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资源植物金荞麦高类黄酮含量愈伤系的获得及其cDNA文库的构建

Keywords: 资源植物,金荞麦,类黄酮,愈伤系,cDNA文库

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Abstract:

用野生金荞麦无菌苗的茎和叶片作外植体,茎在MS添加2.0mg.L-16-BA和0.1mg.L-1NAA的固体培养基上,通过12d的诱导,可获得100%的诱导率;叶在MS添加1.0mg.L-16-BA和0.5mg.L-1NAA的固体培养基上诱导13d,可获得100%的诱导率。茎和叶分别在各自激素组合培养基上连续继代3次后,用目视法直接从大量的稳定愈伤组织中筛选获得4种不同次生代谢能力的细胞系:红色系(Stem1愈伤系)、褐色系(Stem2愈伤系)、浅黄色系(Leaf1愈伤系)和白色系(Leaf2愈伤系)。用分光光度法直接筛选出高类黄酮含量的红色系,其类黄酮含量为6.3804mg.g-1,是含量最低的白色系的3.5倍。用改良的CTAB法提取金荞麦高黄酮含量的红色系1~13d愈伤组织总RNA,用SMARTcDNALibraryConstructionKit构建cDNA文库。经测定原始文库滴度为1.5×106pfu.mL-1,扩增总文库滴度为7.0×109pfu.mL-1,重组率达到98%,插入片段在0.5kb到2kb之间,1kb以上的占66%。通过简并扩增的方式,从总文库中检测到了低峰度表达的R2R3Myb转录调控因子P基因的特异片段。各项指标都表明,已获得较高质量的cDNA文库。金荞麦高类黄酮含量愈伤系的获得及其cDNA文库的构建,不但为野生金荞麦的优异基因资源的保存和其类黄酮次生代谢相关基因的克隆和调控研究打下了的基础,

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