Table_14_Full-length transcriptome sequencing provides insights into alternative splicing under cold stress in peanut.xlsx

IntroductionPeanut (Arachis hypogaea L.), also called groundnut is an important oil and cash crop grown widely in the world. The annual global production of groundnuts has increased to approximately 50 million tons, which provides a rich source of vegetable oils and proteins for humans. Low temperature (non-freezing) is one of the major factors restricting peanut growth, yield, and geographic distribution. Since the complexity of cold-resistance trait, the molecular mechanism of cold tolerance and related gene networks were largely unknown in peanut.
MethodsIn this study, comparative transcriptomic analysis of two peanut cultivars (SLH vs. ZH12) with differential cold tolerance under low temperature (10°C) was performed using Oxford Nanopore Technology (ONT) platform.
Results and discussionAs a result, we identified 8,949 novel gene loci and 95,291 new/novel isoforms compared with the reference database. More differentially expressed genes (DEGs) were discovered in cold-sensitive cultivar (ZH12) than cold-tolerant cultivar (SLH), while more alternative splicing events were found in SLH compared to ZH12. Gene Ontology (GO) analyses of the common DEGs showed that the “response to stress”, “chloroplast part”, and “transcription factor activity” were the most enriched GO terms, indicating that photosynthesis process and transcription factors play crucial roles in cold stress response in peanut. We also detected a total of 708 differential alternative splicing genes (DASGs) under cold stress compared to normal condition. Intron retention (IR) and exon skipping (ES) were the most prevalent alternative splicing (AS) events. In total, 4,993 transcription factors and 292 splicing factors were detected, many of them had differential expression levels and/or underwent AS events in response to cold stress. Overexpression of two candidate genes (encoding trehalose-6-phosphatephosphatases, AhTPPs) in yeast improves cold tolerance. This study not only provides valuable resources for the study of cold resistance in peanut but also lay a foundation for genetic modification of cold regulators to enhance stress tolerance in crops

引言
花生（Arachis hypogaea L.），又名落花生，是全球广泛种植的重要油料与经济作物。当前全球年度花生总产量已增至约5000万吨，可为人类提供丰富的植物油与蛋白质来源。低温（非冻害型）是限制花生生长、产量及地理分布的主要因素之一。由于抗冷性状的复杂性，花生的耐冷分子机制及相关基因网络目前仍未得到充分阐明。

方法
本研究依托牛津纳米孔测序技术（Oxford Nanopore Technology, ONT）平台，对两个耐冷性存在显著差异的花生栽培品种（SLH与ZH12）在10℃低温条件下开展比较转录组分析。

结果与讨论
本研究共鉴定得到8949个新基因位点与95291个新型转录本亚型。与耐冷品种SLH相比，冷敏感品种ZH12中鉴定到的差异表达基因（differentially expressed genes, DEGs）数量更多；而SLH中的可变剪接事件总数则多于ZH12。对共有的差异表达基因进行基因本体（Gene Ontology, GO）富集分析显示，"胁迫响应""叶绿体组分"及"转录因子活性"为富集程度最高的GO条目，表明光合过程与转录因子在花生冷胁迫响应中发挥关键调控作用。
本研究还检测到708个冷胁迫条件下的差异可变剪接基因（differential alternative splicing genes, DASGs），其中内含子保留（intron retention, IR）与外显子跳跃（exon skipping, ES）为最常见的可变剪接类型。此外，研究共鉴定到4993个转录因子与292个剪接因子，其中众多基因在冷胁迫响应过程中呈现差异表达特征或发生可变剪接事件。将两个编码海藻糖-6-磷酸磷酸酶的候选基因（AhTPPs）在酵母中过表达，可显著提升酵母的耐冷性。
本研究不仅为花生抗冷机制研究提供了宝贵的组学资源，也为通过遗传改造冷调控因子以提升作物抗逆性奠定了坚实的理论基础。