大庆湿地噬藻体的分布特征及其与宿主间对应关系研究

Cyanophage play an important role in controlling phytoplankton biomass, community succession and biogeochemical cycles of nutrients. They are also biological resource of ecological significance. Our previous studies suggested there was an abundant auxiliary metabolism genes (AMGs) of cyanophage in Daqing wetland and distribution pattern of cyanophages were not explicit. In this proposed project, different AMGs (g23, pol and psbA genes) will be used for comprehensive information on the biodiversity of cyanophages in Daqing wetland with additional methods of conventional isolation and purification, and PCR-cloning-sequencing methods for complementary analysis. Morphological characteristic of isolated cyanophages and related AMGs will be detected by scanning electron microscopy observation and PCR-cloning–sequencing, to delineate the congruent relationship between morphological characteristics and related AMGs of cyanophage, respectively. DNA-SIP methods will be used to delineate congruent relationship between isolated cyanophages (cyanobacteria) with their hosts (cyanophages). Cyanobacteria and their biodiversity will be detected by isolation and purification, high-throughput sequencing, for in-deep understanding on the abundance of isolated cyanobacterium in cyanobacterial community, distribution pattern of cyanophage in Daqing wetland waters. The projectied results will provide fundamental information for understanding the relationship between cyanophages and cyanobacteria in freshwater ecosystem, and revealing the community structure and function of cyanophages in wetland ecosystem under low temperature climate conditions.

噬藻体在控制浮游生物生物量、群落演替及生物地球化学循环中起重要作用，是一类不可忽视的生物资源。我们前期发现大庆湿地噬藻体存在独特辅助代谢基因（AMGs）类群，但未明确大庆湿地噬藻体的分布特征，为全面了解大庆湿地噬藻体的分布特征及其与宿主对应关系，本项目拟采用传统分离纯化方法结合PCR-克隆测序技术，检测大庆湿地水体噬藻体不同代谢辅助基因（g23、pol和psbA）的多样性；采用电镜观察和PCR-克隆测序技术检测分离纯化后蓝藻病毒的形态特征和其相关代谢辅助基因序列，明确蓝藻病毒形态特征与相关代谢辅助基因的对应关系；采用DNA-SIP技术明确大庆湿地水体噬藻体和蓝藻的对应关系；采用分离纯化和高通量测序技术检测大庆湿地水体蓝藻的多样性，解析蓝藻病毒在大庆湿地水体中分布特征；项目研究结果为解析寒区淡水生态系统蓝藻病毒和蓝藻间相互关系、揭示蓝藻病毒在湿地生态系统中的结构与功能提供重要基础资料。

噬藻体是侵染蓝藻的细菌病毒（噬菌体），在生物地理化学循环、群落演替及微食物环中起重要作用，大庆湿地属于寒区碱盐性湿地，独特的生境可能存在特殊的噬藻体类群。为了全面了解大庆湿地水体中噬藻体的分布特征及其与宿主的对应关系，本项目采用PCR-克隆测序技术检测了大庆湿地水样和可培养噬藻体的分子标记基因（pol、psbA、g23和phoH基因）的基因多样性，4个分子标记基因发现大庆湿地水体中存在24个特有的噬藻（菌）体基因类群，这些基因集群近于其他湿地生态系统基因集群，远于海洋的基因集群；高通量测序技术检测蓝藻和细菌多样性发现，大庆湿地水体中主要蓝藻类群是Cyanobium PCC-6307，丰度较高细菌类群是Limnohabitans和具有潜在病原的Acinetobacter，后者的丰度更高些，蓝藻群落结构在上冻和融化阶段相近，细菌群落也具此规律；分离及初步鉴定获得蓝藻菌株14株，其中5株为新的蓝藻新种，与泽丝藻和席藻同源性在92.0%和96.8%；分离鉴定蓝藻伴生细菌8株，归属于Pseudomonas、Agrobacterium、Peribacillus、Bacillus和Microbacterium；分离噬藻体病毒株25株，可侵染Anabaena PCC7120、Limnothrix sp. FACHB-709、Plectonema boryanum FACHB-240和 Synechococcus FACHB-7942分别有12, 6, 2和5株。在侵染Anabaena PCC7120短尾噬藻体DNA中成功获得三条DNA pol基因，其系统进化地位位于大庆湿地水体特有的噬藻体基因类群中，初步明确了大庆湿地水体中短尾噬藻体分子标记基因与宿主对应关系，其他分子标记基因在分离噬藻体DNA中PCR扩增未获得。以上结果为深入研究水体蓝藻和噬藻体结构与功能提供种质资源和数据支撑。