【作者】 张爱香；

【导师】 王罡；

【作者基本信息】 中国人民解放军军需大学 ， 植物病理学， 2003， 硕士

【摘要】 本研究以宁夏枸杞叶片及果实为材料，采用异硫氰酸胍—酚—氯仿法提取完整的总RNA，利用PolyATract mRNA Isolation System(Promega公司)分离mRNA，然后利用Universal Riboclone ~RcDNA Synthesis System (Promega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Packagene Lambda DNA Packaging System进行了包装，在国内外首次构建出滴度分别为2.78×10~5 pfu／ml、8.4×10~4 pfu/ml，重组效率为88.1％、86.9％的枸杞叶片及果实cDNA文库。用Digoxigenin(DIG)标记龙胆草LycB探针、IPI探针、PSY探针、ZDS探针、LycE探针，并对枸杞叶片及果实cDNA文库进行筛选，获得8个LycB cDNA阳性克隆及7个IPI cDNA阳性克隆。释放质粒后，进行酶切鉴定，LycB阳性克隆cDNA插入片段的大小为2.1kb左右，IPI阳性克降cDNA插入片段的大小约为1.5kb。本研究为利用基因工程手段来调控类胡萝卜素的生物合成奠定了基础。更多还原

【Abstract】 Total RNA of leaf and fruit Lycium Barbarum L. was isolated using guanidinium isothioccyanate and phenol hloroform. mRNA was prepared by PolyATract mRNA Isolation Systems (Promega). Full條ength cDNA was synthesized by Universal RiboClone cDNA Synthesis System(Promega) and then cloned into phage # Excell vector, After Pachage in Packagene lambda DNA Packaging System, the capacities of the libraries were measured. The capacities of the libraries were 2.78 X 105pfu/ml and 8.4 X 104 pfu/ml. the recombination rates reached to 88.1% and 86.9%.Heterologous probe of Lye-. IPI PSY, ZDS, LycE originate from Getina lutea were labeled with digoxingenin-11-dUTP (DIG), were further used to screen the cDNA libraries of leaf and fruit of lycium barbarm. Eiight lycB positive clones were obtained and seven IPI positive clones were obtained. The recombinants with the cDNA insert fragment were released from the positive clones and were digested with EcoR I , The cDNA fragment were 2.1kb and 1.5kb in lycB and IPI positive clones respectively. Thisstudy will lay foundations for regulating carotenoid biosynthesis via genetic engineening.更多还原