Effects Of Salt Stress On Membrane Lipid Composition And Gene Expression In Peanut Seedlings

The aim of this study was to observe the physiological and molecular mechanisms of peanut(Luhua14)related to membrane lipid composition under salt stress and recovery treatment.Transcriptomic analysis was performed with peanut leaves at different treatments to select key genes related with membrane lipid composition.The results are as follows: 1.The effects of salt stress and recovery treatment on physiological parameters in peanut seedlingsIn this experiment,we measured membrane lipid composition,fluorescence parameters,reactive oxygen species,antioxidant enzymes and other physiological parameters under salt stress and recovery treatment.Results showed that the growth of peanut seedlings was inhibited seriously at 250 mM NaCl concentration.In the experiment,we observed the DBI and 18:3 content obviously decreased at 250 mM NaCl concentration.Fo and NPQ both increased at 250 mM NaCl treatment,but decreased obviously in Fv/Fm and ?PSII.The reducing of DBI and unsaturated fatty acid content resulted in decreasing of cell membrane stability.Consequently,salt tolerance of photosystem II and photosynthetic rate decreased pronouncedly.While the decrease in utilization of light energy inevitably resulted in a large amount of excess light energy and resulted in photoinhibition.The photoinhibition would lead to the production of reactive oxygen species in cell and cause oxidative damage to chloroplasts.However,DBI and unsaturated fatty acid content increased and improved cell membrane stability at recovery treatment.The increasing unsaturated fatty acid content alleviated photoinhibition of PSII under salt stress and consequently,photosynthetic rate and Fv/Fm improved slightly.Besides,the activity of SOD and APX increased slightly,which lead to the reducing of O2-· and H2O2 content at recovery treatment.However,the PSI oxidoreductive activity(?I/Io)had no obvious increase at recovery treatment compared with that under salt stress.It seemed that PSI reaction center was damaged seriously and could not be repaired at recovery treatment.These results suggested that the increasing unsaturated fatty acid content could alleviate photoinhibition of PSII under salt stress.2.Analysis and validation of transcriptome sequencing of peanut seedlings under salt stress and recovery treatmentIn the study,total RNA was extracted from the leaves of peanut seedlings at CK,S4(salt stress for 4 days)and R3(transferred from salt stress to standard conditions for 3 days)condition.The qualified RNA samples were sent to Illumina Sequencing Services of BGI(Shen Zhen)where the libraries were produced and sequenced using Illumina HiSeq 2000.The clean reads were generated by removing adaptor sequences,empty reads and low quality sequences.We obtained around 135,144,930 high quality reads.After trimming adapters and removing low quality reads,more than 131,459,048 clean reads were retained for assembly and further analysis.Among all the reads,about 80.02% had Phred-like quality scores at the Q20 level(an error probability of 1%).The analyzing results showed that 1,742 genes showed differential expression levels compare with control.Among these DEGs,898 genes were up-regulated and 844 were down-regulated in leaves under salt stress.There were 390 genes were differentially expressed at R3 condition compared with those at S4 condition.Among these DEGs,323 genes were up-regulated and 67 were down-regulated in leaves.After GO and KEGG analyzing,key genes(CL8534.Conting6,CL8534.Conting7)related to ?-3 fatty acid desaturase were found.These two genes were involved in the synthesis of linoleic acid to linolenic acid.At S4 condition,the expression of these two genes decreased but increased at recovery treatment.These results showed that genes CL8534.Contig6 and CL8534.Contig7 likely played an important role in the process of linoleic acid converting into linolenic acid and improved salt tolerance in peanut seedlings.Up-regulation of two genes caused an increase of unsaturated fatty acid content,which might help improving cell membrane stability and alleviating photoinhibition of PSII caused by salt stress.Besides,12 DEGs were selected and quantitative real-time PCR was performed.The results indicated the RNA-seq data was reliable and can be used for further analysis.3.Preliminary clone of the key genes related to ?-3 fatty acid desaturase in peanut seedlingsBased on the sequence of RNA-seq,the primers of ?-3 fatty acid desaturase genes were designed.The length of 1,199 bp gene fragment was obtained.The homology was 100% according to NCBI database and the gene fragment is part of the ?-3 fatty acid desaturase gene.The expression analyze about the cloned gene fragment was performed at different times and different NaCl concentration.The verification results showed that with the increasing of treatment time and NaCl concentration,the gene expression levels decreased.However,the gene expression levels increased at recovery treatment.Besides,we also measured the activity of ?-3 fatty acid desaturase.Results showed that the activity of ?-3 fatty acid desaturase decreased under salt stress and increased at recovery treatment.These results suggested that down-regulation of genes caused a decrease of unsaturated fatty acid content under salt stress and the up-regulation of genes played a key role in increasing the unsaturated fatty acid content.