| Ajuga lobata is a kind of medicinal plant rich in sterone,alkaloids and other active substances.At present,ecdysone analogue in Ajuga Linn.is mainly applied in the research and development of insect growth regulator and pharmacy.Therefore,the study on ecdysone analogue from the plants of Ajuga Linn,has attracted the attentions of researchers.However,so far there are still bottleneck problems regarding industrial production of phytoecdysteroids on a large scale.In recent years,the researches focus on the extraction and analysis method of ecdysone,ecdysone source plant screening,and identification of new molting hormone analogues,but the research of the gene and protein levels is rarely reported.In this paper,the callus that can produce β-hydroxyecdysone(20E)was used as base material by cell suspension culture,and the influences of NO or H2S on P-hydroxyecdysone synthetic regulation function were analyzed;Then the A.lobata suspension cells induced by NO or H2S signaling molecules were used as material,and the Illumina technology was applied in the transcriptomics sequencing.Digital analysis of gene expression profile was carried out on the H2S,NO,and control samples,respectively;Finally,the transcriptomics and proteomics technology were applied to discuss the molecular response of A.lobata induced by NO or H2S from the differentially expressed genes and the differences in protein levels.The main results are as follows:1.The H2S and NO induced A.lobata high content β-hydroxyecdysone cell lines using cell suspension culture were screened and the results showed that within one training cycle,the dry weight of callus by root induction and β-hydroxyecdysone accumulation were significantly higher than those by leaf induced callus;Five generations of continuous culture of suspended cells showed steady growth in the fourth generation.When the cells were cultured for 6d,2mmol/L NaHS solution and 0.5mmol/L SNP solution were added for 12hr and it was found that both of H2S and NO can promote the synthesis of cells in the β-hydroxyecdysone.Either too long processing time,or too high or too low concentration had inhibition performance on the synthesis of β-hydroxyecdysone in cells.2.The Illumina high-throughput sequencing technologies were applied to A.lobata transcriptomics sequencing and de novo assembly,and functional annotation,classification,and analysis of metabolic pathways were carried out on the obtained Unigenes.The results indicated that N50 value was 934 bp and there were a total of 68,085 A.lobata Unigenes with an average length of 680 bp.Compared to the NCBI NR and Swiss-Prot database,it is found that 46,924 and 28,715 Unigenes are homologous to the genes of other species,respectively.The related 15,495 A.lobata Unigenes can be divided into 25 categories based on COG database.The KEGG database analysis indicated that a total of 26,618 Unigenes participated in 128 kinds of metabolic pathways,and 36 and 64 Unigenes were associated with phytoecdysteroids synthesis in MVA pathway and steride synthetic pathway,respectively.3.The Illumina high-throughput sequencing technologies were used in H2S and NO induced A.lobata cDNA library for the analysis of differences in gene expression profile.The results showed that the significantly differentially expressed genes induced by H2S(|log2Ratio| ≥2)amounted to 3,012,including 1311 upper expressed genes and 1,701 down expressed genes;The differentially expressed genes induced by NO amounted to 755,including 450 upper expressed genes and 305 down expressed genes.There were 181 H2S processed differentially expressed genes with express multiple of more than 10 times(|log2Ratio|>10),in which the number of upper expressed genes and down expressed genes were 140 and 41,respectively;There were 9 NO treated differentially expressed genes with express multiple of more than 10 times(|log2Ratio|>10),and all of them were were upper expressed.The functional annotation on the differentially expressed genes indicated that they were positioned on the membrane structure,performed molecular activity,catalytic activity,and oxidoreductase function,and were involved in biological processes,such as stress response to a stimulus;In addition,the phytoecdysteroids biosynthesis and metabolism,signal transduction,secondary metabolites synthesis of biological processes were corresponded to different number of differentially expressed genes,respectively.Pathway metabolic pathway analysis showed that the differentially expressed genes were extremely significantly commented to the metabolic pathway and biosynthesis of secondary metabolites in two ways.The phytoecdysteroids synthesis of steroid and terpenoid biosynthetic pathways were significantly enriched by the differentially expressed genes.4.The iTRAQ technology was used to appraise and analyze the differences of protein of H2S or NO induced A.lobata.The results showed that there were 364 differences in protein after H2S induced,including 179 upper expressed proteins and 185 down expressed proteins;There were 141 differences in protein after NO induced,including 64 upper expressed proteins and 77 down expressed proteins.The functional annotation on the differences in protein indicated that these proteins were mainly positioned in the middle part of the organelles,participating in catalytic and combination functions.They were involved in metabolism,cell process,stimulate response and other biological processes.Pathway analysis of the differences in protein involved in showed that they were significantly enriched in the metabolic pathway,the biosynthesis of secondary metabolites,benzene propane on the biosynthesis and metabolism of phenylalanine.These metabolic pathways were directly or indirectly involved in A.lobata metabolites synthesis and metabolic processes in the plant body.The transcriptomics and proteomics correlation analysis showed that H2S treatment or NO treatment had a good relevance with the contrast,and H2S treatment had poor correlation with NO treatment.A total of 51 function known and associated with transcriptomics differences proteins and genes after H2S induction were selected,including 8 upper expressed associations and 12 down expressed association;Similarly,A total of 11 function known and associated with transcriptomics differences proteins and genes after NO induction were selected,including 6 upper expressed associations and 2 down expressed association.The differences of these associated protein and genetic,variations were mainly involved in the biological metabolism,stimulate response,stress response,such as catalytic activity of the biological pathways. |
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