基于双向启动子的植物多基因表达系统构建及其在油松瞬时转化中的应用

Engineered plant bidirectional promoter for multigene expression: application in transient transformation of Chinese pine

摘要

【目的】本研究旨在开发一种结构紧凑、表达高效且序列同源性低的双向启动子多基因表达系统，为针叶树本源基因功能验证提供高效平台。【方法】将经验证的植物高效短转录增强子（STE）串联（4 × STE），两端反向连接非重复的CaMV 35S与CsVMV最小核心启动子，从源头上降低序列同源性；同时结合P2A自剪切肽多顺反子策略，构建单个双向启动子驱动5个基因协同表达的系统。通过发根根瘤菌介导的瞬时转化体系，在模式植物烟草及裸子植物油松下胚轴中验证该系统的表达效率与跨物种适用性。【结果】（1）成功构建了结构紧凑的双向启动子，在烟草与油松中均能同时驱动上游与下游基因的高效表达，证实该系统具有跨物种适用性；（2）下游甜菜红素合成途径基因（ CYP76 AD1、 DODA、 GT）成功表达并催化酪氨酸转化为甜菜红素，组织呈现明显红色；（3）上游 GUS基因的表达强度虽低于35S启动子，但仍保持较高水平， LUC亦检测到稳定发光信号。【结论】本研究构建的双向启动子多基因表达系统，有效规避了传统多基因载体中重复序列引发的基因沉默风险，解决了载体结构冗余与表达效率之间的矛盾。该系统为植物多基因遗传改良提供了新型工具，在针叶树等难转化物种的基因功能验证与分子育种中具有重要应用价值。

Abstract

[Objective] This study aimed to develop a compact, efficient and low-sequence-homology bidirectional promoter-based multigene expression system, providing a high-performance platform for native gene functional validation in conifers. [Method] A validated plant short transcriptional enhancer (STE) was tetramerized (4 × STE) and flanked by two non-repetitive minimal core promoters, CaMV 35S and CsVMV, in opposite orientations to minimize source sequence homology. Combined with a P2A self-cleaving peptide-based polycistronic strategy, a single-promoter system for the simultaneous expression of five genes was constructed. The expression efficiency and cross-species applicability of this system were verified in the model plant tobacco (Nicotiana benthamiana) and hypocotyls of the gymnosperm Pinus tabuliformis via Rhizobium-mediated transient transformation. [Result] (1) A compact bidirectional promoter was successfully constructed, driving robust and simultaneous expression of both upstream and downstream genes in tobacco and P. tabuliformis, thereby confirming that the system has certain species applicability. (2) The downstream betalain biosynthesis genes (CYP76 AD1, DODA, GT) were successfully expressed and catalyzed the conversion of tyrosine into betalains, resulting in distinct red pigmentation in tissues. (3) Although the expression intensity of the upstream GUS gene was lower than that driven by the 35S promoter, it remained at a relatively high level, and stable luminescent signals were also detected for LUC. [Conclusion] The bidirectional promoter-based polygenic expression system established in this study effectively avoids the risk of transgene silencing caused by repetitive sequences in traditional polygenic vectors, resolving the trade-off between vector structural redundancy and expression efficiency. This system provides a novel tool for plant polygenic genetic improvement and exhibits significant application potential in gene function characterization and molecular breeding of recalcitrant species, including conifers.

关键词

双向启动子 / 多基因共表达 / 油松 / 短转录增强子 (STE) / 转录调控元件 / P2A

Key words

bidirectional promoter / multigene co-expression / Pinus tabuliformis / short transcriptional enhancer / transcriptional regulatory element / P2A

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基金资助

国家自然科学基金项目(32441043)

中央高校基本科研业务费(QNTD202502)