几种典型赤潮藻中PKS基因的克隆、序列分析与表达情况的研究
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摘要
目的：获得几种典型赤潮藻——东海原甲藻(Prorocentrum donghaiense)、利玛原甲藻(Prorocentrum lima)、塔玛亚历山大藻(Alexandrium tamarense)、海洋卡盾藻(Chattonellia marina)聚酮合成酶基因，进行序列分析和同源性比较；观察聚酮合成酶基因在不同藻类中的表达情况，分析其与藻毒素合成的关系。为赤潮毒素相关基因的分析、鉴定以及赤潮毒素合成机制的研究提供参考和依据。
方法：利用兼并引物，通过PCR技术扩增东海原甲藻、利玛原甲藻、塔玛亚历山大藻、海洋卡盾藻聚酮合成酶基因片段，将基因片段与PMD18-T载体进行TA连接，阳性克隆测序。所得序列在NCBI网站上进行BLAST比对。利用生物软件Dnastar，Dnaman等绘制同源性图谱和系统进化图谱，进行同源性和系统进化分析。根据测得序列设计特异性引物，采用RT-PCR技术观察聚酮合成酶基因在不同赤潮藻中的表达情况。
结果：在东海原甲藻、利玛原甲藻、塔玛亚历山大藻中均扩增出700bp左右条带，与文献报道PKS基因大小一致。海洋卡盾藻中未出现PKS基因条带。表明PKS基因存在于东海原甲藻、利玛原甲藻、塔玛亚历山大藻，而海洋卡盾藻中可能不存在PKS基因。序列分析发现，不同藻中PKS氨基酸序列在其他物种之间的同源性比较高，达到61.43％；系统进化图谱显示，利玛原甲藻和海洋原甲藻聚为一支，塔玛亚历山大藻与一种假单胞菌聚为一个进化分支，东海原甲藻与其他物种分支较远。PKS基因在腹泻性贝毒产毒藻——利玛原甲藻中有显著表达，但在非产毒藻——东海原甲藻和麻痹性贝毒产毒藻——塔玛亚历山大藻中无明显表达。
结论：成功获得典型赤潮藻(东海原甲藻、利玛原甲藻和塔玛亚历山大藻)PKS基因片段，并登录Genbank，登录号为EF521599，EF521600，EF521601。PKS基因在利玛原甲藻中有显著表达，但在东海原甲藻、塔玛亚历山大藻中无明显表达，提示海洋卡盾藻中可能不存在PKS基因，腹泻性贝毒的合成可能与PKS基因的表达有一定关系。
Objective: In order to provide information for the HABs toxins synthesis mechanism, polyketide synthase (PKS) gene were cloned in several typical red tide algaes, such as Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense and Chattonellia marina. The expression of PKS gene in these four algaes were observed. The relationship between the PKS gene and toxins production was analyzed.
Methods: Using degenerate primer, PKS gene in Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense and Chattonellia marina was amplified by PCR. The PCR products were cloned into the PMD18-T vector and sequenced. The homology analysis was conducted and phylogenetic tree was constructed with biotic software of Dnastar and Dnaman. The expression of the PKS gene in different red tide algae were observed by reverse transcriptase PCR (RT-PCR).
Results: 700 bp bands were amplified from Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense, which were consistant with the references in the size of PCR product of PKS gene. These suggested that the PKS gene existed in Prorocentrum lima, Alexandrium tamarense, but not in Chattonellia marina. The amio acids of PKS in different algaes exhibited high homology of 61.43% among other species. Prorocentrum lima grouped strongly with Prorocentrum micans, Alexandrium tamarense and Pseudomonas belong to a cluster, the Prorocentrum donghaiense tended to far away from the large clade. High expressions of PKS gene were observed in DSP toxin-producing Prorocentrum lima in contrast with no expression in toxin-nonproducing Prorocentrum donghaiense and PSP toxin toxin-producing Alexandrium tamarense.
Conclusions: The PKS gene in Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense were obtained successfully. The sequence numbers in Genbank were EF521599, EF521600 and EF521601, respectively. High expressions of PKS gene in Prorocentrum lima and no expressions in Prorocentrum donghaiense, Alexandrium tamarense suggested that the PKS might have an important role in the production of DSP.
