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| Identification and Premliminary Analysis of Salt Stress-responsive Genes in Leaves of Southern Type Alfalfa (Medicago sativa ‘Millennium’) |
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Abstract In order to investigate the molecular mechanism of salt tolerance, the transcriptomes of southern type Alfalfa (Medicago sativa ‘Millenium’) under control condition (WT_CK2) were compared with those under NaCl-treated (WT_CK2) condition. Total RNA was extracted from leaves under control and NaCl-treated conditions, and then was used for RNA Sequencing (RNA-Seq) analysis on the Illumina Hiseq 2000 platform. Meanwhile, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted on the differentially expressed genes (DEGs). qRT-PCR technique was used to validate the expression data of 6 randomly selected DEGs. The results indicated that after filtration of low-quality reads, a total of 62 771 040 and 60 394 756 high-quality reads were acquired, respectively, in control and NaCl-treated samples for the identification of DEGs. Among these high-quality reads, 52.47% (control) and 53.07% (NaCl-treated) could be accurately mapped to the reference genome of Medicago truncatula, thus validating the RNA-Seq data and the reference sequence. A total of 7 497 DEGs were identified, among which 4 078 and 3 419 were detected to be up-and down-regulated, respectively, under NaCl treatment. These genes include 474 transcription factors (TFs) from 46 TF families. GO enrichment analysis showed these DEGs were involved in pathways like binding, catalytic activity, cellular process, metabolic process and response to stimulus. KEGG pathway analysis showed that these DEGs were involved in biosynthesis of secondary metabolites, metabolic pathways, phenylpropanoid biosynthesis, favonoid biosynthesis and plant hormone signal transduction. Important DEGs contained genes involved in osmotic adjustment, ion transport, and pathways involved in biosynthesis and signal transduction. Genes coding calcium-dependent protein kinase, mitogen-activated protein kinase, PP2C family, ABA pathway and hormones (salicylic acid, ethylene and jasmonic acid) signal pathways were up-regulated, TFs from C3H, NAC, WRKY and AP2/EREBP TF families were also differentially up-regulated. On the other hand, genes related to fundamental metabolic mechanism and protein synthesis were generally down-regulated under salt stress. In addition, we identified many candidate genes, such as trehalose-6-phosphate synthase gene, vacuolar Na+/H+ antiporter gene, stress-induced protein kinase gene, calcium-dependent protein kinase gene, sucrose non-fermenting 1-related protein kinase, SnRK, germin-like protein gene, to be related to salt tolerance. Expression analysis of the 6 DEGs with qRT-PCR was in consistent with those of RNA-Seq data, thus furtherly confirming the reliability of RNA-Seq results. This research provides the basic evidence for the molecular mechanisms of salt tolerance in southern type alfalfa.
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Received: 04 May 2015
Published: 11 October 2015
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