| Aquilaria sinensis(Lour.)Spreng.is the main original species of Aquilaria medicinal materials.The research on artificial fragrance technology and the elucidation of fragrance mechanism have attracted much attention.Studies have shown that NaCl can effectively induce the production of fragrance substances.In this process,the types and structures of 2(2-phenylethyl)chromones(PECs)and sesquiterpenes have been discussed.The mechanism of stress-induced aroma formation is relatively insufficient.If multiple "generations" of salt stress can be used to establish a material system with more efficient accumulation of aroma components,it will greatly promote the speed and depth of the research on aroma mechanism.1.ObjectiveBased on the previous research of the research group,it is shown that the salt stress stimulation of three consecutive generations of the isolated roots of A.sinensis can induce a stronger response to the stress marker proline.This study aims to explore whether the PECs material response in the third generation of salt stress has an increasingly stronger trend;and to explore the characteristic information of the third generation of salt stress samples from the three omics levels of metabolites,transcriptome and miRNA,in order to continue to establish or optimize PECs substances provide data for the index’s stress memory system.2.Methods2.1 Material cultivation and salt stress system establishmentAccording to the research methods and results of the research group in the early stage,the continuous culture and salt stress process of the in vitro roots of A.sinensis were repeated,and the continuously processed in vitro root samples were collected as experimental materials for subsequent omics tests.2.2 Analysis of the metabolome of the in vitro roots of A.sinensis for three consecutive generations of salt stressThe third-generation samples were extracted with 70%methanol and detected by UPLC-MS/MS platform.And based on the data of the self-built database of Wuhan Metroville Biotechnology Co.,Ltd.(referred to as Maiwei Metabolism)to identify metabolites and obtain metabolite related data.On the basis of comprehensive analysis of metabolite types and relative content,fold change≥ 2 and fold change≤0.5,and VIP(OPLSDA model variable importance projection)≥1 were used as screening conditions to obtain significant differences in metabolites between groups.Relevant data obtained for subsequent analysis.2.3 Analysis of the transcriptome of the in vitro root of A.sinensis for three consecutive generations of salt stressThe total RNA was extracted with EASYspin Plus Plant Rapid Extraction Kit.After constructing the library,the HiSeq2500 platform was used to obtain sequence information,and the transcriptome data was obtained after quality control and comparison of the data.On the basis of comprehensive analysis of gene expression and functional comparison annotations,FDR<0.05 and |log2FC|>1 were used as screening conditions to obtain significantly different genes between groups,focusing on the pathways related to plant salt stress defense and some secondary metabolite organisms Synthetic pathway.2.4 Analysis of miRNAs in vitro root of A.sinensis for three consecutive generations of salt stressUse EASYspin Plus Plant Rapid Extraction Kit to extract total RNA,use agarose gel electrophoresis to cut the gel to select 18-30nt fragments,build the library and use the HiSeq2500 platform to detect miRNAs.After data quality control and comparison,three consecutive generations of salt stress are obtained miRNA data of the isolated roots of Aquilaria sinensis.On the basis of comprehensive analysis of miRNA expression level,P<0.05 and |log2FC|>1 were used as screening conditions to obtain significant differences in miRNAs between groups.The miRNA-mRNA structure is complementary and the expression trend is negatively correlated as the conditions for screening target genes.2.5 Transcriptome analysis of the in vitro root of A.sinensis for three consecutive generations of salt stressUsing the method of KEGG annotation,the method of comparing miRNA and target gene and the method of Pearson coefficient,the three types of data of metabolome,transcriptome and miRNA are associated to obtain relevant pathway information.3.Results3.1 The metabolome analysis of salt stress for three consecutive generations of isolated rootsThe HPLC-MS platform was used to detect the metabolites and content changes of the samples of each generation after three successive generations of salt stress in the isolated roots of A.sinensis.A total of 722 metabolites were detected and identified,and 152 differential metabolites were screened.The results showed that three consecutive generations of the isolated roots of A.sinensis had a greater impact on the metabolism of amino acids and their derivatives,flavonoids,phenolic acids,organic acids and PECs;there were 18 significant changes in PECs,and 17 of them were classified as first ascending.One of the trends that remain unchanged after high is attributed to a continuous upward trend;there is a strong correlation between the internal substances of PECs;the addition of PECs is negatively correlated with organic acids and PECs,and is related to phenolic acids,amino acids and their derivatives,and flavonoids There is a positive correlation with others;the correlation analysis between PECs,the parent nucleus of PECs and various substances,obtains 25 significant related substances,and most of them have known pathway information.3.2 Analysis of the transcriptome of the in vitro root of A.sinensis for three consecutive generations of salt stressThe HiSeq2500 platform was used to sequence the transcriptome of the isolated roots of A.sinensis after three successive generations of salt stress.A total of 30,923 genes were obtained through parameter analysis,5462 differential genes were obtained after differential analysis,and 8 expression trends of these differential genes were further obtained.Pathway analysis showed that plant hormone signal transduction pathways,starch and sucrose metabolism pathways,energy metabolism pathways,and peroxidase have more differential genes after three consecutive generations of salt stress,which may be the main pathways for multi-generation salt stress defense.The analysis results of plant hormone signal transduction pathway,starch and sucrose metabolism pathway,energy metabolism pathway and antioxidant pathway show that two consecutive generations of salt stress may help to improve the salt tolerance of the isolated roots of A.sinensis The third generation of stress latter part of the metabolic pathway may be disordered.The analysis of the expression trend of CHS genes that may be involved in the biosynthesis of PECs showed that the expression trend of these genes was different from that of PECs,and the significant change trend was the first unchanged and then up-regulated;the structure analysis showed that this type of genes has relatively high High species specificity and structural conservation.3.3 Analysis of salt-stressed miRNA for three consecutive generations of isolated roots of A.sinensisUsing the HiSeq2500 platform,miRNA sequencing was performed on the isolated roots of A.sinensis after three consecutive generations of salt stress.A total of 585 differential miRNAs were obtained,of which 72 unknown miRNAs were found.Analysis of miRNA length showed that 21nt and 24nt miRNAs occupies a large proportion in the body of A.woody,of which 24nt is more.GO analysis showed that multi-generation salt-stressed miRNA target genes are mostly involved in plant signal transduction,biotic and abiotic stress,metabolic processes and oxidative stress processes.The results of miRNA analysis and transcriptome data are more consistent.3.4 Multi-omics integrated analysis of the in vitro root of A.sinensis for three consecutive generations of salt stressIntegrate metabolome and transcriptome,metabolome and miRNA target genes for joint analysis.The results show that the main enrichment pathways of significantly different metabolites include the aromatic amino acid metabolism pathway,the metabolism of cysteine and methionine,and the metabolism of glutathione.4 ConclusionThis study preliminarily reveals the three levels of omics information on the metabolites,gene expression and miRNAs involved in gene expression regulation of the in vitro root metabolites of A.sinensis under salt stress conditions for three consecutive generations,in order to in-depth elucidate the salt stress and the signature aroma substances of A.sinensis.The relationship between metabolism provides a large amount of data and ideas for the subsequent establishment of a stress memory system with PECs as indicators. |
PDF Full Text Request