Transcriptomic profiling and analysis of differentially expressed genes in asparagus bean (Vigna unguiculata ssp. sesquipedalis) under salt stress

Asparagus bean (Vigna unguiculata ssp. sesquipedalis) is a warm season legume which is widely distributed over subtropical regions and semiarid areas. It is mainly grown as a significant protein source in developing countries. Salinity, as one of the main abiotic stress factors, constrains the normal growth and yield of asparagus bean. This study used two cultivars (a salt-sensitive genotype and a salt-tolerant genotype) under salt stress vs. control to identify salt-stress-induced genes in asparagus bean using RNA sequencing. A total of 692,086,838 high-quality clean reads, assigned to 121,138 unigenes, were obtained from control and salt-treated libraries. Then, 216 root-derived DEGs (differentially expressed genes) and 127 leaf-derived DEGs were identified under salt stress between the two cultivars. Of these DEGs, thirteen were assigned to six transcription factors (TFs), including AP2/EREBP, CCHC(Zn), C2H2, WRKY, WD40-like and LIM. GO analysis indicated four DEGs might take effects on the “oxidation reduction”, “transport” and “signal transduction” process. Moreover, expression of nine randomly-chosen DEGs was verified by quantitative real-time-PCR (qRT-PCR) analysis. Predicted function of the nine tested DEGs was mainly involved in the KEGG pathway of cation transport, response to osmotic stress, and phosphorelay signal transduction system. A salt-stress-related pathway of “SNARE interactions in vesicular transport” was concerned. As byproducts, 15, 321 microsatellite markers were found in all the unigenes, and 17 SNP linked to six salt-stress induced DEGs were revealed. These candidate genes provide novel insights for understanding the salt tolerance mechanism of asparagus bean in the future.长豇豆（Asparagus bean, Vigna unguiculata ssp. sesquipedalis）是一种喜温型豆科作物，广泛分布于亚热带及半干旱区域。在发展中国家，其主要作为重要的蛋白源被栽培。盐胁迫作为主要非生物胁迫因子之一，会制约长豇豆的正常生长与产量形成。本研究以两个长豇豆品种（盐敏感基因型与耐盐基因型）为材料，设置盐胁迫与对照两组处理，通过RNA测序（RNA-seq）筛选长豇豆中盐胁迫诱导表达的基因。从对照及盐胁迫处理的转录组文库中，共获得692,086,838条高质量洁净读段，这些读段被注释至121,138个单基因（unigenes）。随后，在两个品种的盐胁迫处理组与对照组间，共鉴定出216个根系来源的差异表达基因（differentially expressed genes, DEGs）以及127个叶片来源的差异表达基因。其中13个差异表达基因被注释为6类转录因子（transcription factors, TFs），分别为AP2/EREBP、CCHC(Zn)、C2H2、WRKY、WD40-like及LIM家族。基因本体（Gene Ontology, GO）富集分析显示，4个差异表达基因可能参与“氧化还原”“物质转运”与“信号转导”生物学过程。此外，本研究通过实时荧光定量PCR（quantitative real-time-PCR, qRT-PCR）验证了9个随机选取的差异表达基因的表达量。经功能预测，这9个验证后的差异表达基因主要富集于阳离子转运、渗透胁迫响应以及磷酸化信号转导系统相关的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路中。本研究还关注到一条与盐胁迫相关的“囊泡运输中SNARE蛋白相互作用”通路。作为本研究的附属产物，我们在所有单基因中鉴定出15321个微卫星标记（microsatellite markers），并发现了17个与6个盐胁迫诱导差异表达基因相关的单核苷酸多态性（single nucleotide polymorphism, SNP）位点。这些候选基因可为未来解析长豇豆的耐盐调控机制提供全新的研究视角。