中国北方海区部分单胞藻的分离、培养及基因条码研究
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摘要
中国海域宽广，海水中单细胞藻种资源极其丰富，然而搜集得到活体纯培养的藻种却非常少。从海水中分离新的单胞藻，获得活体种质，是开展实验研究和开发利用的基础，同时对保护海洋生物资源也具有重要意义。作者从中国北黄海海水中搜集、分离得到了51株单胞藻，探索了一条较为高效的分离纯化方法，用分子系统分析结合形态特点对浮游单胞藻做了初步的鉴定，并对其中的几种赤潮藻做了详细的分子鉴定及培养条件研究，对它们生长条件的研究有助于解释其在自然界大规模暴发的机制。
在青岛沿海分离到一株罕见海水裸藻，依据18S rDNA和16S rDNA序列的系统分析结合形态特点将其鉴定为Eutreptiella gymnastica，为该藻在中国的首次正式详细记录。以广泛使用的光密度值OD_680作为衡量藻体生物量变化的指标，研究了这株藻在不同温度、光照、盐度、氮磷浓度下的生长情况，结果表明它的最适生长条件为：温度20°C,光照强度160μmol/(m~（2+）s)；它可耐受10～31的盐度范围，淡水中无法存活；磷营养是影响其生长的关键元素，PO3-4浓度高于72μmol/L时大大促进藻的生长。
2011年5月山东荣成桑沟湾近海海域暴发赤潮，其规模在桑沟湾历史上罕见，较严重地影响了该海域水产动物的人工养殖。作者对赤潮暴发过程中海水里的一株优势种进行了18S rDNA和ITS序列的分析，将其鉴定为卡罗藻Karlodinium veneficum (=K. micrum)，是一种常见有害赤潮藻，在南方海区曾引发赤潮，在桑沟湾暴发是第一次记录，经分析其种源有可能来自南方。
对两株赤潮藻和一株赤潮藻疑似种的18S rDNA和ITS序列分析结果显示这三株藻分别为锥状斯氏藻（Scrippsiella trochoidea）、赤潮异弯藻（Heterosigmaakashiwo）和共生甲藻（Symbiodinium sp.）。研究了它们在不同培养条件下的生长，结果表明，在批次培养中，共生甲藻、锥状斯氏藻、赤潮异弯藻的生物量都在22℃达到最大，分别为每毫升细胞数9.28×105、7.9×104、4.75×105；强光和长时间光照促进生长，但锥状斯氏藻在强光下更早进入衰退期；三种藻都表现出较强耐受盐度变化的能力，在盐度为15～36的培养液中生长良好；营养实验表明，使用882μmol/L的NO-3和36μmol/L的PO3-4能够维持三种藻的快速生长。
用一对18S rDNA通用引物和一对rbcL通用引物对分离得到的其它单胞藻进行了扩增和分析，结果表明只有部分藻种的18S rDNA能够成功扩增（1720bp左右），rbcL通用引物（扩增rbcL中间部分序列，730bp左右）的扩增成功率较高。由于GenBank中可比对的可靠序列较少，因此根据目前的分子序列分析，仅对少量分离得到的藻种鉴定到种水平，部分到属水平。目前已鉴定的一些藻种包括：布氏双尾藻、中肋骨条藻、茧形藻、海链藻、圆筛藻、角管藻、角毛藻、加拉星平藻、曲舟藻、红胞藻、楔形藻、齿状藻等。
We have broad offshore seawaters in China in which the microalgal resources areextremely rich. However, only few microalgae have been collected and isolated. Tosearch for new microalgae from the sea and conserve their pure living cultures is thebasis of experimental research and exploration. Such research activity will alsobenefit the protection of living marine resources. The author collected and isolated51microalgal strains from coastal water of the North Yellow Sea of China, anddeveloped an effective separation and purification method. These strains wereidentified through molecular and morphological analyses. Some bloom-formingspecies were carefully studied regarding their molecular identification and culturetrials. Understanding the effect of environmental parameters on these species mayhelp us knowing the blooming mechanism of them in natural environment.
