中华绒螯蟹卵巢差减cDNA文库的构建

Construction on Subtractive cDNA Library of Mitten Hand Crab Ovaries

应用抑制性差减杂交技术构建中华绒螯蟹卵巢两个发育时期的差减cDNA文库。正向差减杂交以Ⅲ期卵巢为试验方、Ⅱ期卵巢为驱动方 ,反向差减杂交以Ⅱ期卵巢为试验方、Ⅲ期卵巢为驱动方 ;将所获差减cDNA片段克隆入质粒表达载体 ,转化大肠杆菌JM 10 9。最后获得的正、反向差减文库分别含 86 3、 36 0个重组子。PCR扩增鉴定正、反向差减cDNA文库的插入片段平均大小分别为 36 0和 16 0bp 。

Using suppression subtractive hybridization (SSH),two subtracted cDNA libraries were constructed for mitten hand crab ( Eriocheir japonica sinensis ) ovaries in two periods of developments.Two directional (forward and backward) SSH was performed on cDNAs of mitten hand crab ovaries in periods Ⅱ or Ⅲ.Two directional subtracted cDNA fragments were cloned into PinPoint plasmid vectors,and the vectors were transformed into E coli JM109.Finally,the forward and backward subtracted cDNA libraries (863 and 360 clones,respectively) were obtained.By PCR detection,the length of the inserted fragments of the forward and backward subtracted cDNA libraries was 360 and 160 base pairs on average,respectively.The results showed that the subtracted libraries constructed were suitable for further study on the genes related to the development of the mitten hand crab ovary.