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甜高粱琥珀酸半醛脱氢酶SbSSADH基因克隆及原核表达

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.07.013, PP. 83-90

Keywords: 甜高粱,SSADH,蛋白表达,酶活测定

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Abstract:

γ-氨基丁酸(γ-aminobutyricacid,GABA)代谢旁路是TCA循环的一个代谢支路,广泛存在于动植物和微生物中,植物GABA代谢旁路与植物生物和非生物胁迫响应相关。琥珀酸半醛脱氢酶(SSADH)是GABA旁路的一个关键酶,对GABA途径在生物体内发挥功能起着至关重要的作用。通过检索MaizeGDB和Gramene数据库获得甜高粱SbSSADH基因信息,利用同源克隆的方法获得了甜高粱SbSSADH基因。序列分析结果表明,SbSSADH基因与玉米、甘蔗在核酸序列的相似度为94.77%,但前端约200bp的信号肽部分差异较大。通过以pET-28a为表达载体构建SbSSADH原核表达系统,Rosetta菌株为表达菌,表达出约50kD的SSADH蛋白,将纯化的蛋白进行酶活分析,纯化蛋白具有琥珀酸半醛脱氢酶活性,并且受酶抑制剂ATP和AMP的抑制。

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