A rare marine Euglena was isolated from the sea. Based on the phylogeneticanalyses of18S rDNA and16S rDNA sequences, in combination with morphologicalcharacteristics, this strain was identified as Eutreptiella gymnastica, and it’s the firstdetailed record of this alga in China. To understand its growth characteristics,different culture conditions’ effects on growth were monitored, using OD_680as anestimation of biomass. The results showed that the optimal growth conditions in abatch culture of this isolate were20°C,160μmol/(m~（2+）s) of white light, and a salinityof10–31. Nutrient experiments demonstrated that growth increased dramatically witha PO3-4concentration greater than72μmol/L.
A seldom observed red tide algal species occurred in Sanggou Bay in May of 2011and the local shellfish aquaculture was greatly affected. During this event, theauthor isolated a dominant alga strain and analyzed its18S rDNA and ITS sequences.The phylogenetic trees based on the two sequences both revealed that this speciesclusters with Karlodinium veneficum (=K. micrum). So it is identified as K. veneficum, one ofharmful algal bloom species reported in the Southern Sea of China previously. This isthe first report of this alga to appear in bloom in Sanggou bay and it is deduced thatthis alga was transferred from the south of China.
Using18S rDNA and ITS sequences analyses, two bloom-forming species and asuspected one were identified as Scrippsiella trochoidea, Heterosigma akashiwo andSymbiodinium sp. Different culture conditions’ effects on their growth were monitored,and the results showed that Symbiodinium sp., S. trochoidea and H. akashiwo all gavethe best growth performance at22°C, reaching9.28×105,7.9×104and4.75×105cells/mL respectively in batch cultures. Both elevated irradiance and longerphotoperiods benefited the growth. However, S. trochoidea entered recession phaseearlier under higher irradiance level. All three species were shown to be able totolerate a wide range of salinities (15to36). Concentrations of882μmol/L of NO-3and36μmol/L of PO43-in the medium were proved sufficient for fast accumulation of algalbiomass.
Specific sequences of other newly isolated microalgae were amplified by use of18S rDNA universal primers and rbcL universal primers. The results indicated thatonly part of the algal18S rDNA (about1720bp) could be successfully amplified,while rbcL universal primers (amplifying part of rbcL sequences, about730bp) had ahigh amplification success rate. Due to lack of sufficient and reliable sequences in theGenBank, based on the analyses of present molecular sequences, only a few ofisolated microalgae were identified to species level, some to the genus level. Thesemicroalgae are identified to be Ditylum brightwellii, Skeletonema costatum,Entomoneis, Thalassiosira, Coscinodiscus, Cerataulina, Chaetoceros, Asteroplanuskarianus, Pleurosigma, Rhodomonas, Licmophora, Odontella, etc.
在青岛沿海分离到一株罕见海水裸藻，依据18S rDNA和16S rDNA序列的系统分析结合形态特点将其鉴定为Eutreptiella gymnastica，为该藻在中国的首次正式详细记录。以广泛使用的光密度值OD_680作为衡量藻体生物量变化的指标，研究了这株藻在不同温度、光照、盐度、氮磷浓度下的生长情况，结果表明它的最适生长条件为：温度20°C,光照强度160μmol/(m~（2+）s)；它可耐受10～31的盐度范围，淡水中无法存活；磷营养是影响其生长的关键元素，PO3-4浓度高于72μmol/L时大大促进藻的生长。
2011年5月山东荣成桑沟湾近海海域暴发赤潮，其规模在桑沟湾历史上罕见，较严重地影响了该海域水产动物的人工养殖。作者对赤潮暴发过程中海水里的一株优势种进行了18S rDNA和ITS序列的分析，将其鉴定为卡罗藻Karlodinium veneficum (=K. micrum)，是一种常见有害赤潮藻，在南方海区曾引发赤潮，在桑沟湾暴发是第一次记录，经分析其种源有可能来自南方。
对两株赤潮藻和一株赤潮藻疑似种的18S rDNA和ITS序列分析结果显示这三株藻分别为锥状斯氏藻（Scrippsiella trochoidea）、赤潮异弯藻（Heterosigmaakashiwo）和共生甲藻（Symbiodinium sp.）。研究了它们在不同培养条件下的生长，结果表明，在批次培养中，共生甲藻、锥状斯氏藻、赤潮异弯藻的生物量都在22℃达到最大，分别为每毫升细胞数9.28×105、7.9×104、4.75×105；强光和长时间光照促进生长，但锥状斯氏藻在强光下更早进入衰退期；三种藻都表现出较强耐受盐度变化的能力，在盐度为15～36的培养液中生长良好；营养实验表明，使用882μmol/L的NO-3和36μmol/L的PO3-4能够维持三种藻的快速生长。
用一对18S rDNA通用引物和一对rbcL通用引物对分离得到的其它单胞藻进行了扩增和分析，结果表明只有部分藻种的18S rDNA能够成功扩增（1720bp左右），rbcL通用引物（扩增rbcL中间部分序列，730bp左右）的扩增成功率较高。由于GenBank中可比对的可靠序列较少，因此根据目前的分子序列分析，仅对少量分离得到的藻种鉴定到种水平，部分到属水平。目前已鉴定的一些藻种包括：布氏双尾藻、中肋骨条藻、茧形藻、海链藻、圆筛藻、角管藻、角毛藻、加拉星平藻、曲舟藻、红胞藻、楔形藻、齿状藻等。
