摘要
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 eDNA libraries for gonads of amphioxus were constructed using the suppression subtractive hybridization （SSH） technique, eDNA 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 eDNA fragments were inserted into plasmid vectors, and subsequently the vectors were transferred into E. coli DH5α. As a result, forward and backward subtracted eDNA 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.
出处
《动物学报》
SCIE
CAS
CSCD
北大核心
2008年第3期482-488,共7页
ACTA ZOOLOGICA SINICA
基金
国家自然科学基金资助项目(No.30470938
No.30570208)~~
关键词
文昌鱼
性腺
差减CDNA文库
抑制性差减杂交
Amphioxus, Branchiostomajaponicum, Gonad, Subtractive cDNA library, Suppression subtractive hybridization
