Transcriptome Analysis Of Stress Response In Cynanchum Komarovii And Functional Study Of The Genes CkNF-YB1 And CkEDS1

Cynanchum komarovii A1 Iljinski is a xerophytic desert herb and used as a kind of green herbal pesticide, especically showing the high activity against insect, pathogen, and viruses. It widely distributes in the severely adverse environment and exhibits a high tolerance to drought, low temperatures, and heat, which could survive in deserts with 100 mm rainfall, the high temperature from 67 to 96℃, and the low temperature from-29 to-36℃. At present, the detailed transcriptomic and genomic data for C. komarovii are still insufficient in public databases. To investigate the expression patterns of drought-and cold-responsive genes and explore the mechanisms of stress tolerance in C. komarovii, we performed a transcriptional analysis using RNA-Seq. Our comprehensive transcriptome analysis will provide a valuable resource for further investigation into the molecular adaptation of desert plants under drought and cold condition and facilitate the exploration of stress-tolerant candidate genes. Moreover, in order to study the function of the stress-tolerant genes, we cloned and characterized the CkNF-YBl and CkEDS1 gene from C. komarovii. These results were as follows:1. Approximately 27.5 GB sequencing data were obtained using Illumina sequencing technology. After de novo assembly 148,715 Unigene were generated with an average length of 604 bp. Among these Unigene,85,106 were annotated with gene descriptions, conserved domains, gene ontology terms, and metabolic pathways. In the KOG classification,4,364 unigenes were annotated as "Signal transduction".29,737 unigenes had significant matches and were assigned to 31 main KEGG pathways. Based on KEGG pathway analysis,827 and 2,840 genes were involved in environmental adaption and signaling transduction pathways, respectively. Moreover,426 genes were associated with the biosynthesis of secondary metabolites. We identified 3,134 Unigene as reliable differentially expressed genes (DEGs) with 601 being up-regulated under droungt stress. Importantly, the 26 DEGs were involved in biosynthesis, export, and regulation of plant cuticle, which had a highest rich factor in KEGG enrichment analysis, suggesting that plant cuticle may play a vital role in response to drought stress.2. We generated approximately 30.50 GB of sequencing data using Illumina sequencing technology and assembled a total of 100,006 Unigene, with an average length of 649 bp. By comparing the unigene with public protein databases,47,082 were functionally annotated with gene descriptions, gene ontology terms, and metabolic pathways. In the KOG classification, the unigenes were annotated as "Signal transduction" (1,779) and "Secondary metabolites biosynthesis, transport and catablism" (786). Based on KEGG pathway analysis,17,695 unigenes had significant matches,445 and 305 genes were involved in environmental adaption and signaling transduction pathways, respectively. In total,7,742 Unigene were differentially expressed with 3,081 and 4,661 being up- and down-regulated under cold stress conditions, respectively. Subsequently, based on the results of gene ontology and metabolic pathway enrichment analyses, a series of candidate genes informed various biochemical pathways were selected and discussed including "Plant hormone signal transduction" and "Starch and sucrose metabolism". The pathways "Flavonoid biosynthesis" and "Flavone and Flavonol biosynthesis" had the highest rich factor in the KEGG enrichment analyses. Moreover, we identified 550 cold-inducible transcription factors including members of the MYB, C3H, bHLH, and AP2-EREBP families of transcription factors.3. In this study, a subunit NF-YB(CkNF-YB1), was isolated from C. komarovii and its role was studied. The CkNF-YBl transcript was detected in all vegetative and reproductive tissues with higher levels in root and stem and was greatly induced by ABA and PEG6000 treatment. By constructing CkNF-YB1-GFP vector, CkNF-YB1 is localized in the cell nucleus. Using the yeast one-hybrid system, the results indicated the CkNF-YB1 did not have transcriptional activation activity. Over-expression of CkNF-YB1 in Arabidopsis enhanced drought tolerance, which resulted in more vigorous seed germination (76.00% and 78.00%), and decrease in water loss (the water content of the OE10 and OE13 lines were 65.50% and 66.85%) and relative electrolyte leakage were 36.56% and 35.57% under drought stress compared with wild type plants. Transcription levels of drought induced gene including RD29A, RD29B, CBF4, and KIN1 were substantially higher in transgenic Arabidopsis than in wild-type plants. Taken together, the data provided evidence that CkNF-YB1 positively confers significant tolerance to osmotic and drought stress in transformed Arabidopsis plants.4. In the present study, CkEDSl was cloned and characterized from C. komarovii. The CkEDSl expression pattern displayed the higher levels in root and stem, and was strongly induced by SA treatment. By constructing CkEDS1-GFP vector, CkEDSl is localized in the cell nucleus. Over-expression of CkEDSl in Arabidopsis enhanced the resistance to Verticillium dahliae compared with wild type plants. The disease index in 21dpi of transgenic lines OE12 and OE13 were 19.44 and 22.77, respectively, which were lower than that of the wild type (36.11). The disease index in 2dpi of transgenic lines OE12 and OE13 were 42.78 and 42.22, respectively, which were significantly lower than that of the wild type (72.78). Transcription levels of defense genes including the PR1, PR5, and ICS1 were substantially higher in transgenic Arabidopsis than in wild-type plants. Moreover, the resistance of transgenic cotton against Verticillium wilt was stronger. Our results implicated that CkEDSl plays an important role in disease resistance in transformed plants.