An efficient system composed of maize protoplast transfection and HPLC-MS for studying the biosynthesis and regulation of maize benzoxazinoids
An efficient system composed of maize protoplast transfection and HPLC-MS for studying the biosynthesis and regulation of maize benzoxazinoids
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DOI：
10.1186/s13007-019-0529-2
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发表时间：
2019-11-28
期刊：
影响因子：
5.1
通讯作者：
Wu, Jianqiang
中科院分区：
文献类型：
--
作者：
Gao, Lei;Shen, Guojing;Wu, Jianqiang
背景 食草昆虫对玉米构成重大威胁。苯并恶嗪类化合物是玉米中重要的抗虫次级代谢产物，其生物合成途径已被广泛研究。然而，人们对苯并恶嗪类化合物在玉米中的调控机制知之甚少，部分原因是缺乏突变体资源和遗传转化的阻力。基于叶肉或培养细胞来源的原生质体的瞬时系统已在多种植物物种中得到利用，并已成为快速或高通量检测基因功能的强大工具。然而，这些系统尚未被用来研究次生代谢物的调节。结果优化了从黄化玉米幼苗中分离原生质体并高效转染的方案。此外，还建立了一种运行时间为 10 分钟、高灵敏度的 HPLC-MS 方法来快速检测和定量玉米苯并恶嗪类化合物。将玉米原生质体转染和 HPLC-MS 结合起来，我们通过人工 microRNA 技术过度表达或沉默，筛选了一些可能调节苯并恶嗪类生物合成的基因。结论 结合玉米原生质体转染和 HPLC-MS 分析的能力，该方法可以在选择候选基因进行植物功能分析之前，快速筛选原生质体中玉米苯并恶嗪类化合物的调控和生物合成基因。该方法还可用于研究玉米中其他次生代谢物和其他植物物种（包括那些不适合转化的植物）次生代谢物的生物合成和调节。
Background Insect herbivory poses a major threat to maize. Benzoxazinoids are important anti-insect secondary metabolites in maize, whose biosynthetic pathway has been extensively studied. However, yet little is known about how benzoxazinoids are regulated in maize, partly due to lack of mutant resources and recalcitrance to genetic transformation. Transient systems based on mesophyll- or cultured cell-derived protoplasts have been exploited in several plant species and have become a powerful tool for rapid or high-throughput assays of gene functions. Nevertheless, these systems have not been exploited to study the regulation of secondary metabolites. Results A protocol for isolation of protoplasts from etiolated maize seedlings and efficient transfection was optimized. Furthermore, a 10-min-run-time and highly sensitive HPLC-MS method was established to rapidly detect and quantify maize benzoxazinoids. Coupling maize protoplast transfection and HPLC-MS, we screened a few genes potentially regulating benzoxazinoid biosynthesis using overexpression or silencing by artificial microRNA technology. Conclusions Combining the power of maize protoplast transfection and HPLC-MS analysis, this method allows rapid screening for the regulatory and biosynthetic genes of maize benzoxazinoids in protoplasts, before the candidates are selected for in planta functional analyses. This method can also be applied to study the biosynthesis and regulation of other secondary metabolites in maize and secondary metabolites in other plant species, including those not amenable to transformation.