刺鳅X染色体DNA文库的构建

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

The spiny eel (Mastacembelus aculeatus) is a species of the genus Mastacembelus (Osteichthyes, Perciformes), which mainly live in the fresh water of the south of China. The fish is particularly attractive for cytogeneti-cal 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 Oligonucleotide-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. aculeatus was constructed. The sum of the lengths of the inserted fragments is some 1.08 × 108bp, 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 entire 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 heteroehromatic 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.