Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

中文翻译：

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Hypericum perforatum L. Hyp-1 基因的转基因表达改变防御相关基因的表达并调节对根癌农杆菌的抵抗力

从贯叶连翘中分离出的酚类氧化偶联蛋白 (Hyp-1) 被表征为与贯叶连翘对根癌农杆菌介导的转化的抗逆性相关的防御基因。研究人员和制药行业。然而，通过基因操作提高它们的产量是一项具有挑战性的任务，因为 H. perforatum 对根癌农杆菌介导的转化具有顽固性。在这里，酚类氧化偶联蛋白 (Hyp-1)，一种发病机制相关 (PR) 类 10 家族基因，是从根癌农杆菌处理的 H. perforatum 悬浮细胞的减法 cDNA 文库中选择的。Hyp-1 在防御 A. 在过表达 Hyp-1 的转基因烟草和 Lactuca sativa 中分析了 tumefaciens，并在长春花中对其同源 Hyp-1 基因 CrIPR 进行了沉默。结果表明，农杆菌介导的表达效率在 Hyp-1 转基因植物中大大降低。然而，CrIPR 的沉默诱导 CrPR-5 表达并降低农杆菌的表达效率。在 Hyp-1 转基因烟草植物中还分析了涉及几种防御途径的核心基因的表达。Hyp-1 的过表达导致参与生长素信号传导、基于 microRNA 的基因沉默、活性氧解毒、苯丙烷途径和 PRs 的关键基因大量下调。此外，通过共聚焦显微镜在表皮细胞的细胞核、质膜和细胞质中检测到Hyp-1。总体，