A de novo regulation design shows an effectiveness in altering plant secondary metabolism,Journal of Advanced Research

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A de novo regulation design shows an effectiveness in altering plant secondary metabolism
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2021-06-20 , DOI: 10.1016/j.jare.2021.06.017
Mingzhuo Li ₁ , Xianzhi He ₁ , Christophe La Hovary ₁ , Yue Zhu ₁ , Yilun Dong ₁ , Shibiao Liu ₁ , Hucheng Xing ₁ , Yajun Liu ₁ , Yucheng Jie ₁ , Dongming Ma ₁ , Seyit Yuzuak ₁ , De-Yu Xie ₁

Affiliation

Introduction

Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants.

Objectives

We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering.

Methods

G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism.

Results

PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes.

Conclusion

G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

中文翻译：

从头调控设计显示出改变植物次生代谢的有效性

介绍

转录因子（TF）和顺式调节元件（CRE）控制参与各种生物过程的基因转录。我们假设 TF 和 CRE 可以成为从头调控设计以工程植物的有效分子工具。

目标

我们选择了两种拟南芥 TF 类型和两种烟草 CRE 类型来设计De Novo调控并评估其在植物工程中的有效性。

方法

在四个烟草 JAZ ( NtJAZ ) 启动子中鉴定出 G 盒和 MYB 识别元件 (MRE)。在一个尼古丁途径基因启动子中鉴定出 MRE 样和 G-box 样元件。 TF 筛选导致选择拟南芥生产花青素色素 1 (PAP1/MYB) 和透明种皮 8 (TT8/bHLH)。从商业窄叶 Madole (NL) 和 KY171 (KY) 烟草品种中克隆了两个NtJAZ和两个尼古丁途径基因启动子。进行电泳迁移率变动测定 (EMSA)、交联染色质免疫沉淀 (ChIP) 和双荧光素酶测定来测试 PAP1 (P)、TT8 (T)、PAP1/TT8 一起以及 PAP1 的启动子结合和激活/TT8/透明 Testa Glabra 1 (TTG1) 复合物。设计并合成 DNA 盒，用于将 PAP1 和 TT8 堆叠并表达在一起。按照工业和转基因协议进行了三年的田间试验。完成基因表达和代谢谱分析以表征植物次生代谢。

结果

PAP1、TT8、PAP1/TT8 和 PAP1/TT8/TTG1 复合物结合并激活NtJAZ启动子，但不结合尼古丁途径基因启动子。工程化的红色 P+T 植物显着上调了四个NtJAZ ，但下调了烟草生物碱的生物合成。田间试验表明，在大多数或所有经过改造的 P+T 和 PAP1 基因型的烤烟叶中，五种烟草生物碱和四种致癌烟草特有的亚硝胺显着减少。