Data Mining And Systemic Analysis Of Medicago Falcata Under Abiotic Stress

Legumes are the second most important crops and herbages which ranked after grasses. Medicago falcata, which is closely related to a model species Medicago truncatula, is an important member of grassland legume plants. M. falcata has strong ability to resist cold, drought and salt stresses. It is important to mine key stress resistance genes in M. falcata for molecular breeding stress resistance legumes. In this work, based on high-throughput sequencing technology, we studied the abiotic stressed transcriptome of M. falcata by synthetically using the methods of bioinformatics and molecular biology. We also analysed the important pathways and networks by mining key stress resistance genes.Through the analyses of internal electrolyte leakage, we picked the optimal stress treatment under which electrolyte leakage increased significantly and then present an abiotic stress responsive transcriptome of M. falcata under drought, high salinity and cold treatment by next-generation sequencing platform. Finally a 84.4 Mbp high-quality transcriptome was de novo assembled using optimal parameters. We constructed a relational network of differential expressed transcripts named Transcript-Net, and then we identified two key transcripts, the NAC and the NIP, which situated at the core of the network. Furthermore, we roundly analysed the significant functions and signal pathways of differential expressed transcripts and confirmed the expression patterns of transcripts by qRT-PCR assay. We revealed expression changes of encoding genes at transcriptome level and identified many functions and pathways enrichment of response to abiotic stresses. The GO terms:oxidoreduction, transcripton factor activity and transporters all involved in response to abiotic stresses of M. falcata. In addition, carotenoid metabolism, sugar metabolism and photosynthesis pathway played key roles in stress resistance. A comprehensive analysis of the transcriptome revealed abiotic stress-responsive mechanisms underlying the metabolism and core signalling components of major phytohormones. We identified nod factor signalling pathways during early symbiotic nodulation that are influenced by abiotic stresses. Genetic manipulations of key genes in these pathways might not only enhance stress tolerant ability, but also improve nutrition level of leguminous crops.Moreover, we identified the relationship between miRNAs and targeted transcripts at post-transcriptional level. We validated the patterns of differential expressed miRNAs by qRT-PCR assay. We also predicted the targets of miRNAs and analysed the function enrichment of targets under abiotic stresses. Then we constructed relational network between miRNAs and tragets based on network biology methods. Negative correlation existed between 37 miRNAs and their targets. We found that the largest number of miRNAs targets were transcripts encoding NB-ARC domain, which were important genes involved in disease-resistance. These transcripts were up regulated by miRNAs declared that they might play important role in abiotic response. Additionally, we developed a database called FalcataBase that contains abotic stressed transcriptome data. FalcataBase is available at http://bioinformatics.cau.edu.cn/falcata/. FalcataBase was built as a MySQL relational database system by using the PHP script language. It had a friendly Web interface, which helped in selecting gene markers to improve abiotic stress resistance in legumes.Transporters assist the cell in sensing environmental conditions by forming a complex system of pumps and channels. Based on the complete genome information of M. truncatula, we constructed a novel prediction pipeline and successfully identified transporters from M. truncatula and M. falcata at genomic level. Meanwhile, we developed a database called MTDB that contains transporters information. MTDB is available at http://bioinformatics.cau.edu.cn/MtTransporter/. Based on the M. falcata transcriptome data, we analysed expression patterns of transporter families under abiotic stresses. We identified that ABC transporters and aquaporins played pivotal roles for abiotic stress tolerance. Clones and genetic improvements of these families members could enhance stress tolerant ability of legumes.Many genes we mined play key roles in abiotic stress response. They could provide valuable basis of theory and experiment for revealing mechanism of M. falcata resistance to stresses and molecular biology researches.