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OALib Journal期刊
ISSN: 2333-9721
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拟南芥基因组中注释为异胡豆苷合成酶的基因克隆及异源表达
DOI: 10.3724/SP.J.1145.2013.00224, PP. 224-230
Keywords: 萜类吲哚生物碱,异胡豆苷合成酶,拟南芥,rt-pcr,pcr,异源表达
Abstract:
拟南芥（arabidopsisthaliana）是生物学研究中广泛使用的模式生物.到目前为止，没有证据表明拟南芥中存在复杂生物碱，但对其基因组数据的生物信息学分析表明，有很多基因与萜类吲哚生物碱（tias）生物合成途径中的相关基因有高度的同源性.为了从分子水平研究拟南芥是否具有合成该类复杂生物碱的潜力，首先对拟南芥中可能编码异胡豆苷合成酶（str）的15个基因及其编码的酶进行广泛的生物信息学分析和计算；在此基础上，选择其中5个atstr基因为目的基因，设计特异性引物；从拟南芥中提取总rna，一步法rt-pcr克隆得到上述基因；通过ta克隆将上述基因转入pgm-t载体，筛选、酶切及pcr鉴定，dna测序验证它们的核酸序列；将测序验证的atstr基因经过pcr扩增、质粒构建、转入escherichiacolibl21(de3)中进行异源表达和蛋白纯化；在整体细胞、细胞裂解液、细胞裂解液上清和纯化蛋白共4个层次进行atstr酶功能表征，未发现目标产物或新产物的生成；上述基因的具体功能还有待进一步研究.图8表3参22
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