方法：利用兼并引物，通过PCR技术扩增东海原甲藻、利玛原甲藻、塔玛亚历山大藻、海洋卡盾藻聚酮合成酶基因片段，将基因片段与PMD18-T载体进行TA连接，阳性克隆测序。所得序列在NCBI网站上进行BLAST比对。利用生物软件Dnastar，Dnaman等绘制同源性图谱和系统进化图谱，进行同源性和系统进化分析。根据测得序列设计特异性引物，采用RT-PCR技术观察聚酮合成酶基因在不同赤潮藻中的表达情况。
结果：在东海原甲藻、利玛原甲藻、塔玛亚历山大藻中均扩增出700bp左右条带，与文献报道PKS基因大小一致。海洋卡盾藻中未出现PKS基因条带。表明PKS基因存在于东海原甲藻、利玛原甲藻、塔玛亚历山大藻，而海洋卡盾藻中可能不存在PKS基因。序列分析发现，不同藻中PKS氨基酸序列在其他物种之间的同源性比较高，达到61.43％；系统进化图谱显示，利玛原甲藻和海洋原甲藻聚为一支，塔玛亚历山大藻与一种假单胞菌聚为一个进化分支，东海原甲藻与其他物种分支较远。PKS基因在腹泻性贝毒产毒藻——利玛原甲藻中有显著表达，但在非产毒藻——东海原甲藻和麻痹性贝毒产毒藻——塔玛亚历山大藻中无明显表达。
结论：成功获得典型赤潮藻(东海原甲藻、利玛原甲藻和塔玛亚历山大藻)PKS基因片段，并登录Genbank，登录号为EF521599，EF521600，EF521601。PKS基因在利玛原甲藻中有显著表达，但在东海原甲藻、塔玛亚历山大藻中无明显表达，提示海洋卡盾藻中可能不存在PKS基因，腹泻性贝毒的合成可能与PKS基因的表达有一定关系。
Objective: In order to provide information for the HABs toxins synthesis mechanism, polyketide synthase (PKS) gene were cloned in several typical red tide algaes, such as Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense and Chattonellia marina. The expression of PKS gene in these four algaes were observed. The relationship between the PKS gene and toxins production was analyzed.
Methods: Using degenerate primer, PKS gene in Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense and Chattonellia marina was amplified by PCR. The PCR products were cloned into the PMD18-T vector and sequenced. The homology analysis was conducted and phylogenetic tree was constructed with biotic software of Dnastar and Dnaman. The expression of the PKS gene in different red tide algae were observed by reverse transcriptase PCR (RT-PCR).
Results: 700 bp bands were amplified from Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense, which were consistant with the references in the size of PCR product of PKS gene. These suggested that the PKS gene existed in Prorocentrum lima, Alexandrium tamarense, but not in Chattonellia marina. The amio acids of PKS in different algaes exhibited high homology of 61.43% among other species. Prorocentrum lima grouped strongly with Prorocentrum micans, Alexandrium tamarense and Pseudomonas belong to a cluster, the Prorocentrum donghaiense tended to far away from the large clade. High expressions of PKS gene were observed in DSP toxin-producing Prorocentrum lima in contrast with no expression in toxin-nonproducing Prorocentrum donghaiense and PSP toxin toxin-producing Alexandrium tamarense.
Conclusions: The PKS gene in Prorocentrum donghaiense, Prorocentrum lima, Alexandrium tamarense were obtained successfully. The sequence numbers in Genbank were EF521599, EF521600 and EF521601, respectively. High expressions of PKS gene in Prorocentrum lima and no expressions in Prorocentrum donghaiense, Alexandrium tamarense suggested that the PKS might have an important role in the production of DSP.
引文
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[2] 吴敏仪．pEGFP-C-fos在T24细胞中的表达及在贝毒检测中的应用．暨南大学硕士学位论文，2006：3．
[3] 周名江，李钧，于仁诚．赤潮藻毒素研究进展．中国海洋药物，1999，(3)：33-39．
[4] 于仁诚，周名江．麻痹性贝毒研究进展．海洋与湖沼，1998，29(3)：330-338．
[5] 林燕棠，贾晓平，杨美兰．中国沿海染毒贝类的麻痹性毒素．热带海洋，1999，18(1)：90-95．
[6] 江天久，尹伊伟，骆育敏．广东大亚湾和大鹏湾麻痹性贝类毒素研究．中国环境科学，2000，20(4)：341-344．
[7] Bricelj V.M, Lee J.H, Cembella A.D,et al. Uptake kinetics of paralytic shellfish toxins from the dinoflagellate Alexandrum fundyense in the mussel Mytilus edulis. Mar Ecol. Prog Ser, 1990, 63:177-188.
[8] 丘建文．麻痹性贝毒研究概况．海洋环境科学，1991，10(2)：65-68．
[9] 周成旭，严小军．赤潮生物的毒害机理与毒素生物化学研究．海洋科学，2000，24(5)：23-26．
[10] Snyder R.V., Gibbs P.D.L. Polyketide Synthase Genes from Marine Dinoflagellates. Marine Biotechnology, 2003, 05:1-12.
[11] Lawrence J.E, Cembella A.D. An immunolabeling technique for the detectionof diarrhetic shellfish toxins in individual dinoflagellate cells. Phycologia, 1999, 38(1):60-65.
[12] Hallegraeff G.M. Manual on Harmful Marine Microalgae. UNESCO, France: DCM annuals and Guides No. 33. 1995.182-199.
[13] 陈则玲，付云娜，巩宁．腹泻性贝毒及其高效液相色谱检测方法．海洋通报，2000，19(1)：73-78．
[14] Souto M.L. Influence of amino acids on okadaic acid production, J. toxicon, 2001, 39:659-664.
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