Data associated with RT-qPCR experiments.

Identification and characterization of genes encoding herbicide-detoxifying enzymes is lacking in allohexaploid wheat (Triticum aestivum L.). Gene expression is frequently induced by herbicide safeners and implies the encoded enzymes serve a role in herbicide metabolism and detoxification. Cloquintocet-mexyl (CM) is a safener commonly utilized with halauxifen-methyl (HM), a synthetic auxin herbicide whose phytotoxic form is halauxifen acid (HA). Our first objective was to identify candidate HA-detoxifying genes via RNA-Seq by comparing untreated and CM-treated leaf tissue. On average, 81% of RNA-Seq library reads mapped uniquely to the reference genome and 76.4% of reads were mapped to a gene. Among the 103 significant differentially expressed genes (DEGs), functional annotations indicate the majority of DEGs encode proteins associated with herbicide or xenobiotic metabolism. This finding was further corroborated by gene ontology (GO) enrichment analysis, where several genes were assigned GO terms indicating oxidoreductase activity (34 genes) and transferase activity (45 genes). One of the significant DEGs is a member of the CYP81A subfamily of cytochrome P450s (CYPs; denoted as CYP81A-5A), which are of interest due to their ability to catalyze synthetic auxin detoxification. To investigate CYP expression induced by HM and/or CM, our second objective was to measure gene-specific expression of CYP81A-5A and its homoeologs (CYP81A-5B and CYP81A-5D) in untreated leaf tissue and leaf tissue treated with CM and HM over time using RT-qPCR. Relative to the reference gene (β-tubulin), basal CYP expression is high, expression among these CYPs varies over time, and expression for all CYPs is CM-inducible but not HM-inducible. Further analysis of CYP81A-5A, such as gene knock-out, overexpression experiments, or in vitro activity assays with purified enzyme are necessary to test the hypotheses that the encoded CYP detoxifies HA and that CM upregulates this reaction.

异源六倍体普通小麦（Triticum aestivum L.）中，编码除草剂解毒酶的基因的鉴定与功能表征仍存在明显不足。基因表达常可被除草剂安全剂诱导，这提示其编码的酶在除草剂代谢与解毒过程中发挥关键作用。氯喹氧酯（Cloquintocet-mexyl, CM）是一种常与甲基氯氟吡啶酯（halauxifen-methyl, HM）复配使用的安全剂；后者为合成生长素类除草剂，其植物毒性活性形式为氯氟吡啶酸（halauxifen acid, HA）。本研究的首要目标是通过RNA测序（RNA-Seq），对比未经处理与经CM处理的叶片组织，筛选可解毒HA的候选基因。实验结果显示，平均81%的RNA测序文库读段可唯一比对至参考基因组，76.4%的读段可比对至基因区域。在103个显著差异表达基因（DEGs）中，功能注释结果表明绝大多数DEGs编码与除草剂或异源生物代谢相关的蛋白质。该结论进一步通过基因本体（GO）富集分析得到验证：多个基因被注释到氧化还原酶活性（34个基因）与转移酶活性（45个基因）相关的GO条目。其中一个显著差异表达基因属于细胞色素P450（CYPs）的CYP81A亚家族（记为CYP81A-5A），该家族因可催化合成生长素类除草剂的解毒反应而受到广泛关注。为探究HM和/或CM诱导的CYP基因表达情况，本研究的第二项目标是采用实时定量聚合酶链反应（RT-qPCR），检测未经处理的叶片组织，以及经CM、HM处理不同时长的叶片组织中，CYP81A-5A及其部分同源基因（CYP81A-5B与CYP81A-5D）的基因特异性表达水平。以参考基因β-微管蛋白为内参，结果显示这些CYP基因的基础表达水平较高，且不同CYP基因的表达随时间呈现动态变化；所有检测的CYP基因均可被CM诱导，但不受HM诱导。后续需对CYP81A-5A开展进一步研究，例如基因敲除、过表达实验或纯化酶的体外活性测定，以验证其所编码的CYP可解毒HA，且CM可上调该反应的相关假说。