,7(2):115-122.
[13]Scott A P,Middleton C.Unicellular algae as a food for turbot(Scophthalmus maximus L.)larvae-the importance of dietary long-chain polyunsaturated fatty acids[J].Aquaculture,1979,18(3):227-240.
[14]Rhodes E W,Landers W S.Growth of oyster larva Grassorted virginica of various sizes in different concentrations of the Chrysophyte Isochrysis galbana[J].Proceed Nation Shellfish Assoc,1973,63:53-59.
[15]Zhang L,Li L,Liu J.Comparison of the photosynthetic characteristics of two Isochrysis galbana strains under high light[J].Botanica Mar,2014,57(6):477-481.
[16]Chen J F,Pan Y Y.Studies on the growth of Isochrysis galbana Parke and its nutrient component[J].Oceanologia et Limnologia Sinica,1987,18(1):55-63.[陈椒芬,潘永尧.等鞭藻的生长及其主要营养成分的研究[J].海洋与湖沼,1987,18(1):55-63.]
[17]Chepurnov V A,Chaerle P,Roef L,et al.Classical breeding in diatoms:scientific background and practical perspectives[M]//The Diatom World.Amsterdam:Springer,2011:167-194.
[18]Borowitzka M A.Species and strain selection[M]//Borowitzka M A,Moheimani N R.Algae for Biofuels and Energy.Amsterdam:Springer,2013:77-89.
[19]Guillard R R L,Ryther J H.Studies of marine planktonic diatoms:I.Cyclotella nana Hustedt,and Detonula confervacea(CLEVE)Gran[J].Can J Microbiol,1962,8(2):229-239.
[20]Chen Y,Liu G Q,Li W B,et al.Comparison of three extraction methods for DNA from Chlorella spp.[J].Plant Physiology Communications,2001,37(3):242-244.[陈颖,刘根齐,李文彬,等.3种小球藻DNA提取方法的比较[J].植物生理学报,2001,37(3):242-244.]
[21]Qiao H,Wang G,Zhang X.Isolation and characterization of Chlorella sorokiniana GXNN01(Chlorophyta)with the properties of heterotrophic and microaerobic growth[J].JPhycol,2009,45(5):1153-1162.
[22]Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molec Biol Evol,2011,28(10):2731-2739.
[23]Qiao H,Wang J,Zhang L,et al.An improved direct transesterification method for fatty acid determination of Phaeodactylum tricornutum[J].J Appl Phycol,2015,27(2):697-701.
[24]Duncan D B.Multiple range and multiple F tests[J].Biometrics,1955,11(1):1-42.
[25]Kain J M,Fogg G E.Studies on the growth of marine phytoplankton I.Asterionella japonica Gran[J].J Mar Biolog Assoc UK,1958,37(2):397-413.
[26]Liu J,Sommerfeld M,Hu Q.Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production[J].Appl Microbiol Biotechnol,2013,97(11):4785-4798.
[27]Burgess J G,Iwamoto K,Miura Y,et al.An optical fibre photobioreactor for enhanced production of the marine unicellular alga Isochrysis aff.galbana T-Iso(UTEX LB 2307)rich in docosahexaenoic acid[J].Appl Microbiol Biotechnol,1993,39(4-5):456-459.
[28]Grima E M,Pérez J A S,Camacho F G,et al.Effect of growth rate on the eicosapentaenoic acid and docosahexaenoic acid content of Isochrysis galbana in chemostat culture[J].Appl Microbiol Biotechnol,1994,41(1):23-27.
[29]Grima E M,Camacho F G,Pérez J A S,et al.Biochemical productivity and fatty acid profiles of Isochrysis galbana Parke and Tetraselmis sp.as a function of incident light intensity[J].Proc Biochem,1994,29(2):119-126.
[30]Vazhappilly R,Chen F.Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth[J].J Am Oil Chem Soc,1998,75(3):393-397.
[31]Lin Y H,Chang F L,Tsao C Y,et al.Influence of growth phase and nutrient source on fatty acid composition of Isochrysis galbana CCMP 1324 in a batch photoreactor[J].Biochem Engin J,2007,37(2):166-176.
[32]Sukenik A,Wahnon R.Biochemical quality of marine unicellular algae with special emphasis on lipid composition.I.Isochrysis galbana[J].Aquaculture,1991,97(1):61-72.
[33]Zhu C J,Lee Y K,Chao T M.Effects of temperature and growth phase on lipid and biochemical composition of Isochrysis galbana TK1[J].J Appl Phycol,1997,9(5):451-457.
[34]Pernet F,Tremblay R,Demers E,et al.Variation of lipid class and fatty acid composition of Chaetoceros muelleri and Isochrysis sp.grown in a semicontinuous system[J].Aquaculture,2003,221(1):393-406.
[35]Patil V,K?llqvist T,Olsen E,et al.Fatty acid composition of12 microalgae for possible use in aquaculture feed[J].Aquaculture International,2007,15(1):1-9.
[36]Molina Grima E,Sánchez Pérez J A,García Sánchez J L,et al.EPA from Isochrysis galbana.Growth conditions and productivity[J].Proc Biochem,1992,27(5):299-305.
[37]Fidalgo J P,Cid A,Torres E,et al.Effects of nitrogen source and growth phase on proximate biochemical composition,lipid classes and fatty acid profile of the marine microalga Isochrysis galbana[J].Aquaculture,1998,166(1):105-116.
[38]Alonso D L,del Castillo C I S,Sánchez J L G,et al.Quantitative genetics of fatty acid vaiation in Isochrysis galbana(Prymnesiophyceae)and Phaeodactylum tricorntum(Bacillariophyceae)[J].J Phycol,1994,30(3):553-558.
[39]Flynn K J,Zapata M,Garrido J L,et al.Changes in carbon and nitrogen physiology during ammonium and nitrate nutrition and nitrogen starvation in Isochrysis galbana[J].Europ JPhycol,1993,28(1):47-52.
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