大白菜适于FISH的染色体制片技术研究-中国农学通报.PDF

中国农学通报 2011,27(10):284-288 Chinese Agricultural Science Bulletin 大白菜适于FISH的染色体制片技术研究 李玉玺，轩淑欣，王彦华，李晓峰，李九欢，申书兴 （河北农业大学园艺学院，河北保定071001） 摘 要：采用酶解去壁低渗法进行大白菜染色体制片，研究了影响其有丝分裂相指数和制片质量的根尖 长度、预处理药剂和时间、前低渗和酶解的温度与时间等相关因子，并对大白菜制片质量进行了25S rDNA 的FISH 检测。结果表明：当大白菜生长旺盛的根尖长为1.5~2.0 cm 时中期分裂相指数最高；最佳 预处理药剂为0.002 mol/L 8-羟基喹啉，当预处理时间为2 h 时能获得较多的分裂相和清晰的染色体形 态；0.075 mol/L KCl 中25℃前低渗0.5 h ，然后采用2%纤维素酶和2%果胶酶混合液4℃消化5~6 h 或25℃ 消化2~2.5 h ，染色体分散良好，酶解完全，无细胞质残留，制备的染色体标本可不经预处理直接用于 FISH 杂交。本研究结果为在大白菜上开展FISH 技术的相关研究奠定了基础。 关键词：大白菜；分裂相指数；染色体制片；FISH 中图分类号：S634.1 文献标志码：A 论文编号：2011-0321 StudiesontheChromosomePreparationforFISHinChineseCabbage LiYuxi,XuanShuxin,WangYanhua,LiXiaofeng,LiJiuhuan,ShenShuxing (College of Horticulture,Agriculture University of Hebei ,BaodingHebei071001) Abstract: Adoptingthewalldegestedwithenzymeandlowosmoticmethodtopreparethechromosome productionofChinesecabbage,somerelatedfactorseffectingonthemitoticindexandchromosomepreparation qualitywerestudied,includingtheroottiplength,pretreatmentmedicamentandtime,thetempretureandtime ofpre-lowosmoticandenzymehydrolysis,andfluorescence in situ hybridizationwasalsousedtodetectthe chromosomepreparationqualityusingthe 25SrDNAasprobe.Theresultsshowedthatthebestmethodwas treatingtheroottipswith 0.002 mol/L 8-hydroxyquinolinefor 2 hwhentherootlengthwas 1.5-2 cm, 0.075 mol/L KCl at 25℃ for 0.5 h pre-low osmosis treatment, and the optimum conditions for enzyme hydrolysiswereat 4℃for 5-6 hor 25℃for 2-2.5 hwith 2%cellulaseand 2%pectinase.Thehighquality chromosomepreparationwithscatteringchromosomesandcleanbackgroundwasachievedbyabovemethod, andcouldbeusedtoFISHwithoutneedingpretreatment.Thisstudywillsetupthefoundationfortherelated researchaboutFISHinChinesecabbage. Keywords:Chin