Subcellular Localization of the D27 Protein in Sugarcane ( Saccharum spp. Hybrids) Using an Optimized Protoplast Isolati
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RESEARCH ARTICLE
Subcellular Localization of the D27 Protein in Sugarcane (Saccharum spp. Hybrids) Using an Optimized Protoplast Isolation, Purification, and Transient Gene Expression Protocol Zhuan-di Wu1 • Xin Hu1 • Feng-gang Zan1,2 • Yong-bao Pan2 • David M. Burner1 Zheng-ying Luo1 • Jia-yong Liu1 • Li-ping Zhao1 • Li Yao1 • Yong Zhao1 • Xin-long Liu1 • Hong-ming Xia1 • Kun Yang1 • Jun Zhao1 • Pei-fang Zhao1 • Wei Qin1 • Xue-kuan Chen1,2 • Cai-wen Wu1,2
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Received: 14 April 2020 / Accepted: 31 July 2020 Ó Society for Sugar Research & Promotion 2020
Abstract The transient gene expression of protoplasts in plant is a considerable tool for gene functional research that has been widely used in gene analysis and functional characterization. Therefore, the objectives of this study were to develop a protocol for the isolation and purification of sugarcane protoplasts (Saccharum spp. hybrids), conduct transient PEG-mediated protoplast transfection with D27, and localize the D27 protein in sugarcane protoplasts. Total yield and viability of protoplasts were optimized for enzyme combination, mannitol concentration, and duration and temperature of enzymatic hydrolysis. High production of intact protoplasts (10.94 9 106 protoplasts g-1 FW) and a survival rate of [ 80.0% was achieved through enzymatic hydrolysis at constant temperature of 28 °C, 60–70 rpm min-1 for 8 h in a solution containing 2.0% cellulase R-10, 0.5% macerozyme R-10, 0.6% pectolyase Y-23, 20 mM 2-(N-morpholine) ethanesulfonic acid (MES), 20 mM KCl, and 400 mM mannitol (pH 5.7). Using GFP as the reporter gene, the protoplasts were transformed most efficiently with 25% PEG 4000 for
Zhuan-di Wu, Xin Hu and Feng-gang Zan have contributed equally to this work. & Xue-kuan Chen [email protected] & Cai-wen Wu [email protected] 1
Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences/Yunnan Key Laboratory of Sugarcane Genetic Improvement, Kaiyuan 661699, People’s Republic of China
2
United States Department of Agriculture, Agricultural Research Service, Sugarcane Research Unit, Houma, LA 70360, USA
25 min and the ScD27 protein was localized in the chloroplasts. The localization of ScD27 protein in sugarcane protoplast demonstrated that the newly developed protocol was functionally effective. This optimized sugarcane protoplast isolation, purification, and transient expression protocol lays a foundation for future molecular biology research in sugarcane. Keywords b-Carotene isomerase Protoplasts Saccharum Strigolactones Subcellular localization Transformation
Introduction Sugarcane is a high yield sugar crop in the world that widely grows in tropical area. Due to its complex polyploid genome, there has been little genetic research on this crop. The yield formation of sugarcane is a complex process influenced by physiological, biochemical, and genetic factors as well as growth environment. Strigolactones (SLs) are a group of terpenoid lactone hormones produced in the plant roots that have diverse regulatory effects on plant growth a
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