Analysis Of Leaf Color,Rhizosphere Soil Enzyme Activity And Microbial Community Composition Of Transgenic PaGLK Populus Alba × P.berolinensis

GLK is a class of transcription factors widely found in plants,which plays a key role in regulating chloroplast development and affecting plant fruit quality and leaf color.The PaGLKtransformed P.alba × P.berlinensis has a bright yellow-green leaf color at seedling stage,which has a high ornamental value and is promising for landscaping.In order to promote the industrialization and application of PaGLK transgenic P.alba × P.berlinensis,we used the obtained PaGLK overexpression P.alba × P.berlinensis(OE)and PaGLK repression P.alba ×P.berlinensis(RE)as research materials to carry out growth,leaf color analysis,rhizosphere secretion,rhizosphere soil enzyme activity and microbial community composition,so as to provide reference for the environmental and ecological safety evaluation and industrialization of transgenic P.alba × P.berlinensis.The results are as follows:(1)Molecular detection of 2-year-old trans-PaGLK P.alba × P.berlinensis by PCR and qRT-PCR showed that the introduced target genes and Bar resistance genes were still integrated in the genome of the transgenic lines and could be stably expressed.(2)Determination of relative chlorophyll content and leaf color parameters of transgenic P.alba × P.berlinensis showed that the chlorophyll content of the OE1 line was significantly higher than that of the WT lines,while the brightness and yellow color of the leaves were significantly lower than the WT lines;the RE lines showed a pattern of significantly lower chlorophyll content and higher brightness and yellow color than the WT lines.Analysis of the growth showed that the seedling height of the 2-year-old and 3-year-old transgenic P.alba × P.berlinensis overexpression lines OE was significantly lower than the WT lines,while the seedling height of the 2-year-old RE3 line and the 3 RE lines at 3 years of age were significantly lower than the WT lines.(3)Differential gene analysis based on transcriptome sequencing of OE,RE and WT lines,compared with WT lines,the up-regulated genes of RE lines were mainly related genes such as response to stimuli,response to abiotic stimulus,and response to oxygen-containing compound.Down-regulated genes are mainly photosynthesis,photosynthesis,light reaction,photosynthetic electron transport chain and other related genes;the up-regulated genes in OE lines were mainly in cell process,cell cycle,mitotic cell cycle and other processes.Downregulated genes are mainly involved in protein phosphorylation,transmembrane transport,and response to stimulus.(4)Root exudates of OE,RE and WT lines were analyzed by LC-MS non-targeted metabolomics,showed that 614 differential metabolites were screened and identified.28 differential metabolites were common to the RE＿WT,OE＿WT and RE＿OE3 comparison groups,of which 5 differential metabolites were significantly up-regulated in the RE lines and significantly down-regulated in the OE lines.23 differential metabolites were identified,of which 2 were significantly up-regulated and 21 were significantly down-regulated in the OE lines.The other 23 differential metabolites,two of which were significantly up-regulated and21 were significantly down-regulated in the PaGLK P.alba × P.berlinensis lines.Compared to the WT lines,(S)-ACPA was significantly higher in the RE lines,followed by geniposidic acid,while(S)-ACPA,geniposidic acid and pyranocoumarins were significantly lower in the OE lines.(5)Soil enzyme activities of OE,RE and WT lines were determined by spectrophotometry.the differences between transgenic lines and WT lines were not the same at different growth stages.In mid-September,the activity of catalase and cellulase in rhizosphere of RE lines was higher than that of WT lines.The activity of neutral protease in rhizosphere of OE lines was higher than that of WT lines,and the other four enzyme activities were lower or significantly lower than that of WT lines.(6)The rhizosphere soils of OE,RE and WT lines were used to analyze the differences in bacterial and fungal community composition using high-throughput sequencing technology.The rhizosphere soil bacterial community composition of the tested strains was 971 genera at the genus level.Burkholderia was the dominant group of rhizosphere soil bacterial community of P.alba × P.berlinensis(except OE1).The relative abundance of Burkholderia in RE lines was significantly higher than that in WT lines,while the relative abundance of OE lines was significantly lower than the WT lines.There were 466 genera in the fungal community structure at the genus level.Tomentella was the dominant group in the rhizosphere fungal community of P.alba × P.berlinensis.The relative abundance of Tomentella in the rhizosphere of WT lines was the highest.In addition,the relative abundance of Sphaerosporella in the rhizosphere of OE lines increased significantly,while the relative abundance of Serendipita in the rhizosphere of RE lines was significantly higher than the WT and OE lines.In conclusion,the exogenous gene and target gene in transgenic PaGLK transgenic P.alba× P.berlinensis were still stable.Transgenic activity had a certain effect on rhizosphere soil enzyme activity and microbial composition,but no adverse effect was found.