主要饵料微藻的分子系统学分析
摘要
本文选取收集在中国海洋大学藻种库中绿藻门的三个属(杜氏藻属、四爿藻属、小球藻属)、金藻门的两个属(等鞭藻属、巴夫藻属)、硅藻门的三个属(菱形藻属、褐指藻属、角刺藻属)的一些未明确其确切种类的饵料微藻,扩增并测定其核糖体18SrRNA序列以及部分藻株的核糖体5.8S rRNA和ITS序列,同时从基因库搜索相关微藻的基因序列,对这些微藻的系统发生关系进行了分析,以为这些藻株的分类鉴定提供分子依据。
扩增的绿藻门3个属的18SrRNA序列长度在1671-1729bp之间,分析结果如下:(1)杜氏盐藻属26株盐藻18SrRNA序列的GC含量最低为48.4%,最高为49.0%,在比对的1638个位点中,保守位点1531个,转换和颠换位点92个,插入或缺失位点15个,转换和颠换位点变异率为5.6%,插入或缺失位点变异率为0.9%。系统分析结果表明,26株盐藻可分为三大支,其中D.salina, D.bardawil,D.tertiolecta,D.bioculata,D.primolecta,D.parva共同聚类成一支, D.viridis和D.lateralis strain Nepal分别聚为一支。这与利用ITS1和ITS2进行分析的结果相似。根据序列多样性,7株未明确盐藻中,A43、A57、A83、A85和C43均与D.viridis亲缘关系最近,可以确定为D.viridis的不同株系。而根据18S rRNA序列分析,A121和A124聚类位置处于D.salina和D.viridis之间,根据ITSs序列,两者则与D.viridis更为接近。(2)四爿藻属19株18SrRNA序列GC含量最低为46.8%,最高为47.7%,在比对的1628个位点中,保守位点1551个,转换和颠换位点66个,插入或缺失位点11个,转换和颠换位点变异率为4.1%,插入或缺失位点变异率为0.7%。系统分析表明,四爿藻属19个序列可以分为四个分支,其中C75与T.striata的亲缘关系最近, C73、C74、C152与T.suecica strain KMMCC P-9和T.subcordiformis之间的亲缘关系最近,而C151可能属于T. helgolandica var.tsingtaoensis的一个株系。(3)小球藻属18SrRNA序列的GC含量最高接近50%,在比对的1756个位点中,保守位点1707个,转换和颠换位点49个,无插入或缺失位点,转换和颠换位点变异率为2.8%。种质库的CF126与C.sorokiniana亲缘关系最近。
金藻门两个属的分析结果如下:(1)在比对的23株巴夫藻的18S rRNA序列1714个位点中保守位点1464个,插入或缺失位点70个,多态位点180个,其中简约信息位点147个。构建进化树结果表明,23个巴夫藻相应序列可分为4个大支,8个P.pinguis株系与1个P.pseudogranifera株系优先聚为一支,5个P.gyrans聚为一支,3个Pa. salina株系单独聚类成一支,来自种质库的藻株BF和P. viridis优先相聚又依次与P.virecens和P.lutheri相聚,结果表明,BF可能就是P. viridis的一个株系。(2)7株等鞭金藻的18S rRNA部分序列的比对结果表明,在比对的1399个位点中,保守位点1341个,插入或缺失位点13个,多态位点45个,其中简约信息位点2个。亲缘关系分析结果表明来自种质库的3011、8701与球等鞭金藻(I. galbana)有非常近的亲缘关系,S18、S29与I. aff. galbana 'Tahitian isolate'亲缘关系最近。
PCR扩增种质库的4株硅藻18SrRNA序列长度为1687-1726bp之间,与基因库已有的相关22条序列信息进行比较,亲缘关系分析结果表明,根据18S rRNA可将分属于菱形藻属、褐指藻属和角毛藻属的硅藻完全分开,其中来自种质库的B228和B253与三角褐指藻(P.triconutum)完全聚为一支,说明两者均为三角褐指藻的不同株系,而B13则与角毛藻属的微藻种C.gracilis完全聚类。本研究的结果还表明,小新月菱形藻完全与三角褐指藻聚为一支,而并未与菱形藻属的新月菱形藻聚类,显示该微藻可能与三角褐指藻的亲缘更近。
Some bait-microalgaes were selected from MACC, including Chlorophyta(Dunaliella, Tetraselmis, Chlorella), Chrysophyta(Isochrysis, Pavlova), Bacillariophyta(Nitzschia, Phaeodactylum, Chaetoceros). Their taxonomic position were undefined. To provide molecular basis for the relationship and identification, the18SrRNA and ITSrRNA sequences were amplified and sequenced, phylogenetic relationship among these sequences and some other related algae sequences from GenBank were also analyzed.
