| Transgenic technology has made up for the shortcomings of the traditional tree breeding methods,such as long cycle,tedious process and difficult artificial control of traits.Therefore,a large number of transgenic or genetically modified(GM)tree varieties with enhanced characteristics and new traits were produced.The innovation of forest transgenic technology is accelerating day by day,and remarkable achievements have been made in insect resistance,herbicide resistance,disease resistance,abiotic stress resistance,lignin content reduction and other aspects,which greatly promotes the production and development of forestry and shows great economic benefits.However,the improvement effect,safety and mechanism of exogenous genes of transgenic plants have attracted much attention from scholars all over the world.At present,studies on the effects of transgenic plants on rhizosphere microorganisms and endophytes are mainly focused on annual crops and herbaceous plants,while few studies on trees with a long growth cycle have been conducted.In this study,transgenic Populus alba ×P.berolinensis(exogenous gene JERF36,encoding AP2/EREBP plant transcription factor,related to plant stress resistance)and non-transgenic receptor were used as experimental materials.The diversity of endophytic bacteria and fungi in transgenic poplar and its non-transgenic receptors grown in saline(Daqing)and non-saline(Qiqihar)areas were evaluated by using 16 S r RNA and internal transcribed spacer(ITS)amplicon Illumina Mi Seq sequencing technology.Effects of exogenous genes and environmental conditions on poplar gene expression and genome-wide DNA methylation patterns were studied by transcriptome and whole genome bisulfite sequencing(WGBS).The main conclusions are as follows:1.Transgenic events did not affect the endophytic bacterial and fungal diversity of poplar trees.The bacterial and fungal community structure depends on the p H and the soil organic matter content.The community structure of endophytic bacteria in stem remained stable even under different environmental conditions.Each plant tissue represents a unique ecological niche for the microbial communities.2.According to the 97% sequence similarity cut-off level,the reads of bacteria and fungi were clustered into 1493 and 321 OTUs,respectively.We found that actinobacteria,proteobacteria,bacteroidetes,and firmicutes were the dominant endophytic bacteria,and the fungal community was dominated by dothideomycetes,agaricomycetes,leotiomycetes,and sordariomycetes.Core microbiome associated with the different plant tissues of Poplar and different environmental conditions were determined,bacteria and fungi belonged to 37 OTUs representing 6 phyla and 36 OTUs representing 11 classes,respectively.Meanwhile,14 root endophytic bacteria,5 stem endophytic bacteria,9 root endophytic fungi and 2 stem endophytic fungi were identified to indicate OTU.It provides a basis for further study of host-microbe interaction.3.Transgenic poplar has the ability to enhance biological and non-biological tolerance.Among the differentially expressed genes between transgenic and non-transgenic poplar,30-50% of DEGs contained the regulatory element of exogenous gene JERF36.The up-regulated genes were related to the metabolic pathway of photosynthesis and the resistance to drought stress.A total of 25 DEGs were obtained between transgenic and non-transgenic poplar,and 6 of them were up-regulated at two locations simultaneously,which were annotated into ESTs of drought stress poplar leaves and two key proteins Psb K and Psb I in photocooperative photosystem II.A total of 426 DEGs were obtained between poplars from Daqing and Qiqihar.The upregulated genes in Daqing enhanced poplar's defense against fungal pathogens,while the upregulated genes in Qiqihar enhanced poplar's defense against bacterial pathogens.The DEGs caused by the interaction between genes and environment were mainly involved in the metabolic pathways of poplar such as resistance to drought stress and photosynthesis.4.CG methylation was the main methylation form in the genome of Populus alba ×P.berolinensis,and the methylation level was 31.10%.CG methylation level of each functional element area was the highest,and the variation range was large.The methylation level was lower in the upstream region of promoter,while higher in gene body.CHG and CHH methylation levels were lower and stable.It is most likely to be methylated when the sequence is CAG and CAA at CHG and CHH locus,respectively.The number of DMRs at CHH locus was the most,and fewer at CG and CHG locus.The length of DMRs was mainly concentrated in 10-100 bp,and the longer the DMRs,the lower the frequency.The DMRs was evenly distributed on the chromosomes,and there was no chromosomal bias.5.DMRs were annotated,and the number of differentially methylated genes(DMGs)caused by environmental factors was greater than those caused by transgene.Functional enrichment analysis of DMGs showed that they were mainly concentrated in secondary metabolite synthesis,antibiotic synthesis,RNA transport and microbial metabolism in different environments.6.Transgenic and non-transgenic poplar gene expression in different ecological environments was not significantly regulated by methylation.The correlation between methylation and gene expression was analyzed,and we found that there were positive and negative correlations between gene methylation level and expression level,but none of them reached the significant level. |
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