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为了克隆和转化多枝赖草(Leymus multicaulis)的耐黄矮病、耐蚜虫、耐干旱、耐盐碱等抗逆性基因,利用脉冲电泳分离纯化2Mb以上核DNA,酶切后回收10～200kb DNA片段按大小分5组与TAC载体连接后电转化导入细菌DH10B,在卡那霉素和蔗糖选择压下均有阳性克隆.用两种TAC载体pYLTAC17和pYLTAC747H/sacB分别构建了文库Ⅰ和Ⅱ,约16.5和23.6万个克隆,估计覆盖3～5倍多枝赖草基因组大小.文库以混合克隆形式保存在12×2块深孔96孔板中,每孔1.2mL菌液中含300～600个克隆,40多万个克隆转存到3块384孔板,留24个孔存叶绿体和线粒体DNA探针菌液,可用于后续文库鉴定.此外,从文库Ⅰ和Ⅱ分别挑取2501和2890个单克隆存于14块384孔板中.17块384孔板都用GeneTACTMG3复制了2份,点高密度杂交膜6张,以多枝赖草谷胱甘肽还原酶基因5′RACE-GR和3’RACE-GR为探针初步筛选到19个阳性克隆.两文库为多枝赖草抗性基因的克隆、物理图谱的构建、功能验证等基因组有关研究奠定了基础。
In order to clone and transform the resistant genes of yellowish dwarfish, resistant to aphids, drought resistance and salt tolerance in Leymus multicaulis, the nuclear DNA above 2Mb was isolated and purified by pulse electrophoresis. After digestion, 10-200kb The DNA fragments were ligated with TAC vector in 5 groups and transformed into the bacterium DH10B by electroporation.The positive clones were selected by both kanamycin and sucrose selection.The libraries I and II were constructed with two TAC vectors pYLTAC17 and pYLTAC747H / , About 16.5 and 236,000 clones, which are estimated to cover 3 to 5 times the genome size of Larix plants. The library was kept as a hybrid clone in 12 × 2 deep-well 96-well plates containing 1.2 × 10 ~ 600 clones and more than 400,000 clones were transferred to three 384-well plates and 24 wells were reserved for chloroplast and mitochondrial DNA probes for further library identification.In addition, libraries 2501 and 2890 single clones were deposited in 14 384-well plates, and 17 384-well plates were duplicated with GeneTACTMG3 and 6 high-density hybrid membranes with the 5 ’RACE-GR And 3’RACE-GR as probes, 19 positive clones were screened. Long, physical map construction, functional verification and other related research laid the foundation for the genome.