Study On JA Pathway Responding To Mowing In Zoysia Japonica

Using Zoysia japonica as experimental material,mechanical stress was imposed by pruning the leaves,the genes related to jasmonic acid（JA）synthesis and signal transduction pathway in Z.japonica were systematically explored by means of the whole genome JA related gene identification,then the differentially expressed ones under mechanical stress were selected according to transcriptome analysis.The genes expression patterns were verified by q RT-PCR to reflect the response characteristics of JA pathway in Z.japonica after pruning.By searching for cis-acting elements in response to hormones and identifying of functional transcriptional regulatory sites,the transcriptional factors induced by JA strongly were screened and their transcriptional regulatory target genes were enriched to construct a JA-mediated transcriptional regulatory network.The level of JA under the balance of JA synthesis and signal transduction was detected by JA concentration testing.And the biological function of JA about hydrogen peroxide metabolism after pruning in the physiological layer was verified by biochemical tests of hydrogen peroxide content and peroxidase activity.The main conclusions are listed as follows:●Identification of genes related to JA synthesis and corresponding response monitoring after pruning:1)Referring to the research progress of JA-related functional genes in Arabidopsis thaliana,15At LOX homologous genes,9 At AOS homologous genes,2 At AOC homologous genes,10 At OPR homologous genes,4 At JAR1 homologous genes and 14 At JMT homologous genes were obtained through homologous gene identification within the whole genome of Z.japonica,which collected the main regulatory genes of JA synthesis in Z.japonica.2)Based on reciprocal best hits,the Unigenes of leaf transcriptome under pruning were matched with genes identified above,and the differentially expressed ones were selected for q RT-PCR detection.The results of 5 Zj LOX genes,3 Zj AOS genes,1 Zj AOS gene and 4 Zj AOS genes in JA precursor synthesis showed that there were two types of JA synthesis routes in Z.japonica:rapidly responding one and gradually up-regulated one.In the formation stage of active JA molecule,the expression of a Zj JMT gene was up-regulated sharply in a very short time after pruning,and the synthesis was dominated by a Zj JAR1gene at 2 h after pruning.●Identification of genes related to JA signal transduction and corresponding response monitoring after pruning:3)Based on annotation information in A.thaliana,eight Zj COI1 and eight Zj JAZ were identified by multiple sequence alignment into Z.japonica genome,two genes were selected for q RT-RCR detection,it was found that Zj COI1 gene could be up-regulated by tens of times to tens of thousands of times within2 h after pruning,while Zj JAZ could convert JA signals into two types of downstream signals with positive or negative effects.●Selection of transcription factors that responded to JA signals strongly and the construction of functional networks of target genes:4)64 differentially expressed transcription factors（TF）were classified according to Plant TFDB,49of the above were found possessing cis-acting elements responding to JA signals by Plant CARE.JA and ABA had cross-induction effects on numerous TFs,but the individuals who strongly transduced JA signals had more advantages in quantity and intensity.5)JA concentration gradient test confirmed an oxidative response TF responding to JA signal strongly.Its target genes were obtained by Fun TFBS analysis.Cell component enrichment analysis showed that their expression products were relatively concentrated in chloroplast,peroxisome and cell wall,while biological process enrichment analysis showed that their efficacy revolved around the theme of response to stimuli and was mainly involved in redox metabolism in molecular function.6)Based on the results of gene enrichment,some target genes were picked to construct the functional networks,and it was proved by q RT-PCR and the biochemical metabolism testing of hydrogen peroxide,which suggested that JA could positively regulate the synthesis of lignin precursors,the accumulation of H₂O₂ needed for cross-linking,and the production of laccase and peroxidase catalyzing redox reaction.While,JA could negatively regulate the production of ascorbate peroxidase as an intermediate electron donor for competitive reduction of H₂O₂.7)The q RT-PCR results show that JA synthesis regulatory genes such as Zj LOX8,Zj OPCL and Zj AOX will be regarded as the JA signal targets forming positive feedback regulation to accelerate JA synthesis,which could stimulate the lignin precursor synthesis and the establishment of redox reaction environment above,so that the cell wall of Z.japonica can be repaired quickly after pruning.