枸杞中IPP异构酶相关基因(IPI)的分离

Construction of Leaf cDNA Library of Lycium barbarum and Isolation of IPI Gene

以宁夏枸杞叶片为材料,采用异硫氰酸胍-酚-氯仿法提取完整的总RNA,利用PolyATract mRNA I-solation System(Promega公司)分离mRNA,然后利用Universal RibocloneRcDNA Synthesis System(Pro-mega公司)合成双链cDNA,在cDNA末端连接上EcoRⅠ接头,并与λExCell载体连接,利用PackageneLambda DNA Packaging System进行包装,构建出滴度为2.78×105pfu/mL,重组效率为88.1%的枸杞叶片cDNA文库。用Digoxigenin(DIG)标记龙胆草IPI探针,并对枸杞叶片cDNA文库进行筛选,获得7个IPIcDNA阳性克隆。释放质粒后,进行酶切鉴定,IPI阳性克隆cDNA插入片段的大小为1.5 kb左右。

Total RNA of leaf of Lycium Barbarum L. was isolated using guanidinium isothioccyanate and phenol chloroform, mRNA was prepared by PolyATract mRNA Isolation Systems （Promega）. Full length cDNA was synthesized by Universal RiboClone cDNA Synthesis System（Promega） and then cloned into phage λExcell vector, After Pachage in Packagene Lambda DNA Packaging System, the capacities of the libraries were measured. The capacity of the library was 2.78×10^5 pfu/ml, the recombination rates reached to 88.1%. Heterologous probe of IPI originate from Getina lutea were labeled with digoxingenin-11-dUTP （DIG）, were further used to screen the cDNA libraries of leaf of lycium barbarm. Seven IPI positive clones was obtained. The recombinants with the cDNA insert fragment were released from the positive clones and were digested with EcoR I , The cDNA fragment were 1.5 kb in IPI positive clones.