Analysis Of Oxytropis Ochrocephala Bunge Transcriptome In Response To Abiotic Stresses And The Signaling Pathway Involved In Drought

Oxytropis ochrocephala Bunge ?Fabaceae? is a perennial grass species in China. The plant contains high concentration of swainsonine, which causes serious threat to animal husbandry. O. ochrocephala is tolerant to various environmental stresses and becomes dominant species in plant community. Most of the researches on O. ochrocephala are mainly about chemical composition identification, toxicity analysis and allelopathy, etc. To date, limited genetic information is available in public databases and this hinders our understanding of the stress adaptation mechanism in O. ochrocephala. In this study, we analyzed the transcriptomes of O. ochrocephala under drought, salinity and cold stresses. By means of the bioinformatic methods, a series of stress-related genes were obtained. Their expression patterns were analyzed to investigate the resistance mechanism of drought, salinity and cold stresses. Then the regulatory mechanism of the stress signal NO ?nitric oxide? through the ABA signaling pathway was investigated under drought stress condition. Study the mechanism of O. ochrocephala's adaptation to stress would underpin further locoweed research. The main results are shown as follows:?1? O. ochrocephala has moderate resistance to drought and cold stresses and it can also live under low salinity. The electrolyte leakage and MDA level were lower and the proline content was higher in cold treated plants than in the drought and salinity ones. The activity of SOD and POD increased during drought and cold treatments, but descended at the later stage of salinity stress. Since the changes in physiological and morphological characteristics usually lag behind the gene expression, the plant materials for RNA-seq were collected at 3 h,6 h and 12 h after treatment.?2? The transcriptomes of four O. ochrocephala samples were sequenced using the Illumina Hiseq 2000 system. A total of 23.22G clean reads were assembled into 217,270 transcripts and 118,596 unigenes ?NCBI Accession:PRJNA292613?. There were 88,942 unigenes identified to plant species and the closely related species to O. ochrocephala were Cicer arietinum, Medicago truncutula, Glycine max, etc. In KEGG pathway classifications,10,465 unigenes ?11.76%? were assigned into 276 KEGG pathways. Through GO and KEGG pathway enrichment, differentially expressed genes ?DEGs? were further categorized into different functional groups, including osmotic adjustment, secondary metabolism, unsaturated fatty acid synthesis and metabolism, plant hormone synthesis and signaling pathway, oxidative phosphorylation, transcriptional regulation, etc.?3? From the transcriptome database of O. ochrocephala, we selected 12 candidate reference genes by the local BLAST service. The suitability of these validating reference genes was determined by qRT-PCR analysis. By employing software packages including geNorm, NormFinder and BestKeeper, the reference genes, HIS, ACT7 and ACT101, were assessed as the most suitable set for normalization and 18s?TUA?ACT11 were the least stable reference genes. In order to validate the RNA-seq results, the expression patterns of 30 DEGs were validated by qRT-PCR analysis.80% of the qRT-PCR results were concordant with the differential expression patterns measured by FPKM fold change.?4? The stress inducible genes in O. ochrocephala were chosen and detected the gene expression profiles. These genes included stress-related proteins, secondary metabolism, transcription factors, phytohormones and calcium signals. Among the stress-related genes, OoLEA and OoDHN were up-regulated under the three stress conditions and they act as molecular chaperones. High expression levels of OoPRP and OoGS were observed in the late phase of drought and salinity treatment and they are involved in cell wall components. GLP participates in plant defense. OoGLP had the highest gene expression level under drought stress. OoHSP70 was up-regulated only under salinity stress. The genes involved in lignin and flavonoid biosynthesis ?OoPAL?OoC4H?Oo4CL?OoCHR?OoADH? were up-regulated under all stress conditions. Among the transcription factor genes, OoAREB was involved in the ABA signaling pathway. OoMYB was significantly up-regulated under cold stress. OoNAC was expressed earlier than other genes. OoWRKY and OoERF were involved in the cold stress response. The changing trend of hormone content in O. ochrocephala under the three stress conditions was toward stomatal closure, root elongation and defense response. The expression levels of genes related to phytohormone biosynthesis and signal transduction were increased. Besides, several genes related to calcium signal transduction were up-regulated under the cold stress condition.?5? The open reading frame ?ORF? of OoNCED and OoAREB were cloned and the gene sequences were analyzed by bioinformatic tools. OoNCED encoded 612 amino acids, molecular weight and isoelectric point ?PI? were 68.4 kDa and 8.18 respectively. OoAREB encoded 426 amino acids with a molecular weight of 46.4 kD and the PI of 9.38. The proteins encoded by OoNCED and OoAREB had no signal peptide and transmembrane region. OoNCED was predicted located in the chloroplast and OoAREB in the nuclei. Alpha helix, ?-sheet and random coil were the main secondary structure components of OoNCED and OoAREB. By phylogenetic analysis, the amino acid sequeces of OoNCED and OoAREB were more closely to NCED1 ?ACU86971.1? and AREB ?ABG90380.1? in Caragana korshinskii.?6? The homeostasis of endogenous NO and the correlations between NO and ABA were studied in O. ochrocephala under PEG treatment. NO was mainly localized in root hair, epidermis and vascular tissue in root of O. ochrocephala. The main sources of NO production in root were nitrate reductase ?NR? and non-enzymatic process. And in the whole seedling, NO synthase ?NOA? enzyme was also the source of NO production. OoNOA, OoNR and OoGSNOR regulated the NO equilibrium in O. ochrocephala. The NO accumulated in O. ochrocephala was temporary. The reduction of NO was accompany with the increasing H₂O₂, and there was a balance between ROS ?H₂O₂? and NO production. In O. ochrocephala, drought stress caused a remarkably increase in ABA level. Moreover, ABA biosynthesis inhibitor significantly intensified lipid peroxidation, which indicated the ABA-dependent signaling pathway under drought stress in O. ochrocephala. When ABA biosynthesis was inhibited, the drought-induced NO in the roots was reduced, but ABA content was not affected significantly when endogenous NO was removed. This implies that NO acts downstream of ABA. Moreover, the MDA level also increased when endogenous NO was removed. It is revealed that NO is involved in the ABA-dependent pathway to relieve lipid peroxidation and oxidative damage in O. ochrocephala.