噬藻体分子遗传多样性及与淡水水华蓝藻的互作研究

Cyanophages are viruses that infect cyanobacteria (blue-green algae). They are not only the most abundant biological entities but perhaps the greatest diversity in morphology in aquatic environments and are important mortality agents in aquatic microbial communities. They exert tremendous ecological and evolutionary forces directly on their cyanobacteria hosts. To better understand the cyanophage genetic diversity and cyanophage-host interactions, and to explore the bio-resources of cyanophage, this project involves cultivation and identification of cyanophages infecting bloom-forming cyanobacteria, complete continuous genomes sequencing and analysis of gene function, comparative analyses of transcriptomes and proteomes, interaction and coevolution of cyanophages and the host cyanobacteria. Through meticulous work at the single cell level with cyanobacteria, lytic cyanophages will be obtained from cyanophage communities in the freshwater environment based on it is well established that cyanophage culture techniques. The efficiency of cyanophages infection and burst size will be determined, and the cyanophage will be isolated, purified and identified. The cyanophage genomic DNA samples preparation directly from a host cyanobacteria lysated, and using the method of capturing complete continuous genomes with high accuracy, an automated annotation informatics pipeline that delivers high-quality annotation through comparative genomic approaches. The relationships between the levels of transcripts and the levels of the proteins they encode will be examined comprehensively in the cyanophage isolate, the multiple genes will be tested using gene microarray and using a proteomic strategy combining high-resolving power and reproducibility of two-dimensional differential gel electrophoresis with specific immunological detection (or Western blotting) as well as polypeptide sequencing by high-resolution mass spectrometry. Analyzing this dataset enables us to further characterize of viral proteins that play important roles in the cyanophage attachment (or "adsorption"), entry and structure formation. We'll investigation the biologic consequences of the cyanophage-host interaction by replication of cyanophages in the host cyanobacteria cells using an in situ DNA amplification technique and/or fluorescence in situ hybridization (FISH), gene cloning and expression profiling by amplification of gene and proteins, by quantitative PCR (qPCR. For example, quantitative digital PCR, quantitative PCR microfluidics, quantitative PCR miniaturization) or mass spectrometry-based proteomics will be analyzed. We will strive to find the potential use of cyanophage-encoded genes and to develop new technologies for microbial contro of harmfur algal blooms.

噬藻体是感染蓝藻的病毒，为水环境中最丰富和最具形态多样性的生物类群之一，可导致蓝藻高死亡率，对宿主遗传进化有影响显著。为认识噬藻体的遗传多样性及其与宿主的相互作用，本项目拟对淡水水华蓝藻噬藻体分离培养鉴定、基因组测序与基因功能分析、转录组与蛋白质组比较、噬藻体与宿主蓝藻互作等进行研究。建立淡水水华噬藻体培养技术，筛选溶解水华蓝藻噬藻体毒株；测定毒株感染性及释放量，进行毒株分离纯化；制备毒株基因组DNA，用高精度完整连续基因组捕获方法获得高品质基因组注释；用基因芯片或蛋白质组策略测试噬藻体毒株的转录水平和蛋白水平。进一步鉴定对吸附、侵入及结构形成等起重要作用的噬藻体蛋白；借助原位DNA扩增技术和/或荧光原位杂交、定量PCR或基于蛋白质组谱，对噬藻体在宿主中的复制、基因表达和功能蛋白作综合分析，以了解噬藻体与宿主和水环境互作及其生物学意义。争取研发有应用前景的噬藻体基因产物和微生物控藻新技术

噬藻体(Cyanophage)是感染原核生物蓝藻的病毒。本项目围绕噬藻体基因组结构、分子遗传多样性及与淡水水华蓝藻的互作开展研究。测定了短尾噬藻体A-4L全基因组测序，这是感染模式蓝藻鱼腥藻PCC 7120（Anabaena sp. strain PCC 7120）的噬藻体全基因组的首次报道；还在分离鉴定特异裂解水华蓝藻铜绿微囊藻（Microcystic aeruginosa）的肌尾噬藻体MaMV-DC (Microcystis aeruginosa myovirus from Lake Dianchi)的基础上，剖析了MaMV-DC全基因组，并对其编码的5L (nblA) 基因特征和功能进行了研究。主要结果如下：.A-4L是一株能感染模式蓝藻鱼腥藻PCC 7120的短尾噬藻体，研究了A-4L 在鱼腥藻(Anabaena variabilis)藻苔中形成同心圆噬斑的成因。其感染的潜伏期为 0.5～2 h，释放量约为247 IU / cell ( Infectious Units)。在周期光照条件下，藻苔接种A-4L 3～4 d 后，出现同心圆噬斑，且同心圆数量与攻毒后的天数( n)有相关性，为“n-1”；同心圆间距约为3 mm。A-4L基因组为41750 bp的双链DNA，含有810 bp的长末端直接重复。基因组编码38个开放阅读框。A-4L与来自不同生态系统中的短尾噬藻体进行比较，显示A-4L的末端直接重复序列最长，其基因组结构与淡水短尾噬藻体相似，明显区别于海洋噬藻体。8个核心蛋白构建的串联进化树显示A-4L与淡水噬藻体聚一起，形成了一个独立于海洋噬藻体的进化分支，表明淡水生态系统中的短尾噬藻体与海洋生态系统中的短尾噬藻体之间已经存在明显的进化分歧。.MaMV-DC 基因组为末端循环冗余的169223 bp双链线性DNA，G+C含量约为46.3%。可编码170个预测的开放阅读框（ORFs）和一个转运RNA（tRNA）基因。其中150个在铜绿微囊藻噬藻体Ma-LMM01中存在同源蛋白，29个在铜绿微囊藻中存在同源蛋白。 MaMV-DC 5L基因编码的氨基酸序列与宿主蓝藻的藻胆体降解蛋白NblA高度同源。逆转录PCR与Western blotting检测的结果显示MaMV-DC NblA在噬藻体感染的晚期表达。NblA大量表达后宿主藻的藻蓝蛋白吸收峰被消弱，子