The analysis results of 1671-1729bp 18SrRNA sequences in Chlorophyta showed that: (1) The GC content of 26 Dunaliella strains ranged from 48.4% to 49.0%. There are 1531 conserve loci, 92 transition and transversion loci, 15 insert or deletion loci in total of 1638 loci. The mutation rate of transition and transversion is 5.6%, while the mutation rate of insert or deletion is 0.9%. Analysis results of phylogenetic relationship implied that 26 strains of Dunaliella can be divided into three clads: D.salina, D.bardawil, D.tertiolecta, D.bioculata, D.primolecta and D.parva clustered in one clad, D.viridis and D.lateralis strain Nepal clustered into two clad separately. The result was consistent with ITS1 and ITS2. According to the sequence diversity, seven Dunaliella sp. A43, A57, A83, A85, C43 had closely affinity with D.viridis, could be identified as different strains of D.viridis. A121 and A124 stood between D.salina and D.viridis according to 18SrRNA sequence, and became closer to D.viridis according to ITSrRNA sequence. (2) The GC content of 19 Tetraselmis strains ranged from 46.8% to 47.7%, There are 1551 conserve loci, 66 transition and transversion loci, 11 insert or deletion loci in total of 1628 loci. The mutation rate of transition and transversion is 4.1%, while the mutation rate of insert or deletion is 0.7%. Analysis results of phylogenetic relationship showed that 26 strains of Tetraselmis can be divided into 4 clads, C75 had closely affinity with T.striata, C73, C74, C152 were much closer to T.suecica strain KMMCC P-9 and T.subcordiformis; C151 could belong to T. helgolandica var.tsingtaoensis. (3) The highest GC content of Chlorella 18SrRNA sequences is close to 50%. There are 1707 conserve loci, 49 transition and transversion loci, 0 insert or deletion loci in total of 1756 loci. The mutation rate of transition and transversion was 2.8%, while the mutation rate of insert or deletion was 0. Strain CF126 was most closely related to C.sorokiniana.
The analysis results of two Chrysophyta genus showed that: (1) 18SrRNA sequence of 23 strains of Pavlova, There are 1464 conserve loci, 180 transition and transversion loci, 70 insert or deletion loci in total of 1714 loci. The phylogenetic relationship analysis showed that 23 strains of Pavlova can be divided into four clades, 8 strains of Pa.pinguis and 1 strain of Pa.pseudogranifera clustered firstly in a clade, 5 strains of Pa.gyrans clustered in one clade, 3 strains of Pa. salina clustered in one clade, BF of MACC and a strain of Pa.viridis clustered firstly, then clustered further in one clade with Pa.virecens and Pa.lutheri. (2) Match results of 7 Isochrysis strains showed that 1399 sites were compared, the number of conserve sites are 1341, the number of transition and transversion sites are 45, the number of insert or deletion sites are 13. Analysis results of phylogenetic relationship show that 3011, 8701 and Isochrysis galbana (I. galbana) had a very close affinity, S18, S29 were closer to I. aff. Galbana 'Tahitian isolate'.
The18SrRNA sequence length of 4 Diatom strains were 1687-1726bp. Phylogenetic relationship among these sequences and 22 related algae sequences from GenBank were compared. The results implied that all the strains of Nitzschia, Phaeodactylum, Chaetoceros can be devided through 18S rRNA. B253, B228 and P.triconutum clustered in one clade, which suggested B253, B228 were different strains of P.triconutum, B13 and C.gracilis clustered in one clade. The result also indicated that it was Nitzschia closterium f.minutissima which clustered in one clade with P.triconutum entirely, not N.closterium. Nitzschia closterium f.minutissima had a closer affinity with P.triconutum.
