Lei Shanshan, Zhu Yaolong, Jia Weiyu, Zhang Jing, Chi Yingjun, Xu Bin
College of Agro-Grassland Science, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.
Plant Methods. 2024 May 9;20(1):67. doi: 10.1186/s13007-024-01192-5.
With the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes' effects on heat and oxidative stress responses.
In this study, a perennial ryegrass (Lolium perenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and HO-induced oxidative stress on protoplasts' viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1/LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts' viability rates after heat stress.
This protoplast-based rapid stress regulatory gene identification assay was briefed as 'PRIDA' that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.
随着组学数据的不断积累，人们期望有一种高效且省时的瞬时检测方法来表达目标基因。叶肉原生质体保留了大多数应激生理反应和细胞活动，如同完整植株一样，为研究目标基因对热应激和氧化应激反应的影响提供了一种替代性瞬时检测方法。
在本研究中，建立了一种基于多年生黑麦草（Lolium perenne L.）叶肉原生质体的检测方法，以有效上调或下调目标基因。可使用RT-qPCR对目标基因的相对表达水平进行定量，并且可以定量测量热应激和过氧化氢诱导的氧化应激对原生质体活力的影响。通过鉴定黑麦草中潜在的热传感器基因LpTT3.1/LpTT3.2证明了该检测方法的实用性，过表达这些基因会显著改变热应激后原生质体的活力率。
这种基于原生质体的快速应激调控基因鉴定检测方法简称为“PRIDA”，它将补充稳定遗传转化研究，以快速鉴定多年生黑麦草和其他草种中的候选应激调控基因。
