巴西橡胶树染色体制片方法的改良及FISH信号检测

Modified Method of Chromosome Preparation and Detection of FISH Signal in Brazilian Rubber Tree(Hevea brasiliensis)

本研究以巴西橡胶树热研7-33-97品种幼叶为材料,通过改良配制纤维素酶与果胶酶混合酶的溶剂、酶解时间等参数,对酶解去壁低渗方法进行了相应改良。结果表明,在37℃下,采用磷酸盐缓冲液(NaH2PO4,31.21 g/L;Na2HPO4, 71.64 g/L;Na Cl, 8 g/L;pH=5.5)作为溶剂,配制终浓度为5%纤维素酶、4%果胶酶的酶混合液时,酶解时间为1.0 h,大大缩短了酶解时间。用该法制备的中期标本,染色体形态分散、清晰、细胞膨大良好,荧光原位杂交信号检测后,信号正常。本研究可为橡胶树细胞遗传学、基因定位提供细胞学方法。

In this study,young leaves of Brazilian rubber tree(Hevea brasiliensis)of Reyan 7-33-97 variety were used as materials.The method of enzymatic hydrolysis and low permeability for cell wall removal was modified by improving the solvent and time of enzymatic hydrolysis of mixed cellulase and pectinase.The results showed that enzymolysis time was 1.0 h by using final concentration of 5%cellulase and 4%pectinase enzyme mixture prepared at 37℃,using phosphate buffer(NaH2 PO4,31.21 g/L;Na2 HPO4,71.64 g/L;NaCl,8 g/L;pH=5.5)as the solvent,this modification greatly shortened the time of enzymatic hydrolysis.The metaphase samples prepared by this method showed that the chromosome morphology was dispersive,clear and the cells were well expanded.The signal of the fluorescence in situ hybridization was well detected.This study can provide a kind of cytological approach for studies of cytogenetics and gene localization of rubber tree.