Identification And Functional Analysis Of Stress Responsive Genes Involved In Jasmonic Acid Synthesis Of Peanut

The peanut is one of the most important oil crop and cash crop worldwides.With the global climate change,the rainfall capacity in the peanut planting area in China was decreasing,and the area of arid,semi-arid and saline land has been enlarged.Drought and salt stress has a serious impact on peanut quality and yield.Jasmonic acid(JA)is a kind of novel plant hormone.Studies have indicated that JA played an important role in response to stresses such as drought,salinity,high temperature,diseases and insect pests.The peanut transcriptome sequencing under drought and salt stress was performed to analysis the abundance differentially expressed genes(DEG).The genes responded to both stresses were identified,and genes belonged to the Jasmonic acid(JA)synthesis pathway(α-linolenic acid metabolism)were significantly upregulated.Then analyse the dynamic changes of genes involved in the JA biosynthetic pathway,and two key genes were screened from the peanut genome database.Finally,ues the plant transgenic technology and VIGS technology,the specific function of AhAOC9 and AhAOS9 were verified.The main results are as follows:(1)Peanut transcriptome analysis under drought and salt stress.Data from transcriptome sequencing under salt and drought stress were analyzed,and 177 up-regulated DEGs and 108 down-regulated DEGs under two stresses were obtained.GO and KEGG enrichment analysis results showed that DEGs was main classified into the pathway of "starch and sucrose metabolism","phenylpropane biosynthesis" and “α-linolenic acid metabolism”.Five genes involve in Jasmonic acid synthesis,LOX gene(Araip.849ER),AOS gene(Aradu.1BH3V),AOC gene(Araip.673NH),OPR gene(Araip.17941)and JMT gene(Araip.I4CFI),were significantly up-regulated within the DEGs under the drought and salt stress(2)Optimization of JA content determination method and distribution of JA under normal or stress condition.In order to reveal the relationship between Jasmonic acid content and resistance to stress of peanut,a method for determination of peanut Jasmonic acid content with high separation efficiency,high sensitivity and good stability was established in this study.The detection range was 5~1000 ng/g.The LOD is 0.50 ng/g.Then,JA content under normal condition was determined,it showed thatstem tissue have the highest level of JA content,followed byleaf and root tissues,and the podding stage was the most sensitive to drought stress,followed by flowering and seedling stage.Under the drought stress,the JA content kept the same trends in different tissues,JA content had a reduction after reaching the paek value within 4-6 h,and increased with the extra time,and JA content in seedling stage increased more than flowering and seedling stag.Also,thevelocity of JA in resistant variety has significantly higher than that in sensitive variety.These results indicate that Jasmonic acid participates in the peanut stress response.(3)Genome-wide identification and expression pattern of genes involve in peanut JA synthesis.The total of 71 LOX genes,18 AOS genes,17 AOC genes,20 OPR genes,and 33 JMT genes were respectively identified in peanut genome.Bioinformatics analysiswas aimed at revealing the conserved domains,phylogenetic relationships,chromosomal distribution,gene structure,and protein conserved motifs.Those genes were high conserved.AOS and AOC gene have higher conservative and high similarity.The genes LOX,OPR and JMT were found the tandem repeats.All the five type genes often arranged as one cluster.Expression pattern of these genes showed tissue-specific,showed some degrees of expression in leaves,stems,roots,flowers,pods,and seeds at different development stages or different locations(4)Functional analysis of gnens involvd in peanut JA synthesis.We cloned the AhLOX28,AhLOX16,AhAOS9,AhAOC2,AhAOC4,AhAOC9,AhOPR2,AhOPR6,and AhJMT12 genes were cloned and then the full-length CDS sequence were aligned with 66.56%-75.42% similarity between the homologs.The expressed level was observed in Arabidopsis,we found that the transgenic AhAOS9 and AhAOC9 genes exhibit the resistance to stress.7 days after salt stress treatment(200 mmol/L NaCl),the Arabidopsis plants displayed irregular phenotypes: wilting and yellowing,and the resistance of Arabidopsis to salt stress in AhAOC9 over-experession plants and AhAOS9 over-experession plants was higher than the wild control.7 days after natural drought stress,Arabidopsis plants showed the similar symptoms as salt stree,while the damage degree were milder.The VIGS gene silencing vector was constructed to transform into peanuts,plants with silenced AhAOS9 and AhAOC9 showed weaker than the control under normal conditions.The transformed plants showed the higher levels of wilting and more severe leaf curl after drought treatment than control.All results indicate that the JA synthesis-related genes,AhSOS9 and AhAOC9,played the important roles in peanut resistant to drought and salt stress.In summary,this study identified key genes involve in Jasmonic acid synthesis at the whole genomic level,analyzed its expression patterns.Two peanut genes response to drought and salt stress were screened,then the functions were verified.These results of this study will help for elucidating the molecular mechanism of peanut resistant to stress and genetic breeding for stress tolerance.