日本血吸虫中国大陆株雌性特异性DNA片段及重复序列的分离与鉴定

FEMALE-SPECIFIC DNA SEQUENCES AND REPETITIVE ELEMENTS ISOLATED AND IDENTIFIED FROM SCHISTOSOMA JAPONICUM(CHINESE MAINLAND STRAIN)

目的 分离鉴定日本血吸虫基因组 DNA的雌性特异性片段及 DNA重复序列。方法 分别提取雌、雄日本血吸虫基因组 DNA ,制备检测子和驱动子 ;利用代表性差异分析 (RDA)及 PCR进行扣除杂交 ,获得目的 DNA片段 ;对所获目的 DNA片段用低熔点凝胶回收 ;转质粒载体、重组质粒载体的阳性克隆筛选、测序 ;用 Southern blotting对目的 DNA片段进行鉴定。结果 RDA及 PCR获得 5个目的 DNA片段 ;并转质粒载体测定了 5个目的 DNA片段 (P1～ P5 )的序列。 Southern blotting显示 DNA片段 P1和 P2均与雌、雄基因组 DNA强烈杂交 ;DNA片段 P3与雌性基因组 DNA的杂交强度明显高于与雄性基因组 DNA的杂交强度 ;DNA片段 P4和 P5仅与雌性基因组 DNA杂交 ,但不与雄性基因组 DNA杂交。结论 DNA片段 P1和 P2为日本血吸虫 DNA重复序列 ,并以多拷贝存在于日本血吸虫基因组 DNA中 ;DNA片段 P3存在于日本血吸虫性染色体 W和 Z上 ,在性染色体 W的拷贝数多于在性染色体 Z上的拷贝数 ;DNA片段 P4和 P5仅存在于日本血吸虫性染色体 W上 ,为日本血吸虫雌性特异性片段。

Objective To isolate and identify the female-specific DNA sequences and repetitive elements from genomic DNA of Schistosoma japonicum.Methods The genomic DNA of female and male Schistosoma japonicum were extracted and the tester and driver were prepared. The target DNA sequences were obtained by subtractive hybridization with Representational Difference Analysis (RDA) and PCR, and retrieved with the low melting point gel. The target DNA sequences were joined to the plasmid vector. The positive clones were screened with recombinant plasmid vector and the DNA sequences were tested. The target DNA sequences were identified by Southern blotting. Results Five DNA elements were isolated by subtractive hybridization with RDA and PCR from genomic DNA of Schistosoma japonicum. The sequences of 5 DNA elements were detected. The results of Southern blotting showed that both of the DNA element P1 and P2 hybridized with the female and male genomic DNA of schistosomes, also hybridized with the female and male PCR-amplified product from the female or male schistosome strongly. The DNA element P3 hybridized with the female and male genomic DNA of schistosome, also hybridized with the PCR-amplified product from the female or male schistosome, but the intensity of hybridizating with the female genomic DNA was stronger than that with the male genomic DNA. The DNA element P4 and P5 hy-bridized with the female genomic DNA and the female PCR-amplified product only. Conclusion The DNA element P1 and P2 are the DNA repetitive elements in the genomic DNA of Schistosoma japonicum with multi-copies. It is possible that P3 exists in the sex chromosome W and Z of Schistosoma japonicum, and the copy number existing in the sex chromosome Z is lower than that in the sex chromosome W. The DNA element P4 and P5 are the female-specific sequences of Schistosoma japonicumexisting in the sex chromosome W.