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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(s)-去甲乌药碱合成酶的分子克隆与异源表达[英文]
DOI: 10.3724/SP.J.1145.2013.00061, PP. 61-68
Keywords: 分子克隆,rt-pcr,异源表达,(s)-去甲乌药碱合成酶,苄基异喹啉
Abstract:
至今在拟南芥中尚未发现复杂生物碱，但是其基因组测序则表明有35个以上基因可编码(s)-去甲乌药碱合成酶（ncs）.本研究首先以经过生化表征、机理清楚的来自罂粟、日本黄连、黄唐松草和花菱草的ncs为参考，与拟南芥中注释的ncs进行了广泛的生物信息学分析与比对，从中选择5个atncs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atncs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个层次进行了atncs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.
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