枸杞中番茄红素β-环化酶基因(LycB)的分离

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 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进行包装，在国内外首次构建出滴度为2.78×105 pfu/ml，重组效率为88.1%的枸杞叶片cDNA文库。用Digoxigenin(DIG)标记龙胆草LycB探针，并对枸杞叶片cDNA文库进行筛选，获得5个LycB 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。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。

Construction of Leaf cDNA Library of Lycium barbarum andIsolation of LycB Gene

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 2.78×105pfu/ml. the recombination rates reached to 88.1%.Heterologous probe of LycB originate from Getina lutea were labeled with digoxingenin-11-dUTP (DIG), were further used to screen the cDNA libraries of leaf of lycium barbarm. Five lycB positive clones was obtained. The recombinants with the cDNA insert fragment were released from the positive clones and were digested with EcoRⅠ, The cDNA fragment were 2.1kb in LycB positive clones. This study will lay foundations for regulating carotenoid biosynthesis via genetic engineening.