The Transcriptome Analyses Of Two Melon(Cucumis Melo L.) Cultivars Under Salt Stress

Melon (Cucumis Melo L.) is one of the world’s ten important fruit, which is asloan important facility cultivation crop in China. Cucumis Melo L. is mediumsalt-tolerant plants, whose salt resistance is second to pumpkin among all thecucurbitaceous plants, it has the potential to become a resource with desalination andhigh yield of crop. The researches about salt resistance of melon mainly focused onthe physiological and biochemical aspects before, however, the molecular mechanismwere rarely reported. The development of melon genome and the improvement of thevarious molecular techniques, make the further research and exploration of thesalt-tolerant molecular mechanisms of melon by means of new biological methodpossible, which is helpful to clarify the molecular mechanisms of melon.This study first used high-throughput sequencing technologies (Illumina) tostudy the two varieties of the melon under normal blade (control) and300mM NaCl;The ion flow, chlorophyll fluorescence intensity of photosynthesis, transpiration rate,stomatal conductance, plant dry weight of these two varieties are measured before andafter the NaCl treatment.The transcriptome data、physiological and biological data were combined tomake a comprehensive analysis on the changes of melon gene expression under saltstress and excavate the responsive genes. Morever, we focused on the functionprediction and metabolic pathways of these genes. The transcriptome sequencingresults were verified by RT–PCR, as well as the expression pattern of12differentgenes that coding different functional protein under the salt-treatment for1h,24h,1week.Main conclusions:1. The influence of salt stress of different melon seedling on photosynthesis,chlorophyll fluorescence parameters of biomass, ion flow were studied, as to provide theoretical basis for screening salt-tolerant genes. High concentration NaCl reduce thevalue of Pn, Gs, Tr, Fv/Fm, ФPSII, while increase F0and Ci. The influence of saltstress in ‘YL’ is less than in ‘BXC’, the main reason for the decline in photosyntheticrate was stomatal limitation under high salt stress.300mmol/L NaCl turns the influxof Na+、K+into efflux status, meanwhile, the amount of influx of Cl-has beenincreased, which indicated the defense mechanism of melon.2. Four pools of mRNA samples, one from ‘BXC’ and one from ‘YL’, both at CK and300mmol/L NaCl, were used to build libraries for high-throughput parallelsequencing using Illumina sequencing technology. Based on these studies, over40million short reads were generated, each read had a100-bp length. The total amountof data was about7.67G. These short reads were assembled by Cufflinks, resulting in116,504genes. After mapping these data to melon genome database, we found thatour transcriptome dataset had a high degree of consistency (85.77%). The amount ofgenes in ‘BXC’ and ‘YL’ were17607and22126, respectively, accounted for54.5%and67.8%of the total genes.3. Differences in tag frequencies in the ‘BXC’ and ‘YL’ libraries were used to estimatedifferences at gene expression levels among the four libraries.1171and1487transcripts genes were significantly different respectly(︱log2Ratio︱≥1and P≤0.05) in ‘BXC’ and ‘YL’; Occupied6.65%and6.72%of the total number ofexpressed genes respectively. There were a large number of overlapping genesbetween ‘BXC’ and ‘YL’, including232common up-regulated genes and379common down-regulated genes. Meanwhile, there were1436specific differentlyexpressed genes between ‘BXC’ and ‘YL’.4. To identify the putative functions of the gene sequences, we performed Blast2GOprocess (E-value <1e-6) against the NCBI non-redundant databases (NR) databases.Based on their sequence homology, a total of16875genes (57.1%) were categorizedinto three main categories.5. Based on the Blast2GO statistics results, among232common up-regulated genes,155(66.8%) mapped to GO terms in the NR database in NCBI, among306common down-regulated genes,127(41.5%) mapped to GO terms; Among1436specificdifferential genes,894(62.3%) mapped to GO terms. That is to say,1176differentialgenes mapped to the GO terms successfully. In GO,1176annotated genes which hadhomology were classed into three categories. Most of them were involved inbiological process, such as cell processes, metabolic processes and so on. As formolecular function, most of annotated genes were involved in binding (includingmany connection forms such as ion binding) and activity (including all kinds ofcatalytic enzyme, transmembrane transporter, electron carrier). These cules indicatedthat these genes play important role in the growth and development of melon andphysiologically regulated melon under salt stress.6. To identify the biological pathways that were active in muskmelon,2166differentially expressed genes were mapped to the reference canonical pathways inKEGG and were assigned to26KEGG pathways. The pathways most represented inthe unique sequences were metabolic pathways (179), biosynthesis of secondarymetabolites(113). Related genes that took part in the process of plant hormone signaltransduction, the biosynthesis of secondary metabolites, photosynthesis, starch andsucrose metabolism, dicarboxylic acid metabolism, indicating that these genes playsimportant roles in the regulation of salt stress.10genes were mapped to2Phenylalanine pathways, which enriched the regulation data of secondary metabolitesunder salt stress. The differentially expressed genes of YL were enrich in auxin,cytokinins, abscisic acid and brassinolide in hormone signal transduction pathway, thediscovery of these genes will provides reference data for the research of hormonesignal transduction pathways and its regulatory mechanism of the melon under saltstress.7. We selected12responsive genes (incluing transcription factors, nitrate transporter,aquaporins, potassium channel protein) for qPCR validation. The results showedconsistent expression patterns with the observed findings in transcriptome analysis,which indicating the veracity of the Illumina sequences data from the melontranscriptome. 8. The different chlorophyll fluorescence parameters between these two melonvarieties indicated that regulation mode at transcription level under salt stress mayalso be different between these two varieties. By identifying the responsivetranscription factor under salt stress and comparing the their expression differences, todeepen the understanding of expression characteristics of transcription factor of melon,these results provided the reference in order to further study the function of thesetranscription factors.9. We screened the differently expressed genes related to photosynthetic system, sugarmetabolic pathways, cell wall organization, antioxidant system, transcriptionregulatory factors, transporters, ion channel proteins, heat shock protein and the lateembryonic development rich protein are involved in Photosynthesis, osmoticregulation, cell protection, protect and repair protein structure, active oxygen removal,transcriptional regulation and signal transmission. By combaining physiological datawith these responsive genes, we obtained the regulation mechanism that raleted to theabove physiological indexes.10. qPCR method is used to analyze the patterns of expression of these12responsivegenes at three time points after salt stress (1h,24h,1week). Significant attentionwas paid on genes realted to ion transporter, transcription factor. Analysis were doneto study their response characteristics under salt stress by their response geneexpression pattern.