摘要
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PolyATractmRNA Isolation System(Promega公司)分离mKNA,然后利用Universal Riboclone RcDNA SynthesisSystem(Promega公司)合成双链cDNA,在cDNA末端连接上EcoR Ⅰ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2.78×10 5pfu／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。
Total RNA of leaf of Lycium Barb arum 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 EcoR I , The cDNA fragment were 2.1kb in LycB positive clones. This study will lay foundations for regulating carotenoid biosynthesis via genetic engineening.
出处
《中国农学通报》
CSCD
2005年第1期46-49,共4页
Chinese Agricultural Science Bulletin
基金
"枸杞中番茄红素环化酶基因的分离"由吉林省科委专项课题"转基因高产VA前体(β-胡萝卜素)酵母体系的建立"资助(编号20020641)。
