Expression profiling of heat stress response in peanut using oligonucleotide microarrays

To gain insights into molecular mechanisms of tolerance to heat stress, we conducted a transcript profiling experiment to identify heat-responsive genes in contrasting peanut mini core accessions, either un-acclimated or acclimated to heat stress. Plants at reproductive stage were exposed to 28 °C (control), 45 °C for 15 d (un-acclimated) or 45 °C for 1 d followed by 7 d recovery and 15 d stress (acclimated). Two contrasting genotypes showing diverse response to stress were selected based on a bioassay involving chlorophyll fluorescence yield under elevated respiratory demand and membrane thermostability. Transcript profiling was performed using 4 x 44k custom oligo microarrays containing 22k peanut EST sequences. The microarray analysis identified 710 stress-induced and 770 stress-repressed putative heat-responsive transcripts in the tolerant genotype. Gene enrichment analysis was performed using Blast2GO program and genes with homology to known proteins were categorized into detailed molecular functional groups. Majority of stress-responsive genes assigned to KEGG pathways belonged to starch, sucrose and galactose metabolism followed by amino acid metabolism, and secondary metabolite biosynthesis. Differentially expressed transcripts from samples obtained from first year’s experiment were validated in the samples from second year by quantitative real-time PCR. Transcripts of eight genes involved in terpenoid and flavanoid biosynthesis were induced after second and seventh day, respectively, in leaves under heat stress. Metabolite analysis confirmed increases in metabolites of selected pathways under heat stress. The heat up-regulated genes in tolerant COC041 mini-core accession are potential candidate genes for engineering stress-tolerant peanuts and unraveling molecular mechanisms of peanut adaptation to heat stress. Overall design: We used Agilent peanut microarrays to identify putative heat stress-responsive genes. Directly heat-stressed leaf tissues of the peanut genotypes COC041 (tolerant) and COC166 (susceptible) were used in the study. Three replications of microarray experiments were carried out by hybridizing the cRNA from different time points and stress conditions in a loop design on 4 x 44k microarray.

为深入探究耐热应激的分子机制，本研究开展了转录组分析实验，旨在识别对比花生微型核心基因库中在未适应或适应热应激条件下的热响应基因。在生殖阶段，植物分别暴露于28°C（对照组）、45°C持续15天（未适应组）或45°C持续1天后恢复7天，再进行15天应激处理（适应组）。根据涉及在高呼吸需求下叶绿素荧光产量和膜热稳定性的生物测定，选取了两种对胁迫反应不同的基因型。采用包含22k花生EST序列的4 x 44k定制寡核苷酸微阵列进行转录组分析，微阵列分析在耐性基因型中鉴定出710个应激诱导和770个应激抑制的潜在热响应转录本。利用Blast2GO程序进行基因富集分析，将具有已知蛋白质同源性的基因分类至详细的分子功能组。大多数分配至KEGG通路的热响应基因属于淀粉、蔗糖和半乳糖代谢，其次是氨基酸代谢和次生代谢物生物合成。第一年实验获得的样本中的差异表达转录本通过第二年样本的定量实时PCR得到验证。在热应激条件下，参与萜类和黄酮生物合成的8个基因的转录本分别在第二和第七天被诱导。代谢物分析证实，在热应激条件下，所选通路的代谢物水平增加。在耐性COC041微型核心基因库中，热上调的基因是工程化耐热花生和揭示花生适应热应激分子机制的潜在候选基因。总体设计：本研究采用Agilent花生微阵列以鉴定潜在的耐热应激基因。研究中使用了花生基因型COC041（耐性）和COC166（易感）直接热应激的叶片组织。通过在4 x 44k微阵列上以循环设计杂交不同时间点和应激条件下的cRNA，进行了三次重复的微阵列实验。