扩增的绿藻门3个属的18SrRNA序列长度在1671-1729bp之间,分析结果如下:(1)杜氏盐藻属26株盐藻18SrRNA序列的GC含量最低为48.4%,最高为49.0%,在比对的1638个位点中,保守位点1531个,转换和颠换位点92个,插入或缺失位点15个,转换和颠换位点变异率为5.6%,插入或缺失位点变异率为0.9%。系统分析结果表明,26株盐藻可分为三大支,其中D.salina, D.bardawil,D.tertiolecta,D.bioculata,D.primolecta,D.parva共同聚类成一支, D.viridis和D.lateralis strain Nepal分别聚为一支。这与利用ITS1和ITS2进行分析的结果相似。根据序列多样性,7株未明确盐藻中,A43、A57、A83、A85和C43均与D.viridis亲缘关系最近,可以确定为D.viridis的不同株系。而根据18S rRNA序列分析,A121和A124聚类位置处于D.salina和D.viridis之间,根据ITSs序列,两者则与D.viridis更为接近。(2)四爿藻属19株18SrRNA序列GC含量最低为46.8%,最高为47.7%,在比对的1628个位点中,保守位点1551个,转换和颠换位点66个,插入或缺失位点11个,转换和颠换位点变异率为4.1%,插入或缺失位点变异率为0.7%。系统分析表明,四爿藻属19个序列可以分为四个分支,其中C75与T.striata的亲缘关系最近, C73、C74、C152与T.suecica strain KMMCC P-9和T.subcordiformis之间的亲缘关系最近,而C151可能属于T. helgolandica var.tsingtaoensis的一个株系。(3)小球藻属18SrRNA序列的GC含量最高接近50%,在比对的1756个位点中,保守位点1707个,转换和颠换位点49个,无插入或缺失位点,转换和颠换位点变异率为2.8%。种质库的CF126与C.sorokiniana亲缘关系最近。
金藻门两个属的分析结果如下:(1)在比对的23株巴夫藻的18S rRNA序列1714个位点中保守位点1464个,插入或缺失位点70个,多态位点180个,其中简约信息位点147个。构建进化树结果表明,23个巴夫藻相应序列可分为4个大支,8个P.pinguis株系与1个P.pseudogranifera株系优先聚为一支,5个P.gyrans聚为一支,3个Pa. salina株系单独聚类成一支,来自种质库的藻株BF和P. viridis优先相聚又依次与P.virecens和P.lutheri相聚,结果表明,BF可能就是P. viridis的一个株系。(2)7株等鞭金藻的18S rRNA部分序列的比对结果表明,在比对的1399个位点中,保守位点1341个,插入或缺失位点13个,多态位点45个,其中简约信息位点2个。亲缘关系分析结果表明来自种质库的3011、8701与球等鞭金藻(I. galbana)有非常近的亲缘关系,S18、S29与I. aff. galbana 'Tahitian isolate'亲缘关系最近。
PCR扩增种质库的4株硅藻18SrRNA序列长度为1687-1726bp之间,与基因库已有的相关22条序列信息进行比较,亲缘关系分析结果表明,根据18S rRNA可将分属于菱形藻属、褐指藻属和角毛藻属的硅藻完全分开,其中来自种质库的B228和B253与三角褐指藻(P.triconutum)完全聚为一支,说明两者均为三角褐指藻的不同株系,而B13则与角毛藻属的微藻种C.gracilis完全聚类。本研究的结果还表明,小新月菱形藻完全与三角褐指藻聚为一支,而并未与菱形藻属的新月菱形藻聚类,显示该微藻可能与三角褐指藻的亲缘更近。
Some bait-microalgaes were selected from MACC, including Chlorophyta(Dunaliella, Tetraselmis, Chlorella), Chrysophyta(Isochrysis, Pavlova), Bacillariophyta(Nitzschia, Phaeodactylum, Chaetoceros). Their taxonomic position were undefined. To provide molecular basis for the relationship and identification, the18SrRNA and ITSrRNA sequences were amplified and sequenced, phylogenetic relationship among these sequences and some other related algae sequences from GenBank were also analyzed.
