Laboratory of Genetics, Wageningen University & Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
College of Horticulture Science & Technology, Hebei Normal University of Science & Technology, No 360, West of HeBei street, Qinhuang Dao, China.
BMC Plant Biol. 2022 Jul 22;22(1):360. doi: 10.1186/s12870-022-03739-x.
Metals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary Zn/Cd/Ni hyperaccumulating species, is used as a model plant for metal hyperaccumulation and phytoremediation studies. Current reverse genetic techniques to generate mutants based on transgenesis is cumbersome due to the low transformation efficiency of this species. We aimed to establish a mutant library for functional genomics by a non-transgenic approach, to identify mutants with an altered mineral profiling, and to screen for mutations in bZIP19, a regulator of Zn homeostasis in N. caerulescens.
To generate the N. caerulescens mutant library, 3000 and 5000 seeds from two sister plants of a single-seed recurrent inbred descendant of the southern French accession Saint-Félix-de-Pallières (SF) were mutagenized respectively by 0.3 or 0.4% ethyl methane sulfonate (EMS). Two subpopulations of 5000 and 7000 M2 plants were obtained after 0.3 or 0.4% EMS treatment. The 0.4% EMS treatment population had a higher mutant frequency and was used for TILLING. A High Resolution Melting curve analysis (HRM) mutation screening platform was optimized and successfully applied to detect mutations for NcbZIP19, encoding a transcription factor controlling Zn homeostasis. Of four identified point mutations in NcbZIP19, two caused non-synonymous substitutions, however, these two mutations did not alter the ionome profile compared to the wild type. Forward screening of the 0.4% EMS treatment population by mineral concentration analysis (ionomics) in leaf material of each M2 plant revealed putative mutants affected in the concentration of one or more of the 20 trace elements tested. Several of the low-Zn mutants identified in the ionomic screen did not give progeny, illustrating the importance of Zn for the species. The mutant frequency of the population was evaluated based on an average of 2.3 knockout mutants per tested monogenic locus.
The 0.4% EMS treatment population is effectively mutagenized suitable for forward mutant screens and TILLING. Difficulties in seed production in low Zn mutants, obtained by both forward and reverse genetic approach, hampered further analysis of the nature of the low Zn phenotypes.
当锌或镉等金属通过受污染的土壤或食物以高浓度暴露时，对植物和人类是有毒的。念珠藻是一种非凡的锌/镉/镍超积累物种，被用作金属超积累和植物修复研究的模式植物。由于该物种的转化效率低，目前基于转基因的反向遗传技术生成突变体较为繁琐。我们旨在通过非转基因方法建立一个用于功能基因组学的突变体文库，以鉴定具有改变的矿物质特征的突变体，并筛选锌稳态调节剂 bZIP19 在念珠藻中的突变。
为了生成念珠藻突变体文库，分别用 0.3%或 0.4%的乙基甲烷磺酸酯（EMS）对来自法国南部圣费利克斯-德帕利耶斯（SF）单一种子重复自交后代的两个姐妹植株的 3000 粒和 5000 粒种子进行诱变。用 0.3%或 0.4%EMS 处理后获得两个 5000 和 7000 株 M2 植物的亚群。用 0.4%EMS 处理的种群具有更高的突变频率，并用于 TILLING。优化了高分辨率熔解曲线分析（HRM）突变筛选平台，并成功应用于检测控制锌稳态的转录因子 NcbZIP19 的突变。在 NcbZIP19 中鉴定出的四个点突变中，有两个导致非同义取代，但与野生型相比，这两个突变并没有改变离子组特征。通过对每个 M2 植物叶片材料中的矿质浓度分析（离子组学）进行正向筛选，发现 0.4%EMS 处理的种群中存在一个或多个 20 种痕量元素浓度受影响的假定突变体。在离子组学筛选中鉴定出的一些低锌突变体没有产生后代，这说明了锌对该物种的重要性。基于每个测试单基因座的 2.3 个敲除突变体的平均值，评估了种群的突变频率。
0.4%EMS 处理的种群有效地发生突变，适合正向突变筛选和 TILLING。通过正向和反向遗传方法获得的低锌突变体在种子生产方面存在困难，这阻碍了对低锌表型本质的进一步分析。