We have broad offshore seawaters in China in which the microalgal resources areextremely rich. However, only few microalgae have been collected and isolated. Tosearch for new microalgae from the sea and conserve their pure living cultures is thebasis of experimental research and exploration. Such research activity will alsobenefit the protection of living marine resources. The author collected and isolated51microalgal strains from coastal water of the North Yellow Sea of China, anddeveloped an effective separation and purification method. These strains wereidentified through molecular and morphological analyses. Some bloom-formingspecies were carefully studied regarding their molecular identification and culturetrials. Understanding the effect of environmental parameters on these species mayhelp us knowing the blooming mechanism of them in natural environment.
A rare marine Euglena was isolated from the sea. Based on the phylogeneticanalyses of18S rDNA and16S rDNA sequences, in combination with morphologicalcharacteristics, this strain was identified as Eutreptiella gymnastica, and it’s the firstdetailed record of this alga in China. To understand its growth characteristics,different culture conditions’ effects on growth were monitored, using OD_680as anestimation of biomass. The results showed that the optimal growth conditions in abatch culture of this isolate were20°C,160μmol/(m~（2+）s) of white light, and a salinityof10–31. Nutrient experiments demonstrated that growth increased dramatically witha PO3-4concentration greater than72μmol/L.
A seldom observed red tide algal species occurred in Sanggou Bay in May of 2011and the local shellfish aquaculture was greatly affected. During this event, theauthor isolated a dominant alga strain and analyzed its18S rDNA and ITS sequences.The phylogenetic trees based on the two sequences both revealed that this speciesclusters with Karlodinium veneficum (=K. micrum). So it is identified as K. veneficum, one ofharmful algal bloom species reported in the Southern Sea of China previously. This isthe first report of this alga to appear in bloom in Sanggou bay and it is deduced thatthis alga was transferred from the south of China.
Using18S rDNA and ITS sequences analyses, two bloom-forming species and asuspected one were identified as Scrippsiella trochoidea, Heterosigma akashiwo andSymbiodinium sp. Different culture conditions’ effects on their growth were monitored,and the results showed that Symbiodinium sp., S. trochoidea and H. akashiwo all gavethe best growth performance at22°C, reaching9.28×105,7.9×104and4.75×105cells/mL respectively in batch cultures. Both elevated irradiance and longerphotoperiods benefited the growth. However, S. trochoidea entered recession phaseearlier under higher irradiance level. All three species were shown to be able totolerate a wide range of salinities (15to36). Concentrations of882μmol/L of NO-3and36μmol/L of PO43-in the medium were proved sufficient for fast accumulation of algalbiomass.
Specific sequences of other newly isolated microalgae were amplified by use of18S rDNA universal primers and rbcL universal primers. The results indicated thatonly part of the algal18S rDNA (about1720bp) could be successfully amplified,while rbcL universal primers (amplifying part of rbcL sequences, about730bp) had ahigh amplification success rate. Due to lack of sufficient and reliable sequences in theGenBank, based on the analyses of present molecular sequences, only a few ofisolated microalgae were identified to species level, some to the genus level. Thesemicroalgae are identified to be Ditylum brightwellii, Skeletonema costatum,Entomoneis, Thalassiosira, Coscinodiscus, Cerataulina, Chaetoceros, Asteroplanuskarianus, Pleurosigma, Rhodomonas, Licmophora, Odontella, etc.
引文
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