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生命科学深入研究至关重要，可提供系统性标记工具，用于活体微观生物活动的可视化观测，革新了基础研究领域。棉花体细胞再生效率低下，给棉花遗传转化工作带来了极大阻碍。借助荧光报告系统则可有效筛选转基因体细胞胚，但目前可应用于棉花的荧光标记系统仍十分有限。为优化棉花的荧光报告系统、拓展可用荧光蛋白的覆盖范围，本研究选取了涵盖红、绿、黄、青色荧光的11种荧光蛋白，并在棉花中进行了异源表达。除mRuby2与G3GFP外，其余9种荧光蛋白（mCherry、tdTomato、sfGFP、Clover、EYFP、YPet、mVenus、mCerulean及ECFP）均可在转基因愈伤组织与体细胞胚中稳定且高效表达，并能在由筛选获得的体细胞胚发育而来的棉花各组织器官中稳定遗传。此外，表达tdTomato的转基因棉花在白光下会呈现粉红色，该特征不仅存在于愈伤组织与体细胞胚组织中，也可在成熟植株的各类器官中观测到，可为棉花遗传转化过程提供可视化标记，加快转基因事件的评估效率。进一步地，本研究构建了表达mCherry标记的活体细胞器标记物的转基因棉花，其覆盖7种特定亚细胞区室：质膜、内质网、液泡膜、线粒体、质体、高尔基体及过氧化物酶体。本研究同时提供了一套简便高效的策略，可借助细胞器标记物快速鉴定棉花细胞中未表征蛋白的亚细胞定位。最后，本研究利用气孔特异性表达启动子（ProKST1、ProGbSLSP及ProGC1）驱动Clover的表达，构建了首个棉花气孔荧光报告系统。本研究优化的荧光标记系统可用于转基因体细胞胚筛选与功能基因标记，有望提升棉花体细胞再生效率，并实现棉花体内多种细胞过程的活体可视化监测。