刺鳅X染色体DNA文库的构建  

THE CONSTRUCTION OF X CHROMOSOME LIBRARY OF SPINY EEL(MASTACEMBELUS ACULEATUS)

作　　者：陈戟[1] 赵刚[1,2] 臧亚婷[1] 余其兴[1] 刘江东[1] 

机构地区：[1]武汉大学生命科学学院,武汉430072 [2]湖北中医学院生物学教研室,武汉430061

出　　处：《水生生物学报》2009年第4期571-576,共6页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金青年科学基金项目(30400044)资助

摘　　要：刺鳅(Mastacembelus aculeatus)是具有明显X和Y异形性染色体分化的淡水鱼。本实验室通过显微切割(Mi-crodissection)和兼并引物PCR(DOP-PCR)方法,从雌性刺鳅中期染色体分裂相中分离获得X染色体并扩增其DNA,利用T载体和电转化方法,建立了刺鳅X染色体DNA质粒文库。该文库插入片段的平均长度约为500bp,理论上包含X染色体98%以上的序列。当用荧光原位杂交(FISH)来验证文库的专一性时发现,在无竞争性DNA杂交条件下,整个X和Y染色体上都表现出强烈的杂交信号,并且常染色体上也出现一些随机散布信号;当含有竞争性DNA时,常染色体上的信号消失,仅性染色体上异染色质区域保留有较强信号。就此,本文对刺鳅性染色体上的序列类型进行了探讨。The spiny eel （Mastacembelus aculeatus）is a species of the genus Mastacembelus （Osteiehthyes, Perciformes）, which mainly live in the fresh water of the south of China. The fish is particularly attractive for eytogenetical study owing to possessing well differentiated X and Y sex chromosomes. In the present study, the X chromosomes of M. aculeatus were microdissected from the metaphases of chromosomes of the female. Then, the fragments of X chromosomes were put into a micro tube and amplified using Degenerated Oligonueleotide-Primed PCR （DOP-PCR）. Thereafter, the product of PCR was connected to T vector and the plasmids were electric transformed into E. coli. As a result, the library of X chromosome of M. aeuleatus was constructed. The sum of the lengths of the inserted fragments is some 1.08 × 10^8 bp, with an average length of 500bp. It is believed that the library covered more than 98% sequences of the whole X chromosome theoretically. To check the credibility of the library, fluorescence in situ hybridization （FISH） was applied. The fragments of the library were labeled with biotin by PCR. Then the probes were hybridized to the chromosome metaphases of both sex of M. aculeatus in the absence or the presence of competitor DNA. Strong signals were detected on the en- tire sex chromosomes as well as signals dispersed on all autosomes with the condition of FISH in the absence of competitor DNA. Whereas competitor DNA was added, the signals disappeared except those showed on heterochromatie regions of X and Y chromosomes. Hence, we assorted the repetitive sequences in the X chromosome of M. aculeatus into three types. It was interpreted that the signals of FISH in the presence of competitor DNA showed the distribution of type Ⅱ repetitive sequences on the sex chromosomes of M. aculeatus.

关 键 词：刺鳅 性染色体 染色体文库 荧光原位杂交 

分 类 号：Q785[生物学—分子生物学]