Data_Sheet_1_Optimizing the Protein Fluorescence Reporting System for Somatic Embryogenesis Regeneration Screening and Visual Labeling of Functional Genes in Cotton.DOCX

Protein fluorescence reporting systems are of crucial importance to in-depth life science research, providing systematic labeling tools for visualization of microscopic biological activities in vivo and revolutionizing basic research. Cotton somatic cell regeneration efficiency is low, causing difficulty in cotton transformation. It is conducive to screening transgenic somatic embryo using the fluorescence reporting system. However, available fluorescence labeling systems in cotton are currently limited. To optimize the fluorescence reporting system of cotton with an expanded range of available fluorescent proteins, we selected 11 fluorescent proteins covering red, green, yellow, and cyan fluorescence colors and expressed them in cotton. Besides mRuby2 and G3GFP, the other nine fluorescent proteins (mCherry, tdTomato, sfGFP, Clover, EYFP, YPet, mVenus, mCerulean, and ECFP) were stably and intensely expressed in transgenic callus and embryo, and inherited in different cotton organs derive from the screened embryo. In addition, transgenic cotton expressing tdTomato appears pink under white light, not only for callus and embryo tissues but also various organs of mature plants, providing a visual marker in the cotton genetic transformation process, accelerating the evaluation of transgenic events. Further, we constructed transgenic cotton expressing mCherry-labeled organelle markers in vivo that cover seven specific subcellular compartments: plasma membrane, endoplasmic reticulum, tonoplast, mitochondrion, plastid, Golgi apparatus, and peroxisome. We also provide a simple and highly efficient strategy to quickly determine the subcellular localization of uncharacterized proteins in cotton cells using organelle markers. Lastly, we built the first cotton stomatal fluorescence reporting system using stomata-specific expression promoters (ProKST1, ProGbSLSP, and ProGC1) to drive Clover expression. The optimized fluorescence labeling system for transgenic somatic embryo screening and functional gene labeling in this study offers the potential to accelerating somatic cell regeneration efficiency and the in vivo monitoring of diverse cellular processes in cotton.

蛋白质荧光报告系统对于深入的生命科学研究至关重要，它们为在体内可视化微观生物活动提供了系统的标记工具，并彻底革新了基础研究。棉花的体细胞再生效率低下，这给棉花的转化带来了困难。利用荧光报告系统进行转基因体胚筛选颇为有益。然而，目前棉花中可用的荧光标记系统仍然有限。为了优化棉花的荧光报告系统，使其具备更广泛的可用荧光蛋白，我们选取了涵盖红、绿、黄和青色荧光的11种荧光蛋白，并在棉花中进行了表达。除了mRuby2和G3GFP外，其他九种荧光蛋白（mCherry、tdTomato、sfGFP、Clover、EYFP、YPet、mVenus、mCerulean和ECFP）在转基因愈伤组织和胚中稳定且强效地表达，且在经过筛选的胚衍生出的不同棉花器官中得以遗传。此外，表达tdTomato的转基因棉花在白光下呈现粉红色，不仅限于愈伤组织和胚组织，还包括成熟植物的各种器官，为棉花遗传转化过程提供了可视标记，加速了转基因事件的评估。进一步地，我们构建了表达mCherry标记的细胞器标记的转基因棉花，这些标记覆盖了七个特定的亚细胞区室：质膜、内质网、液泡膜、线粒体、叶绿体、高尔基体和过氧化物酶体。我们还提供了一种简单且高效的策略，用于快速确定棉花细胞中未表征蛋白的亚细胞定位。最后，我们利用气孔特异性表达启动子（ProKST1、ProGbSLSP和ProGC1）构建了第一个棉花气孔荧光报告系统，以驱动Clover的表达。本研究中优化的转基因体胚筛选和功能基因标记的荧光标记系统，具有加速棉花体细胞再生效率和监测棉花中各种细胞过程的体内过程的潜力。