黑曲霉3.795glaA基因及其5'调控区的克隆和序列分析  

Cloning and Sequencing of Glucoamylase Genefrom Aspergillus niger 3795

作　　者：乔殿华[1] 钟丽婵[1] 唐国敏[1] 杨开宇[1] 

机构地区：[1]中国科学院微生物研究所

出　　处：《中国生物化学与分子生物学报》1998年第3期254-257,共4页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金;联合国科教文组织资助

摘　　要：黑曲霉Ｔ２１是由黑曲霉３．７９５经诱变育种获得的糖化酶高产菌株，为阐明其高产的分子机制，由黑曲霉３．７９５克隆了糖化酶结构基因及其５′旁侧序列，并与黑曲霉Ｔ２１的相应序列进行了比较．由黑曲霉３．７９５菌丝体分离染色体ＤＮＡ，Ｓｏｕｔｈｅｒｎ杂交分析表明，糖化酶结构基因位于～２．５ｋｂ的ＥｃｏＲⅠ－ＥｃｏＲⅤ染色体ＤＮＡ片段上，在此ＥｃｏＲⅠ位点上游约１．０ｋｂ处有一ＳａｌⅠ位点．为构建糖化酶结构基因及其５′旁侧序列的基因组文库，该染色体ＤＮＡ分别用ＥｃｏＲⅠ＋ＥｃｏＲⅤ和ＥｃｏＲ＋ＳａｌⅠ消化，琼脂糖凝胶电泳分离并回收长度在１．０ｋｂ左右和２．５ｋｂ左右的ＤＮＡ片段，分别与ｐＵＣ１９载体连接后转化入Ｅ．ｃｏｌｉＤＨ５．用原位杂交方法筛选到了携带糖化酶基因编码区及其１５０５ｂｐ５′旁侧序列的阳性克隆．对克隆片段的ＤＮＡ序列进行了测定并与黑曲霉Ｔ２１的相应序列进行了比较，结果表明，在糖化酶基因编码区及其１５０ｂｐ３′非编码区内，未发现碱基差异，但在－３４０～－１５０５的５′上游区内发生了９个位置的碱基变化，包括缺失、插入和替换．这些结果表明，黑曲霉Ｔ２１与３．７９５的糖化酶产量的差异与其结构基因无关，但可能与其?To elucidate the molecular mechanism of the high yield of glucoamylase in Aspergillus niger T21 strain,a mutant from A.niger 3795 strain by random mutagenesis,the glucoamylase structural gene and its 5′flanking sequence from A.niger 3795 were cloned,sequenced and compared with those from A.niger T21.Chromosomal DNA was isolated from the mycelia of A.niger 3795.Southern blot analysis indicated that the glucoamylase gene was situated on a 25 kb EcoRⅠEcoRⅤ fragment,and a SalⅠ site was located about 10 kb upstream from the EcoRⅠ site.To construct the genomic libraries of the glucoamylase structural gene and its 5′flanking sequence,the chromosomal DNA from A.niger 3795 strain was digested with EcoRⅠ+EcoRⅤ and EcoRⅠ+SalⅠ respectively.The fragments of around 25 kb and 10 kb were isolated through agarose gel electrophoresis from corresponding digestion and ligated into pUC19 vector separately.The resultant plasmids were transformed into E.coli DH5.The positive clones carrying the glucoamylase gene (glaA) coding region or a 1 505 bp 5′flanking region of glaA gene were selected respectively by in situ hybridization.The sequence of glaA gene from A.niger 3795 strain was determined and compared with that from A.niger T21 strain reported previously.The results showed that in the glucoamylase coding region and its 150 bp 3′ noncoding region,no difference was observed.However,within the 5′ upstream region of -340～-1 505 base changes did occur at 9 loci,including deletion,insertion and substitution.These results revealed that the difference in glucoamylase productivity between these two strains is not related to the structural gene but maybe related to the mutation of the 5′flanking sequence,which may cause an upregulation of glaA gene expression at transcriptional and/or posttranscriptional level.

关 键 词：黑曲霉 糖化酶 序列分析 克隆 

分 类 号：Q949.327.1[生物学—植物学] Q78