本研究以日本文昌鱼(Branchiostoma japonicum)性腺cDNA为材料,应用抑制性差减杂交技术构建文昌鱼雌雄性腺的差减cDNA文库。正向差减杂交以卵巢为试验方、精巢为驱动方,反向差减杂交以精巢为试验方、卵巢为驱动方,将所获差减cDNA片段克隆插入质粒表达载体,转化大肠杆菌DH5α,最后获得的正、反向差减文库分别含459、243个重组子。PCR扩增鉴定正、反向差减cDNA文库的插入片段,其中90%左右的克隆皆能扩增出有效产物,插入片段范围为200-600bp,符合差减文库PCR产物片段的大小。随机选取30个阳性克隆测序分析,得到26有效基因片段,进一步从中选取16个序列,用实时定量PCR对文库质量进行验证,结果表明所建文库能够达到富集雌雄性腺差异表达基因的目的。文昌鱼性腺差减文库的构建,为进一步分离、鉴定性腺分化和发育相关基因奠定了基础。In the present study,two subtracted cDNA libraries for gonads of amphioxus were constructed using the suppression subtractive hybridization(SSH) technique.cDNA from ovary was adopted as tester for the construction of a forward library,and then as a driver for the reverse library construction.Two-directional subtracted cDNA fragments were inserted into plasmid vectors,and subsequently the vectors were transferred into E.coli DH5α.As a result,forward and backward subtracted cDNA libraries containing 459 and 243 clones respectively,were obtained and subjected to PCR analysis.Results confirmed that about 90% of the clones contained inserts of 200-600 bp in the two libraries,in accord with our prediction.Thirty of the positive clones were selected and sequenced randomly,and,of those sequenced clones,twenty-six gene fragments were obtained.Then,sixteen sequenced genes were further analyzed via Real-time PCR method.The results indicated that these genes were expressed differently between male and female gonads.The subtractive cDNA libraries of amphioxus gonads provide a foundation for further studies of the genes related to sexual differentiation and gonadal development.国家自然科学基金资助项目(No.30470938,No.30570208)~
