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大豆科学  2007 

菜豆几丁质酶基因的克隆、序列分析及其表达

DOI: 10.3969/j.issn.1000-9841.2007.02.001, PP. 121-126

Keywords: 菜豆,几丁质酶基因,克隆,序列分析,表达

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Abstract:

根据GenBank中收录的菜豆几丁质酶基因mRNA序列设计一对引物,以菜豆品种五常油豆总DNA为模板,通过PCR扩增获得一个约1.1kb的DNA片段Bchi,将其克隆入pUC18载体。测序和序列分析结果表明,该序列全长1088bp,含有一个981bp的完整开放读码框,无内含子,编码327个氨基酸,编码产物在结构上具有ClassIa几丁质酶的结构特征:N-端具有一个26个氨基酸的信号肽,其后是富含8个半胱氨酸、长41个氨基酸的几丁质结合区和由249个氨基酸组成的高度保守的催化区,C-端为由11个氨基酸组成的液泡定位多肽。序列与结构上的同源性分析表明Bchi基因是菜豆ClassIa几丁质酶基因。此序列已在GenBank中注册,登录号为AY357300。以pQE-30为原核表达载体,构建成pQE-Bchi重组表达载体,转化表达受体菌E.coliM15,经IPTG诱导后表达出一个约35kD的蛋白,与推测的Bchi基因编码产物的大小一致,表明Bchi基因在大肠杆菌中能够表达,是一个具有表达功能的基因。

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