黑曲霉糖化酶高产和低产菌株糖化酶基因调控区的克隆及其分析比较  

CLONING, SEQUENCING AND COMPARISON OF THE 5' FLANKING REGIONS OF glaA GENE FROM HIGH AND LOW GLUCOAMYLASE-PRODUCING STRAINS OF ASPERGILLUS NIGER

作　　者：钟丽婵[1] 乔殿华[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,北京100080

出　　处：《微生物学报》1996年第3期181-186,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金资助项目

摘　　要：以PCR合成的糖化酶高产菌株黑曲霉(Asp. niger)T21糖化酶基因5’近端非编码区588bp(EcoRI-BamHI)的序列为探针,从T21染色体DNA中克隆到近2.0kb的糖化酶基因5’端非编码区序列,并以此序列为探针从糖化酶低产菌株黑曲霉3.795(T21的诱变出发株)的染色体DNA中克隆到1.5kb的糖化酶基因5’端非编码区序列。该二序列的分析测定结果表明,其结构特征与文献报道的黑曲霉糖化酶基因5’端非编码区的基本一致,被称为“核心启动子”(Core promoter)的TATAAAT框及GCAAT框,分别在翻译起始点的-109bp及-178bp处。此外,在曲霉amdS,amyB基因中已发现有调控功能的CCAAT序列存在于-449bp和-799bp处。高产和低产菌株糖化酶基因5’端非编码区序列的分析比较结果表明,有9个部位的碱基发生了变化。此实验结果为进一步研究黑曲霉糖化酶基因在转录水平上的调控规律打下了基础。A 2kb DNA fragment of the 5' flanking region of glaA gene was cloned from the chromosomal DNA of Aspergillus niger T21, a glucoamylase over producing strain, using a 588bp fragment ( EcoR I -BamH I) of the 5' proximal flanking region as a probe, which had been synthesized previously by PCR from the chromosomal DNA of T21 covering EcoR I -BamH I stretch. The cloned 2kb DNA fragment was then used as the probe for cloning 1.5kb of the 5' flanking region of glaA gene from the chromosomal DNA of Asp. niger 3.795, a low glucoamylase-producing strain from which A.niger T21 was selected through mutation. Sequence analysis showed that both the 5' flanking regions of glaA gene contained the 'core promoter' of TATAAAT box and GCAAT sequence at nt -109 and -178 from the start codon respectively. In addition, two CCAAT sequences existed at nt-799 and-449, which had been proven to play a functional role for expression and regulation of Aspergillus amdS and amyB gene. From comparison of the 5' non-coding regions of glaA gene from high and low glucoamylase-producing strains, nine sites with different bases were found. These results would prove to be valuable for further study on the regulation of transcription of the Aspergillus niger glaA gene.

关 键 词：黑曲霉 糖化酶 5'端非编码区 基因克隆 

分 类 号：Q949.327.1[生物学—植物学]