西南印度洋中脊热液区真菌群落生态分布及其反硝化作用能力研究

Since the discovery of hydrothermal vent systems, microbial community structure and its ecological function in hydrothermal ecosystems are one of hotspots of deep-sea scientific researches. Denitrification is an important process in the global nitrogen cycle, which has been generally considered a prokaryotic process for more than a century and has been extensively studied in bacteria, but fungi were reported to exhibit denitrifying activity recently. However, few studies focused on the ecological distribution and denitrification ability of fungal community in hydrothermal vent ecosystems. In this proposal, hydrothermal samples of the Southwest Indian Ridge (SWIR) were used as materials to investigate the diversity and distribution of deep-sea fungi and their denitrification ability. First, we will investigate the fungal diversity and abundance of hydrothermal samples at the SWIR by traditional isolation and culture method, high-throughput sequencing and quantitative real-time PCR approaches. Then, the diversity and community structure of fungal denitrifiers will be studied by direct cloning and sequencing of genes encoding to nitrite reductase (nirK), the key functional enzyme involved in the denitrifiction process. Finally, identification and characterization of N2O-producing fungi in hydrothermal vent ecosystems will be conducted to investigate the dominant genera of N2O-producing fungi in this community. The results of this work not only extend our knowledge of fungal diversity and their great biotechnological potentiality in SWIR hydrothermal vents, but also provide important insight on diversity, ecology, and physiology of fungal denitrifiers in SWIR hydrothermal vent ecosystems, which will advance the knowledge about the function of fungi in the nitrogen cycle of marine ecosystems.

深海热液区及其栖息的微生物群落结构和生态功能自发现以来便是科学界研究的热点。反硝化作用是氮循环中的一个重要组成部分，之前人们普遍认为它是由原核生物主导，后续研究表明真菌同样具有反硝化能力。鉴于目前国内外对热液环境中深海真菌群落生态分布及其参与的反硝化作用了解较少，本项目拟以西南印度洋洋中脊热液样品为研究对象。先运用分离培养方法、高通量测序和荧光定量PCR等技术来阐明该环境中深海真菌的多样性和丰度；再基于真菌的nirK基因来分析热液环境中反硝化真菌的免培养多样性；最后，对纯培养菌株通过反硝化作用产生N2O的能力进行分析，获得反硝化真菌的纯培养多样性及其优势种属。通过本项目的实施，不仅可以全面了解西南印度洋洋中脊热液区真菌资源储备情况及其开发潜力，而且还可获得该环境中的反硝化真菌多样性及其反硝化能力的数据，为进一步分析深海真菌在海洋氮循环中的作用提供良好的数据支持和研究基础。

深海环境热液区及其栖息的微生物群落结构及其所参与的生物地球化学循环中一直以来都是深海科学研究的热点。反硝化作用是氮循环中的一个重要组成部分，之前人们普遍认为它是由原核生物主导，后续研究表明真菌同样具有反硝化能力。鉴于目前国内外对热液环境中深海真菌群落生态分布及其参与的反硝化作用了解较少，本项目以西南印度洋洋中脊热液样品和南大西洋一处热液中脊梁的硫氧化物为研究对象。运用分离培养方法、克隆建库、高通量测序和荧光定量PCR等技术发现热液环境中深海真菌主要以子囊菌门和担子菌门为主。使用6种培养基从中一共分离获得138株真菌，归属于9 个属（14个种）：支顶孢属Acremonium，链格孢属Alternaria，枝孢属Cladosporium，青霉属Penicillium，Cyphellophora，拟茎点霉属Phomopsis，Zasmidium，红酵母属Rhodotorula和隐球菌属Cryptococcus。以 ITS2基因为分子标记，通过高通量测序获得到有效序列 757,467 条。以 97%相似度水平划分 OTU，共得到 723个 OTUs，其中652 个OTUs属于子囊菌门Ascomycota, 35个OTUs属于担子菌门Basidiomycota, 剩下来的36 个OTUs属于unknown fungi。进一步使用qPCR对ITS2基因分析发现，这些样品中的丰度约在1.93±0.32×104 到2.10±0.07×106 copies/g sample(湿重)。此外基于真菌的nirK基因和P450nor基因克隆建库的方法来分析热液环境中反硝化真菌的免培养多样性，发现主要以曲霉属，青霉属和枝孢属为主；最后，对纯培养菌株通过反硝化作用产生N2O的能力进行分析，获得反硝化真菌的纯培养多样性及其优势种属也大多为曲霉属，枝孢属和青霉属。通过本项目的研究，我们获得了深海热液区真菌资源多样性，而且还获得该环境中的反硝化真菌多样性及其反硝化能力的数据，为进一步分析深海真菌在海洋氮循环中的作用提供良好的数据支持和研究基础。