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坛紫菜TPS基因的克隆及其原核表达

DOI: 10.7668/hbnxb.2013.05.012, PP. 66-73

Keywords: 坛紫菜,TPS基因,原核表达,重组菌,耐盐性

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Abstract:

以叶状体总DNA为模板,用3对DNA引物进行PCR扩增,获得了坛紫菜6-磷酸海藻糖合成酶基因(PhTPS)3个重叠的片段,大小分别为1.3,1.1,0.7kb。用pMD18-T载体克隆这些片段,经测序与序列拼接获得了该基因完整的ORF序列,其长度为2727bp。在已经构建条斑紫菜TPS基因原核表达载体pET22b-PyTPS的基础上,用PhTPS基因替代该表达载体中的PyTPS基因,获得了PhTPS基因的原核表达载体pET22b-PhTPS。重组菌BL21(pET22b-PhTPS)经IPTG诱导,SDS-PAGE显示获得了约100kDa的特异性蛋白条带;目测及OD600的测定结果表明重组菌的耐盐性明显高于对照菌,即PhTPS基因的表达提高了重组菌的耐盐性。为利用PhTPS基因进行作物耐盐等抗逆转基因改良提供了依据。

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