适于荧光原位杂交的大薯叶片染色体制片技术

Chromosome Preparation of Leaf Cells of Dioscorea alata L.for FISH

为克服利用根尖制备染色体标本的局限性,拟通过优化酶解去壁低渗法中的材料幼嫩程度、预处理方法以及酶解时间,建立大薯嫩叶染色体制备技术体系。结果表明:取刚展开的大薯嫩叶,用0.002 mol·L^-18-羟基喹啉于4℃下预处理2 h,用3.5%纤维素酶和1.75%的果胶酶混合溶液于37℃下解离1 h,能获得较好的大薯染色体制片。最后,以大薯45S rDNA序列为探针,对有丝分裂间期和中期细胞进行荧光原位杂交检测,杂交信号清晰稳定。本研究建立的适于荧光原位杂交的大薯嫩叶染色体制备技术可为分子细胞遗传学研究提供一种简便实用的细胞学方法。

In order to overcome the limitations of root tip for chromosome preparation,tender leaves of Dioscorea alata L.Da40 were used to prepare chromosome samples by using enzymatic hydrolysis and wall degradation hypotonic method.Some important factors during the preparation,such as leaf age,pretreatment methods,enzymatic hydrolysis time,were discussed to optimize the chromosome preparation.The results showed that using the immediately expanded tender leaves pretreated with the solution of 0.002 mol·L^-18-hydroxyquinoline at 4℃for 2 hours,and then enzymolyzed with the mixture of 3.5%cellulase and 1.75%pectinase for 1 hour at 37℃were proved to be the best for the chromosome preparation.Finally,fluorescence in situ hybridization was used with a probe of 45 S rDNA sequence to detect the signal of 45 S rDNA on chromosome,and these signals were recognizable and stable.These results indicated that tender leaf could be used for chromosome preparation of D.alata L.plants for FISH analysis,which provided a convenient and practical cytological method for the molecular cytogenetic study of D.alata L..