Diversity And Transcription Characteristics Of Root-associated Methanotrophs And Nitrogen-fixing Bacteria Of Emergent Plants In Wuliangsuhai Wetland

Emergent plants are active components in wetland ecosystems and have a profound impact on the ecological and environmental functions.Such plants are connected to the three-phase interface of water,gas and solid.As a link between the sediment and the atmosphere,the well-developed aeration tissue inside the emergent plants can mediate the oxygen and other gases in the atmosphere into the sediment,and promote the methane and other greenhouse gases produced in the sediment to diffuse into the atmosphere.In this process,root-associated methanotrophs will directly affect methane emission.However,root-associated methanotrophs play an important role in reducing methane emission in wetlands.At present,the research on root-associated methanotrophs of emergent plants in natural wetland mainly focuses on the functional diversity at DNA level,there are few reports on the function at the transcription level and the coupling of carbon and nitrogen.In this study,three emergent plants including Phragmites australis,Typha angustifolia and Schoenoplectus triqueter,which are widely distributed in Wuliangsuhai wetland were taken as the research object.The RNA(or reverse to cDNA)which was extracted from the roots of three emergent plants were used to study the community structure,diversity and the abundance of dominant bacteria of aerobic methanotrophs and nitrogen-fixing bacteria by the methods of the cloning library,high-throughput sequencing and metatranscriptomic sequencing.The conclusion is as follows:(1)High-throughput sequencing of 16 S rRNA gene based on three plant root DNA showed that although the proportion of methanotrophs and nitrogen-fixing bacteria in the total bacterial community of the whole root system was not high(0.3-0.8% and 0.22-1.85%),they were found in all three plant roots,which provides a premise for our subsequent study of the transcription activity of functional flora.(2)High-throughput sequencing of pmoA based on DNA and cDNA was used to analyze the methanotrophic community.Sequencing of cDNA pmoA amplicons confirmed that the structure of active methanotrophic was not always consistent with DNA.A type I methanotroph,Methylomonas,was the most active group in P.australis,whereas Methylocystis,a type II methanotroph,was the dominant group in S.triqueter.In T.angustifolia,these two types of methanotroph existed in similar proportions.However,at the DNA level,Methylomonas was predominant in the roots of all three plants.(3)Sequencing of a cDNA clone library based on nifH,a functional gene encoding a nitrogen-fixing reductase,showed that the composition of the root nitrogen-fixing bacterial community of the three plants differed.P.australis and S.triqueter were dominated by the genus Methylosinus,while Typha angustifolia were dominated by Rhizobium.In addition,in order to understand the physiological function of root-associated denitrifying bacteria,the denitrifying related functional genes(nirS and nirK)were amplified by PCR.The results showed that the nir S gene was not amplified in the roots of three plants,indicating that its transcription level was very low.Nir K gene was not amplified in the roots of Phragmites australis,but there were weak bands in Typha angustifolia and Schoenoplectus triqueter.According to the sequencing results of nir K gene,the proportion of unclassified denitrifying bacteria and Rhizobium was higher.(4)The analysis of ? diversity showed that the three plants root-associated bacterial communities,methanotrophs and nitrogen-fixation functional flora were clustered into three clusters according to the plant type both at the level of DNA and cDNA.It showed obvious host dependence.Different types of emergent plants affected the species and distribution of bacteria(including functional flora)on their roots by affecting their root microhabitat.(5)The results of pmoA and nifH qPCR showed that there was no significant difference in the copy numbers of the two genes between the three plants at the transcription level,but in comparison,the copy numbers of pmoA and nifH in the roots of Phragmites australis were higher than the other two.(6)To understand the functional transcriptional properties of the carbon and nitrogen cycle in roots,a new approach was attempted: macrotranscriptome sequencing was performed after removal of plant mRNA from total RNA in roots of Phragmites australis.The taxa abundance of Methylosinus and Methylomonas accounted for 7.8% and 5.3% in macrotranscriptome sequencing,respectively,which is much higher than the relative abundance(<1%)from sequencing based on 16 S rRNA gene amplicons at the DNA level.In addition,they accounted for 80% of the expression of nitrogen fixing genes(nifH/D/K),and belonging to the dominant nitrogen fixing bacteria.Moreover,transcription of the functional gene mcrA/B of the methanogenic archaea Methanospirillum was detected.Therefore,root-associated methanotrophs in Phragmites australis may use the methane produced by methanogens as carbon source to oxidize and complete nitrogen fixation,so as to provide more nitrogen nutrition for the survival of host plants.In this study,the physiological activities of carbon and nitrogen cycling functional flora(methane oxidation,nitrogen fixation,etc.)in the roots of emergent plants in natural wetlands were studied at the transcription level by using a variety of sequencing techniques,and found that the root-associated methanotrophs of three emergent plants in the natural wetland had nitrogen fixation activity,which was the first time to find that methanogens play an important role in methane oxidation and nitrogen fixation in the roots of wild emergent plants in natural wetlands after the report of the nitrogen-fixation methanotrophs of the rice roots in the artificial wetland.At the same time,the root-associated methanotrophs with nitrogen fixing activity were detected in all these three plants involved in this study,which indicated that they existed widely of the emergent plants' roots.These functional flora on the microhabitat of plant roots form an interdependent relationship with the plant host,which not only ensures their own growth and reproduction,but also promotes the growth of plants.In addition,denitrification functional groups with physiological activities were detected in the roots of Typha angustifolia and Schoenoplectus triqueter.These results provided microbiological data for the reduction of greenhouse gas emission and eutrophication in wetlands by using root functional flora.It was by metatranscriptomic sequencing that made it possible to study some functions of root-accociated bacteria from the whole metabolic pathway,and it had opened up a new research direction for comprehensively understanding the functional flora of root system and discovering the coupling relationship between the functions.