Embrapa Savannah, Planaltina, DF, Brazil.
National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brazil.
Planta. 2022 Sep 6;256(4):69. doi: 10.1007/s00425-022-03980-6.
The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (β-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher β-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression of this reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.
pUceS8.3 是一个组成型基因启动子，具有在受到害虫攻击的植物组织中异位和强基因过表达或生物活性分子的潜力，包括线虫诱导的巨型细胞或虫瘿。大豆（Glycine max）是全球最重要的农业商品之一，也是主要的蛋白质和油源。在此，我们鉴定了大豆泛素结合（E2）酶基因（GmUBC4；Glyma.18G216000），该基因在植物寄生线虫攻击 Anticarsia gemmatalis 和 Meloidogyne incognita 诱导的虫瘿时显著上调。使用转基因拟南芥和 G. max 系在线对 GmUBC4 启动子序列及其不同模块进行了功能表征。其全长转录调控区（启动子和 5´-UTR 序列，命名为 pUceS8.3 启动子）能够在转基因拟南芥系的不同组织中驱动更高水平的 uidA（β-葡萄糖醛酸酶）基因表达，与三个缩短的模块和 p35SdAMV 启动子相比。值得注意的是，当完整的 pUceS8.3 启动子驱动该报告基因表达时，在 M. incognita 诱导的巨型细胞中显示出更高的β-葡萄糖醛酸酶（GUS）酶活性。此外，在转基因大豆系中，pUceS8.3 启动子成功控制了线虫特异性 dsRNA 分子的过表达。这里使用的 RNAi 基因构建体旨在转录后下调先前从 Heterodera glycines 和 M. incognita 中鉴定的 pre-mRNA 剪接因子基因。在感染寄生前第二阶段（ppJ2）线虫后，从六个含有 RNAi 基因构建体的转基因大豆系中选择了进行分子特征分析。与野生型对照植物相比，在这些转基因大豆系中观察到寄生后 M. incognita 产生的卵数减少了 71%至 92%。本数据表明，pUceS8.3 是一个能够有效驱动转基因大豆系线虫诱导的巨型细胞中 dsRNA 过表达的基因启动子，并可成功应用于生物技术资产，以产生对根结线虫和其他害虫具有改良抗性的转基因作物。
