| Selenium is an important trace element,which is present in animals,plants and microorganisms and is an active ingredient of glutathione peroxidase in animals and humans,and has anti-oxidation,anti-aging effects and enhances human immunity.However,selenium supplementation should not be excessive,because excessive intake of selenium can lead to poisoning,hair loss,disloeation and other phenomena.At present,the research on selenium mainly focuses on the distribution of selenium,selenium-enriched cultivation,the form of selenium in plants,and the effects of selenium on plant physiology and biochemistry.However,there are few studies on the molecular mechanism of selenium in plants.Pueraria lobata(willd.)Ohwi,a perennial woody vine of the genus Leguminosae,is a large and important Chinese medicinal material.It was recognized as a dual-use Plant by the Ministry of Health in 2002,also a selenium-enriched plant.P.lobata is used as a dry root to be used as a genus of puerarin.It has the functions of improving brain memory,improving and treating cardiovascular and cerebrovascular diseases,and its main biological active ingredient is isoflavones.In this study,the seedlings ofP.lobata were treated by using different concentrations of sodium selenite,and then the selenium content,and some physiological indexes of the plants were determined,and the optimum concentration of sodium selenite was screened.Then,the transcriptome was used to study the differential expression of the geranium functional gene under the action of sodium selenite.The signaling pathway and expression regulation mode of the differentially expressed genes were identified,and the fianctional genes related to selenium metabolism and isoflavone metabolism were found according to the annotation information.Finally,small RNA sequencing technology was used to reveal the differential expression of genomic DNA and its target genes under the action of sodium selenite,revealing the complexity of differentially expressed microRNA(miRNA)target gene responses in radicle,and providing insight into the miRNAs that respond to selenium stimulation in Gesong root.Thus,the molecular mechanism of Ge's root response to selenium stimulation was revealed,which provided a theoretical basis for the in-depth study of selenium-rich kudzu.Theoretical basis.The main results are as follows:1.The seedlings of.lobata were treated with different concentrations of sodium selenite,and then the selenium content in P.lobata was determined.The content of selenium in leaves and stems of Rlobata seedlings was determined with the increase of sodium selenite treatment concentration.It is expressed as a mode of increasing first and then decreasing,and the content of selenium in Rlobata reaches a maximum at a concentration of 25 mg/L.2.According to the above results,six different concentrations of sodium selenite were used to treat the P.lobata seedlings,and then the root length,soluble sugar and malondialdehyde content,superoxide dismutase,and the above results were measured.It is clear that 25 mg/L is a suitable treatment concentration for sodium selenite.3.Treat the Rlobata seedlings with 25 mg/L sodium selenite,set up three biological replicates,collect the geranium roots on days 0,1,3,5,7,and 9 after treatment,and extract RNA using the modified Trizol method.The transcriptome sequencing of the above samples was performed using the second-generation sequencing Illumina HiSeq X ten platform,and 18 files with a number of bases greater than 6,200,000,000 were obtained.After Trimmomatic software quality control,323,717 transcripts were obtained by Trinity software splicing.After clustering and redundancy,the longest sequence was retained,and 150,567 Unigenes with an average length of 624.31 were obtained.The N50 was 1,135 bp and the average GC content was more than 46%..Functional annotation of the sequenced assembled Unigene sequence revealed that 59,266,64,759,5,416,58,101,33,401,31,796 genes were respectively NR,GO,KEGG,Swissprot,KOG,and Pfam were annotated.In the KEGG database,a total of 9,746 genes were identified in 215 pathways in 23 secondary catalogues,including 29 selenium metabolism-related genes and 41 isoflavones and their upstream synthetic pathway genes.A total of 4,246 differentially expressed genes were obtained using 0 days as a control group.Among these genes,we found that 19 genes were differentially expressed in the selenium metabolic pathway,while 9 genes were differentially expressed in the isoflavones and their upstream pathways,and these 9 genes were up-regulated.In this study,many transcription factors were found to be co-expressed with selenium metabolic pathways and isoflavone metabolic pathway genes with |R|>0.75.we fuond 301 transcription factors were associated with selenium metabolism genes,and 341 transcription factors were related to isoflavone metabolism genes.4.Small RNA sequencing was performed using Illumina HiSeq X ten platform In total,88 conserved miRNAs were identified using psRobot software which belong to 26 miRNA families and 166 non-conserved miRNAs were obtained,which could be classfied to 118 miRNA families.A total of 2541 target genes were predicted for these miRNAs with a predicted value of less than 3.0.Functional enrichment of target genes for differentially expressing miRNA revealed that the top five in GO terms were "integral component of membrane","nucleus","ATP binding","transcription","DNA-templated",and "plasma".The KEGG enrichment analysis showed that the target genes of 125 differentially expressed miRNAs were enriched into 74 metabolic pathways,of which 69,23,20,and 13 target genes were enriched in"Metabolism","Genetic Information Processing","Environmental Information Processing,,and "Cellular Processes",respectively.It indicated that the differential expression of miRNA target gene response in juvenile roots was complicated after treatment with sodium selenite. |
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