摘要
【目的】以胡桃楸雄花序为材料,建立染色体形态良好、染色体分散且无细胞质背景的胡桃楸高质量染色体制片技术,为深入开展胡桃楸分子细胞遗传学研究提供参考。【方法】通过卡宝品红压片观察花药发育进程,采集花药细胞处于分裂旺盛阶段的雄花序,分别采用1 MPa一氧化二氮(N2O,笑气)、0.7 mmol·L^(-1)环己酰胺、2 mmol·L^(-1)8-羟基喹啉对花序进行不同时间预处理。剥开花序取出花药,酶解后制成悬液,在55℃的烤片机上涂片。以45S rDNA和5S rDNA为探针,对中期染色体进行原位杂交。【结果】1)当雄花序颜色鲜绿、长度达到约1.5 cm、花药顶端变为红色时,花药细胞分裂旺盛,可以观察到大量小孢子母细胞和中期分裂相。因此,为了获得丰富的有丝分裂中期分裂相,取材最佳时机是雄花序中上部的花药顶端变红时,可以保证雄花序中大部分花药处于旺盛分裂期。2)2 mmol·L^(-1)8-羟基喹啉预处理4、6、8 h的染色体都均凝缩不充分,边缘不清晰,拖尾明显,且大部分着丝粒无法辨认;0.7 mmol·L^(-1)环己酰胺预处理4 h的染色体较长,凝缩不充分,拖尾严重,预处理6 h的染色体凝缩适当,形态清晰,大部分着丝粒可以辨认,预处理8 h的染色体边缘清晰,但凝缩过度,着丝粒不明显;1 MPa N2O预处理2 h的染色体长度适中,但凝缩不充分,边缘模糊,处理3、4 h的染色体凝缩程度都较高,染色体较为粗短,染色体间形态差异不明显;此外,N2O预处理4 h的染色体中,有部分会出现粘连拉丝的情况。因此,胡桃楸雄花序最合适的预处理条件为0.7 mmol·L^(-1)环己酰胺处理6 h。3)胡桃楸染色体数为32,染色体基数为16(2n=2x=32)。45S rDNA和5S rDNA探针在胡桃楸染色体上均产生明亮的FISH信号。45S rDNA的杂交信号位于1对中着丝粒染色体的着丝粒附近,2条染色体上的信号强度相近;5S rDNA的杂交信号位于另外1对中着丝粒染色体的�
【Objective】This study aims to establish a high-quality chromosome preparation method for Juglans mandshurica with dispersed and good morphology chromosome using the male inflorescence of J.mandshurica as materials,so as to lay the foundation for further research on the molecular cytogenetics of J.mandshurica.【Method】The process of anther development was observed using carbol fuchin staining.Male inflorescences at the vigorous anther cell division stage were collected and pretreated with 1 MPa nitrous oxide(N2O),0.7 mmol∙L^(-1) cycloheximide,and 2 mmol∙L^(-1)8-hydroxyquinoline for different durations,respectively.The inflorescence was peeled off to take out the anthers that were enzymatically digested to obtain suspensions.Then suspensions were spread on a slide using a needle on a heater at 55°C.The mitotic metaphase chromosomes were used for fluorescence in situ hybridization(FISH)with 45S rDNA and 5S rDNA probes.【Result】1)When the male inflorescence became a fresh green color,reached a length of about 1.5 cm,and the tip of the anther turned red,the anther cell division was vigorous and a large number of microsporocyte mother cells and mitotic metaphase chromosomes could be observed.Therefore,the optimal sampling time was when the anthers in the upper part of the male inflorescence turned red,which can ensure that most of the anthers were in the vigorous cell division period.2)Chromosomes treated with 2 mmol∙L^(-1)8-hydroxyquinoline for 4,6,and 8 hours were condensed insufficiently,had unclear margins,and exhibited obvious trailing.Most of the centromeres could not be recognized.Chromosomes treated with 0.7 mmol∙L^(-1) cycloheximide for 4 hours were long and condensed insufficiently,with severe trailing.Chromosomes treated for 6 hours showed appropriate condensation and good morphology,with most of the centromeres recognizable.Chromosomes treated for 8 hours had clear margins but excessive condensation,with unclear centromeres.Chromosomes treated with 1 MPa N2O for 2 hours had appropriate le
作者
倪润欣
宁仪杭
王子玥
刘光欣
甄艳
席梦利
Ni Runxin;Ning Yihang;Wang Ziyue;Liu Guangxin;Zhen Yan;Xi Mengli(State Key Laboratory of Tree Genetics and Breeding Co-Innovation Center for the Sustainable Forestry in Southern China Nanjing Forestry University,Nanjing 210037)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2024年第10期50-55,共6页
Scientia Silvae Sinicae
基金
国家自然科学基金项目(31670603)。
关键词
胡桃楸
雄花序
染色体制片
荧光原位杂交(FISH)
Juglans mandshurica
male inflorescence
chromosome preparation
fluorescence in situ hybridization(FISH)