The analysis results of 1671-1729bp 18SrRNA sequences in Chlorophyta showed that: (1) The GC content of 26 Dunaliella strains ranged from 48.4% to 49.0%. There are 1531 conserve loci, 92 transition and transversion loci, 15 insert or deletion loci in total of 1638 loci. The mutation rate of transition and transversion is 5.6%, while the mutation rate of insert or deletion is 0.9%. Analysis results of phylogenetic relationship implied that 26 strains of Dunaliella can be divided into three clads: D.salina, D.bardawil, D.tertiolecta, D.bioculata, D.primolecta and D.parva clustered in one clad, D.viridis and D.lateralis strain Nepal clustered into two clad separately. The result was consistent with ITS1 and ITS2. According to the sequence diversity, seven Dunaliella sp. A43, A57, A83, A85, C43 had closely affinity with D.viridis, could be identified as different strains of D.viridis. A121 and A124 stood between D.salina and D.viridis according to 18SrRNA sequence, and became closer to D.viridis according to ITSrRNA sequence. (2) The GC content of 19 Tetraselmis strains ranged from 46.8% to 47.7%, There are 1551 conserve loci, 66 transition and transversion loci, 11 insert or deletion loci in total of 1628 loci. The mutation rate of transition and transversion is 4.1%, while the mutation rate of insert or deletion is 0.7%. Analysis results of phylogenetic relationship showed that 26 strains of Tetraselmis can be divided into 4 clads, C75 had closely affinity with T.striata, C73, C74, C152 were much closer to T.suecica strain KMMCC P-9 and T.subcordiformis; C151 could belong to T. helgolandica var.tsingtaoensis. (3) The highest GC content of Chlorella 18SrRNA sequences is close to 50%. There are 1707 conserve loci, 49 transition and transversion loci, 0 insert or deletion loci in total of 1756 loci. The mutation rate of transition and transversion was 2.8%, while the mutation rate of insert or deletion was 0. Strain CF126 was most closely related to C.sorokiniana.
The analysis results of two Chrysophyta genus showed that: (1) 18SrRNA sequence of 23 strains of Pavlova, There are 1464 conserve loci, 180 transition and transversion loci, 70 insert or deletion loci in total of 1714 loci. The phylogenetic relationship analysis showed that 23 strains of Pavlova can be divided into four clades, 8 strains of Pa.pinguis and 1 strain of Pa.pseudogranifera clustered firstly in a clade, 5 strains of Pa.gyrans clustered in one clade, 3 strains of Pa. salina clustered in one clade, BF of MACC and a strain of Pa.viridis clustered firstly, then clustered further in one clade with Pa.virecens and Pa.lutheri. (2) Match results of 7 Isochrysis strains showed that 1399 sites were compared, the number of conserve sites are 1341, the number of transition and transversion sites are 45, the number of insert or deletion sites are 13. Analysis results of phylogenetic relationship show that 3011, 8701 and Isochrysis galbana (I. galbana) had a very close affinity, S18, S29 were closer to I. aff. Galbana 'Tahitian isolate'.
The18SrRNA sequence length of 4 Diatom strains were 1687-1726bp. Phylogenetic relationship among these sequences and 22 related algae sequences from GenBank were compared. The results implied that all the strains of Nitzschia, Phaeodactylum, Chaetoceros can be devided through 18S rRNA. B253, B228 and P.triconutum clustered in one clade, which suggested B253, B228 were different strains of P.triconutum, B13 and C.gracilis clustered in one clade. The result also indicated that it was Nitzschia closterium f.minutissima which clustered in one clade with P.triconutum entirely, not N.closterium. Nitzschia closterium f.minutissima had a closer affinity with P.triconutum.
引文
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