菜豆几丁质酶基因的克隆、序列分析及其表达

CLONING,SEQUENCING AND EXPRESSION OF CHITINASE GENE FROM KIDNEY BEAN

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Class Ia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Class Ia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。

In this study,we designed pair of primers,which based on the mRNA sequences of chitinase gene of bean collected in GenBank,to amplify this gene in the genome of Wuchang bean by PCR.We got a DNA fragment of about 1.1kb,which was named Bchi,then we cloned this DNA fragment in pUC18 vector.By sequencing,we found that this fragment was 1088bp and had a ORF of 981bp,which had no introns.This sequence encoded 327 amino acids,whose product had the structure trait of Class Ia chitinase: There was a signal peptide of 26 amino acids on the N end followed by chitin-binding domain of 41 amino acids and highly conservative catalytic region of 249 amino acid;on the C end,there was VTP of 11 amino acids.The homogeneity in structure and sequence showed that this DNA fragment was the gene of Class Ia chitinase of bean,which was registered in GenBank,the accession number :AY357300.We put this gene into the prokaryote express vector of pQE-30 and made it express in EcoliM15 by IPTG inducing.At last,we got a protein of 35kD,which was identical to the prediction product of this gene,and this showed that Bchi gene was a functional one,which could express in E.coli.