海洋球石藻Emiliania huxleyi(Prymnesiophyceae)病毒主要外壳蛋白基因的克隆与序列分析

CLONE AND SEQUENCE OF MAJOR CAPSID PROTEIN IN COCCOLITHOPHORID EMILIANIA HUXLEYI VIRUS

在实验室纯培养条件下,用过滤的海洋球石藻特异性病毒(Emiliania huxleyi virus,EhV)EhV-99B1株感染球石藻(E. huxleyi,Eh),建立病毒与宿主之间稳定的感染体系,病毒裂解滤液经卷式切向流超滤和PEG8000浓缩、CsCl密度梯度离心,获得足量高纯度的病毒颗粒。根据已报道的其它EhV株系主要外壳蛋白(MCP)基因内保守片段设计合成一对特异引物,从EhV-99B1病毒基因组中克隆到了长度约为300bp的病毒外壳蛋白基因保守片段。该片段与pBS-T载体连接后转化Escherichia coli DH5α,对筛选到的阳性克隆进行序列测定与分析。结果表明,该克隆片段与GenBank中EhV163(AF453851)分离株的同源性最高,该区域内的核苷酸与对应推导的氨基酸序列同源性均为100%,证实获得的DNA片段是EhV-99B1的外壳蛋白基因;与EhV203(AF453855)分离株的核苷酸及氨基酸序列的同源性较低,分别为93%和100%。表明该病毒在自然海域中分布广泛并具有一定的多态性,同时在进化上也存在相当的复杂性。因此,MCP基因可以作为一种新的分子遗传标记以区分自然群落中EhV的不同基因型,对于理解海洋球石藻类病毒与宿主之间复杂的相互作用关系将是一个十分有价值的工具。

A stable in culture system of Emiliania huxleyi （Eh） host-Emiliania huxleyi-specific virus （EhV-99B1 strain） was developed, in which lysed cultures were passed sequentially through 0.45 μm and 0.2 μm filters for removing large cellular debris. The virus filtrates were concentrated by tangential flow ultrafiltration with a 50kMW size cut-off unit （Prep/Scale TFF-1, PTQK50, Millipore） to a final volume of 20-50ml; and the viral particles were then purified with CsCl density gradient centrifugation. Partial EhV gene fragment of major capsid protein （MCP） ha about 300bp was cloned from EhV-99B 1 strain by PCR using a pair of specific primers designed from conserved region of the MCP genes of Coccolithovirus. The PCR product was cloned into pBS-T vector and the linked DNA was transformed into Escherichia coli DH5α. The positive clones were sequenced and analyzed. The results show high identification between the EhV 163 （AF453851） virus isolate and the clone in 100% homology of both nucleotide and deduced amino acid sequences for MCP, which indicates that the cloned fragment is highly identical with known Coccolithovirus MCP genes. However, low identification was between the EhV 203 （AF453855） virus isolate and the clone, in 93% of the nucleotide homology and 100% deduced amino acid sequences for MCP, which indicates that the MCP gene of EhV strains has some polymorphism and exists much complexity in evolutionary relationship. Therefore, the major capsid protein （MCP） could be used as a new genetic tool to differentiate viral genotypes in natural communities. In addition, MCP as a diagnostic marker for E. huxleyi-specific viruses will be an important tool to study complex interaction between some EhV and their hosts.