Identification of rapidly-induced genes in the peanut (Arachis hypogaea) response to water deficit and abscisic acid. Arachis hypogaea

Purpose: to screen the candidate genes involved in the peanut drought stress response, we conducted global transcriptome analysis of peanut plants challenged with water deficit and ABA, using the Illumina HiSeq2000 sequencing platform. Methods: a sequences library of Yueyou7 were constructed at first. Then the profile of diffentialy expressed genes (DEGs) under three different treatments (control, water deficit without ABA, and water deficit with ABA) were conducted based on above sequence library. For sequencing library construction, plants were grown under normal conditions, as described previously , Seeds were planted in soil and kep in the greenhouse at temperature of 25-30℃ and water well. Three tissues (leaves, roots, and stems) were collected at three development stages (four-leaf, flowering and podding stages), respectively. Then all of these tissues were mixed to extract the total RNA for sequence library construction. For DEGs study, two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 µmol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel. After treatments, Total RNA was extracted from 100 mg of plant material, and RNA integrity was checked by gel electrophoresis. Also RNA quality was checked and RNA was quantified using the Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) and Nanodrop ND-1000 (Thermo Scientific, Waltham, USA). Results: we generated 4.96×107 raw sequence reads and assembled the high quality reads into 92,390 unique genes. Compared with the control, we found that 621 genes (≥1.5 fold change) responded rapidly to water deficit and 2665 genes (≥1.5 fold change) responded rapidly to ABA. We found 279 genes that overlapped between water deficit and ABA responses, 264 genes that showed the same trend in expression while 15 genes expression that showed opposite trend. Among the identified genes, 257 showed high induction by ABA (>5 fold), and 19 showed high induction by drought (>5 fold). In addition, we identified 100 transcription factor genes among the ABA-inducible genes but only 22 transcription factor genes among the water deficit-inducible genes. Conclusions: we identified genes differentially expressed in the early response to water deficit or ABA. These genes were annotated with GO functional categories for water deficit (33 categories) or ABA (31 categories). We found that only 19 genes were highly induced by water deficit, but 257 genes were highly induced by ABA. Our previous work has examined many of these genes and our future work will reveal their functions and relationships. These data will facilitate functional genomic studies and have established a biotechnological platform for examination of the early drought- and ABA-responsive transcriptome regulatory network in peanut. Overall design: Two-week-old plants were divided into three groups with three independent replication: (1). Water deficit without ABA groups. Plants directly steeped in water containing 30% PEG600 for 30 min in this groups. (2) Water deficit with ABA groups. Plant was subjected to 100 µmol/L ABA for 30 min and then steeped in water containing 30% PEG6000 for 30 min in this groups, (3) Control. Plants steeped in H2O. All treatments were conducted in parallel.

### 研究目的
为筛选参与花生干旱胁迫应答的候选基因，本研究依托Illumina HiSeq2000测序平台，对受水分胁迫与脱落酸(ABA)处理的花生植株开展全转录组分析。

### 研究方法
首先构建粤油7号(Yueyou7)的测序文库，基于该文库分析三种不同处理（对照组、无ABA水分胁迫组、含ABA水分胁迫组）下的差异表达基因(differentially expressed genes, DEGs)谱。
测序文库构建流程：将植株种植于正常培养条件下，如前文所述：将种子播种于土壤中，置于温度25-30℃的温室并充分浇水。分别在三个发育时期（四叶期、开花期、结荚期）采集叶片、根、茎三种组织，随后混合所有组织以提取总RNA，用于测序文库构建。
差异表达基因分析流程：将两周龄的植株分为三组，每组设置三次独立生物学重复：(1) 无ABA水分胁迫组：将植株直接浸没于含30% PEG600的水溶液中处理30分钟；(2) 含ABA水分胁迫组：先将植株用100 μmol/L的脱落酸(ABA)处理30分钟，随后浸没于含30% PEG6000的水溶液中处理30分钟；(3) 对照组：将植株浸没于超纯水中。所有处理同步平行开展。处理完成后，从100 mg植物材料中提取总RNA，通过凝胶电泳检测RNA完整性；同时使用Agilent 2100生物分析仪（Agilent Technologies，圣克拉拉，加利福尼亚州，美国）和Nanodrop ND-1000分光光度计（Thermo Scientific，沃尔瑟姆，美国）检测RNA质量并进行定量。

### 研究结果
本研究共获得4.96×10^7条原始测序读段，将高质量读段组装为92390个唯一基因。与对照组相比，共有621个基因（表达倍数变化≥1.5）快速响应水分胁迫，2665个基因（表达倍数变化≥1.5）快速响应ABA。其中，279个基因在水分胁迫与ABA应答中存在重叠：264个基因的表达趋势一致，15个基因的表达趋势相反。在鉴定到的基因中，257个基因可被ABA显著诱导（表达倍数>5），19个基因可被干旱胁迫显著诱导（表达倍数>5）。此外，在ABA诱导基因中鉴定到100个转录因子基因，而在干旱胁迫诱导基因中仅鉴定到22个转录因子基因。

### 研究结论
本研究鉴定了在水分胁迫或ABA早期应答中差异表达的基因，并对这些基因进行了基因本体(GO)功能注释，其中水分胁迫相关基因分为33个功能类别，ABA相关基因分为31个功能类别。研究发现仅19个基因可被干旱胁迫高度诱导，而257个基因可被ABA高度诱导。本团队前期工作已对其中多数基因进行了研究，后续工作将揭示它们的功能与相互关系。本数据集将为功能基因组学研究提供助力，并为解析花生早期干旱与ABA应答转录调控网络构建了生物技术平台。

### 实验整体设计
将两周龄的植株分为三组，每组设置三次独立生物学重复：(1) 无ABA水分胁迫组：将植株直接浸没于含30% PEG600的水溶液中处理30分钟；(2) 含ABA水分胁迫组：先将植株用100 μmol/L的脱落酸(ABA)处理30分钟，随后浸没于含30% PEG6000的水溶液中处理30分钟；(3) 对照组：将植株浸没于超纯水中。所有处理同步平行开展。