利玛原甲藻中聚酮合酶基因克隆与分析
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国家自然科学基金-广东省联合基金重点资助项目(U0733006);广东省自然科学基金资助项目(031885);广东省自然科学基金重点资助项目(8251063201000001);国家重点基础研究发展规划973资助项目(2001CB409710)


Cloning and analysis of PKS gene from Prorocentrum lima
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    摘要:

    为探讨聚酮合酶 (polyketide synthase, PKS)基因与藻毒素合成的关系,揭示PKS基因在赤潮毒素合成中的作用,采用兼并引物,通过PCR技术获得利玛原甲藻(Prorocentrum lima)可能存在的I型PKS基因;并对所获得PKS基因的同源性进行了分析,构建了基于PKS氨基酸序列的系统进化树;采用RT-PCR技术分析了PKS基因在利玛原甲藻中的表达状况;并通过多聚腺苷酸RNA的扩增、细菌的分离鉴定、限制性内切酶酶切、Southern blotting等技术对PKS基因进行了分析。结果表明,利玛原甲藻中PKS基因与海洋原甲藻聚为一支,在利玛原甲藻中有显著表达;以Oligo(T)引物进行RT-PCR扩增时,可出现18S rRNA和PKS基因相应条带;限制性内切酶酶切和Southern blotting结果显示,该基因中存在明显的甲基化;16S rRNA基因序列分析显示,从利玛原甲藻培养液中分离到的细菌与海洋放线菌假诺卡氏菌属(Pseudonocardia)基因序列同源性达到99%,该菌株中并不存在PKS基因。结果显示,所获得的PKS基因是利玛原甲藻聚酮合酶基因,基因序列已提交GenBank (EF521601);PKS可能在腹泻性贝毒合成中起着关键作用。

    Abstract:

    In order to provide information for the HAB toxin synthesis mechanism and explore the role of polyketide synthase in the synthesis of HABs toxins, potential polyketide synthase (PKS) gene in Prorocentrum lima was amplified by PCR using degenerate primers. The homology analysis of the PKS gene in related species was conducted and phylogenetic tree was constructed using software of DNAStar and DNAMAN. The expression of PKS in P. lima was determined using reverse transcriptase polymerase chain reaction (RT-PCR). Multiple approaches including amplification of polyadenylate RNA, resistance to methylation-sensitive restriction enzymes, Southern blotting, and isolation of bacteria from cultures were performed to eliminate the possibility of PKS in associate bacteria. Results showed that type I polyketide synthase may be present in Prorocentrum lima, which grouped strongly with P. mican. High level expression of PKS in Prorocentrum lima were observed. 18S rRNA and PKS gene were amplified successful by RT-PCR using Oligo(T). The analysis of 16S rRNA sequece showed 99% homology between bacteria from P. lima culture and marine actinomycete Pseudonocardia. However, PKS gene was not obtained by PCR and no expression of PKS was detected by RT-PCR in the bacteria. These results suggested that the PKS gene was P. lima originated but not bacteria. PKS might play an important role in the production of diarrheic shellfish poisoning toxins. The sequence of PKS has been accepted by GenBank (accession NO: EF521601).

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汤敬谦,李挺,杨维东*,刘洁生,李宏业.利玛原甲藻中聚酮合酶基因克隆与分析.生态学报,2009,29(5):2383~2390

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