Zhou Jie, Li Dongyue, Zheng Chao, Xu Rumeng, Zheng Ersong, Yang Yong, Chen Yang, Yu Chulang, Yan Chengqi, Chen Jianping, Wang Xuming
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ministry of Agriculture Key Laboratory for Plant Protection and Biotechnology, Zhejiang Provincial Key Laboratory of Plant Virology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China.
College of Plant Protection, Northwest A&F University, Yangling, China.
Front Plant Sci. 2020 Dec 11;11:602680. doi: 10.3389/fpls.2020.602680. eCollection 2020.
Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter () in rice and its application for accurate transgene expression. Our results indicate that the high expression of the promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a β-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by -mediated selection. Distinct β-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.
以期望的方式精确表达转基因对于植物基因工程和基因功能解析很重要，但要获得不受用于表达选择标记基因的组成型启动子（如 cauliflower mosaic virus (CaMV) 35S 启动子）干扰的特异性转基因表达是一项挑战。因此，迫切需要避免这些不适当调控的解决方案。在本研究中，我们报道了水稻中一个愈伤组织强启动子（）的特性及其在精确转基因表达中的应用。我们的结果表明，该启动子在愈伤组织中的高表达能够实现与 CaMV 35S 启动子相当的潮霉素高效筛选，而其在其他组织中的表达较低。为了评估可能的渗漏效应，在通过介导的筛选产生的转基因水稻植株中观察了由六个涉及激素信号传导、病原体反应、细胞命运决定和增殖的特异性启动子驱动的β-葡萄糖醛酸酶报告基因的表达。在为每个启动子获得的大多数转基因系中一致发现了明显的β-葡萄糖醛酸酶表达。此外，我们应用这些特异性标记系来研究根细胞对外源细胞分裂素和生长素处理的反应。结果表明，细胞分裂素对根生长的抑制在高浓度和低浓度下受到不同的调控。总之，我们已经确立了使用愈伤组织特异性启动子依赖性筛选来减轻水稻中转基因错误表达的可行性。通过实现高效转化，将很容易获得具有可靠转基因表达的水稻植株以用于广泛应用。
