Abstract：To explore the effect of gene expression on the fate of root tip cells, different types of root cells need to be grouped for further analysis. In the present study, the protoplasts of young root tips of Zea mays were obtained by enzymatic hydrolysis, and analyzed and sorted by fluorescence staining and flow cytometry. The results showed that the optimum method for obtaining protoplasts of 4~5 d young maize root tip tissue by enzymatic hydrolysis included combination of 1.0% cellulase and 0.3% pectinase, optimum osmotic pressure with 10% mannitol, optimum enzymatic hydrolysis time with 4 h and optimum pH with 5.8. The protoplasts extracted under the above optimum conditions were purified and the impurities such as cell fragments in the protoplast suspension were effectively removed. The purified protoplasts were stained by Propidium iodide (PI) and Fluorescein diacetate (FDA), and the observation under fluorescence microscopy showed that the protoplasts were highly active. For further sorting by flow cytometry, different fluorescent dyes were used. The staining effect of Hoechst33342 on active protoplasts was better than that of 4', 6-diamidyl-2-phenylindole (DAPI), most protoplasts in the system were stained with high resolution. At the same time, for Hoechst33342, the optimum dyeing temperature was 38 ℃, and the optimum dyeing pH was 10. The study provides basic data for further related researches on root tip cells in plants.
[1]董燕, 宋程程, 黄鹤.单细胞测序技术研究进展[J].化学工业与工程, 2015, 32(1):71-78
[2] 冯玉兰.浅析植物生理化研究中的DAPI技术[J]. 科技风, 2011, (24):36-36.
[3]郭艳萍, 任成杰, 李志伟, 等.玉米胚乳细胞原生质体的分离与流式纯化[J].作物学报, 2014, 40(3):424-430
[4]李旭全, 杨兵, 路岸瑞, 等.利用不同染料复合染色判定家蚕血细胞的存活状况[J].蚕业科学, 2011, 37(4):000671-675
[5] 廖嘉明.2010.热应激下拟南芥原生质体生长、发育的初步研究[D].硕士学位论文，重庆大学, 导师：王伯初.(Liao J M.2010. Preliminary Study on the Growth and Development of Protoplast of Arabidopsis Thaliana under Heat Stress[D]. Thesis for M. S., Chong Qing University, Supervisor: Wang B C)
[6]刘德培, 梁植权.细胞分化与基因表达调控[J].中国生物工程杂志, 1994, 14(1):23-25
[7]徐晓雪, 户乃丽, 孙丽娜, 等.两种碘化丙啶染色 定量方法检测 -细胞凋亡结果的比较[J].首都医科大学学报, 2014, 35(6):823-826
[8]朱海英, 张朵, 谢东甫, 等.荧光染料与干细胞研究[J].生命的化学, 2005, 25(1):52-53
[9]刘锡娟, 丁慧荣, 张宏.用和染色法检测的流式细胞方法探讨[J].北京大学学报医学版, 2010, 42(4):480-484
[10]Birnbaum K, Jung J W, Wang J Y, et al.Cell type–specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines[J].Nature Methods, 2005, 2(8):615-619
[11]Bargmann B O R, Birnbaum K D.Fluorescence Activated Cell Sorting of Plant Protoplasts[J].Journal of Visualized Experiments Jove, 2010, 36(36):e1673-e1673
[12]Boffey S A, Ellis J R, Selldén G, et al.Chloroplast Division and DNA Synthesis in Light-grown Wheat Leaves[J].Plant Physiology, 1979, 64(3):502-5
[13]Crane C A, Panner A, Murray J C, et al.PI(3) kinase is associated with a mechanism of immunoresistance in breast and prostate cancer[J].Oncogene, 2009, 28(2):306-
[14] Pena T C D L, Brown S C.Flow cytometry in plant cell biology[M]// Plant Cell Biology: A practical approach. 2001:85-106.
[15]Puite K J, Broeke W R R T.DNA staining of fixed and non-fixed plant protoplasts for flow cytometry with hoechst 33342[J].Plant Science Letters, 1983, 32(1):79-88
[16]Rommel C, Bodine S C, Clarke B A, et al.Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)KAktmTOR and PI(3)KAktGSK3 pathways[J].Nature Cell Biology, 2001, 3(11):1009-
[17]Sánchez W C, García-Ponce B, Sánchez M D L P, et al.Identifying the transition to the maturation zone in three ecotypes of Arabidopsis thaliana roots[J].Communicative & Integrative Biology, 2018, 11(1):00-00
[18]YANGHONGYUAN, XINLIWU et al.Fluorescent vital staining of plant sexual cell nuclei with DNA-specific fluorochromes and its application in gametoplast fusion[J].Cell Research, 1993, 3(2):121-130
