【摘要】 免疫荧光技术在植物染色体研究中具有重要意义,但在甘蔗等复杂多倍体作物中的应用仍存在技术挑战。本研究以甘蔗近缘野生种滇蔗茅(Erianthus rockii,‘Yunnan83-224’)、河八王(Narenga porphyrocoma,‘Guangdong64’)和斑茅(Tripidium arundinaceum,‘Yunnan2012-6’)为材料,系统优化了染色体免疫荧光实验体系。通过对预处理时间、甲醛固定时间等关键参数的梯度优化,确定了对二氯苯-α-溴代萘处理1.5 h和4%多聚甲醛固定5 min为最适条件。利用水稻(Oryza sativa)着丝粒特异性组蛋白H3变体(centromeric histone H3, CENH3)多克隆抗体成功检测到各材料中着丝粒蛋白的特异性信号:滇蔗茅30个信号、河八王30个信号、斑茅40个信号,信号数目与各物种染色体数目一致。进一步建立免疫荧光与荧光原位杂交联合分析(immunofluorescence and fluorescence in situ hybridization, Immuno FISH)技术,实现了着丝粒蛋白CENH3与着丝粒DNA序列So1的共定位分析,证实甘蔗近缘种中着丝粒结构的保守性。本研究建立的优化体系为甘蔗及其近缘种的分子细胞遗传学研究提供了可靠的技术平台,为深入解析复杂多倍体甘蔗的染色体行为和着丝粒功能奠定了重要基础。更多还原

【Abstract】 Immunofluorescence technology is of great significance in plant chromosome research;however,its application in complex polyploid crops such as sugarcane remains technically challenging.This study focused on systematically optimizing a chromosome immunofluorescence experimental system using wild relatives of sugarcane:Erianthus rockii ’Yunnan83-224’,Narenga porphyrocoma’Guangdong64’,and Tripidium arundinaceum ’Yunnan2012-6’.Through gradient optimization of key parameters,including pretreatment and formaldehyde fixation durations,the optimal conditions were determined to be a 1.5 h treatment with p-dichlorobenzene-α-bromonaphthalene,followed by a 5 min fixation in 4% paraformaldehyde.Using a polyclonal antibody against the centromeric histone H3 variant(CENH3) in Oryza sativa,we successfully detected specific signals for centromeric proteins in each material:30 signals in Erianthus rockii,30 signals in Narenga porphyrocoma,and 40 signals in Tripidium arundinaceum,which are consistent with the chromosome numbers of the respective species.Furthermore,a combined immunofluorescence and fluorescence in situ hybridization(ImmunoFISH) technique was established,enabling the co-localization analysis of the centromeric protein CENH3 with the centromeric DNA sequence Sol.This confirmed the structural conservation of centromeres in these sugarcane-related genera.The optimized system established in this study provides a reliable technical platform for molecular cytogenetic research on sugarcane and its relatives,laying a crucial foundation for the in-depth analysis of chromosome behavior and centromere function in complex polyploid sugarcane.更多还原