Analysis Of The Involvement Of Intracellular Redox Status In Regulation Of Response And Adaptation Of Jatropha Curcas To Drought Stress Based On High-throughput Sequencing

Jatropha curcas L.is an energy plant species with great development potential,and drought is an important environmental factor that limits the development of J.curcas.When the plants are subjected to drought stress,the content of reactive oxygen species increases,and activities of antioxidant system decrease,resulting in an imbalance of the intracellular redox state.Therefore,clarifying the mechanism of redox state regulation in J.curcas will be helpful to improve their adaptation to drought stress.In our lab's previous study,the transcriptome data of J.curcas during drought hardening and following drought stress were obtained analyzed by Illumina Hiseq 2000 high-throughput sequencing,Based on these data,and the corresponding differentially expressed genes related to redox status were further significantly enriched and analyzed and their pathways were constructed.Ascorbic acid and glutathione contents were determined and the spatiotemporal expression of key enzyme genes were analyzed to clarify the regulation of intracellular redox status on response and adaptation of Jatropha curcasseedlings to drought stress.The main results are summarized as follows:1.The three-week-old J.curcas seedlings were used as experimental materials,and 10% PEG6000 was used for drought-hardening treatment for 2 days,then drought stress was applied for 3 d.RNA-seq sequencing was performed after the leaf materials were mixed uniformly,and use the seedlings without any treatment as controls.Sequencing results showed,that there were 73 differentially expressed genes related to glutathione metabolism,a key substance in the redox status regulation;44differentially expressed genes related to ascorbate metabolism;and 39 thioredoxin differentially expressed genes;There are 33 differentially expressed genes of glutaredoxin.These genes havedifferential expression changes to different extent during drought hardening and the following droughtstress.Ascorbic acid,glutathione,thioredoxin,and glutaredoxin are key components and core proteins that regulate the redox status of plants,and their metabolic changes indicate that the intracellular redox state is involved in the response and adaptation of J.curcas seedlings todrought hardening and stress.2.Through the determination of ascorbic acid and glutathione content and the activity of glutathione peroxidase?GPX?in J.curcas,it was found that during drought-hardening and the following drought stress,J.curcas seedlings with drought-hardening had much higher total content of glutathione and ascorbic acid than that those without drought-hardening.The determination of GPX enzyme activity showed that the activity was increasing during the whole drought stress.The J.curcas seedlings with drought-hardening had relatively low enzyme activity in the early stage of drought stress,and then increased rapidly in the later period.Compared with the seedlings without drought-hardening,they showed stronger adjustment ability.The background expression of glutathione peroxidase gene?XM₀12231531.2?and ascorbate peroxidase gene?XM₀12226880.2?in leaves was relatively highest and the fold change was significant.Based on this results,the both of genes were cloned and bioinfomatic analysis were performed.These results showed that glutathione and ascorbic acid played an important role in regulating the response and adaptation of J.Curcas seedlings to drought stress.3.Cluster analysis of genes of glutaredoxin?GRX?and thioredoxin?TRX?in the digital expression profiling showed that the overall gene change was not significant during the drought-hardening period,but there were significantchangesduring the subsequentdroughtstress.The quantitative analysis of the glutathione and thioredoxin gene family showed that the expression level of GRX after drought-hardening was significantly higher tha n that without drought-hardening,and the up-regulation of the thioredoxin family gen e expression enhancened significantly.In addition,a high-expression level protein gene,glutaredoxin C9,was selected for cloning.Sequence analysis revealed that the protein encoded by this gene has a typical “CCXX” motif unique to higher plants.It has a total length of 726 bp,a coding region of 438 bp,and encodes 145 amino acids.It is closely related to the phylogenetic relationships of ramie,rubber tree,and cassava.A highly induced expression by drought stress indicates that it belongs to a drought-responsive gene.On conclusion,these results from redox * ubstances,enzyme activities and gene expression of the corresponding key enzymes in J.curcasindicated thatdrought-hardening enhanced the activity of redox state regulation and the ability to remove active oxygen species,then the drought resistance of J.curcas was further improved.These results will have scientific significance and potential application prospects to some extent,and can also provide drought-resistant genetic resources for genetic improvement of other crops and trees.