扩展青霉PF898碱性脂肪酶基因5'端侧翼区域的克隆及调控功能的研究
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摘要
通过LA-PCR方法，从扩展青霉PF898基因组DNA中扩增得到2000 bp的碱性脂肪酶基因5’端侧翼区域的单一产物，并进行序列测定及提交GenBank数据库(GenBank accession NO.DQ677520)。该序列分别有类似于TATA盒、CAAT盒、GC盒等元件，这些元件均是真核启动子序列的基本结构特征。
对该碱性脂肪酶基因5’端侧翼区域进行片段缺失扩增，得到系列亚克隆片段，连接到以绿色荧光蛋白(GFP)为报告基因的pGlow-TOPO质粒中，构建了系列重组表达质粒，并成功转化大肠杆菌E.coil TOP10。荧光显微观察表明阳性转化子均发出绿色荧光，它们启动绿色荧光蛋白表达的功能得到确认。
进一步将脂肪酶基因5’端侧翼区域的系列亚克隆片段定向连接到启动子探针型载体pSUPV4中，构建能在大肠杆菌E.coil DH5a中启动卡那抗性基因表达的重组质粒。结果表明阳性克隆子均赋予大肠杆菌卡那抗性，但抗性的强度不同，卡那霉素抗性水平最高表现为3500μg／mL，最低表现为1000μg／mL。这些结果充分说明了不同的启动子片段对卡那抗性基因的启动表达能力不同。这些结果与启动GFP报告基因表达的结果相吻合，9个亚克隆片段启动子的功能得到最终确认。
A 5' flanking region DNA encoding alkaline lipase from Penicillium expansumPF898 with about 2000 bp was specially amplified by LA-PCR, then sequenced andsubmitted into the GenBank data (GenBank accession NO. DQ677520). The sequencecontained a novel promoter region with TATA box、CAAT box and GC box-like elementsin the identical positions shared by the eucaryote promoter sequence regions incomparison with the sequences in the GenBank.
The amplified sub-cloned products of 5' flanking region were ligated into the vectorwhich contained the green fluorescence protein (GFP) gene as the reporter gene toconstruct the expression plasmids. The recombined plasmids were transfered toEscherichia.coil TOP10. Many Escherichia.coil cells with the recombined plasmid couldemit fluorescence. The result indicated that the 5' flanking region had the promoter genesegment, which could promote GFP gene expression in Escherichia.coil cells.
The sub-cloned products of 5' flanking region were ligated into the promoter-probevector pSUPV4 with kanamycin-resistant gene (Kan~r) as the reporter gene to construct therecombined plasmids, which were transfered to Escherichia.coil DH5a to determine thepromoter function of the sub-cloned PCR produces. Escherichia.coils with therecombinant Kan~r gene promoted by the above fragments showed the different resistancesto Kanamycin in the range of 1000-3500μg/mL. The result was similar to the analysisresult of promoter function using GFP as the reporter gene, showing that the promoterfunction was sured.
对该碱性脂肪酶基因5’端侧翼区域进行片段缺失扩增，得到系列亚克隆片段，连接到以绿色荧光蛋白(GFP)为报告基因的pGlow-TOPO质粒中，构建了系列重组表达质粒，并成功转化大肠杆菌E.coil TOP10。荧光显微观察表明阳性转化子均发出绿色荧光，它们启动绿色荧光蛋白表达的功能得到确认。
进一步将脂肪酶基因5’端侧翼区域的系列亚克隆片段定向连接到启动子探针型载体pSUPV4中，构建能在大肠杆菌E.coil DH5a中启动卡那抗性基因表达的重组质粒。结果表明阳性克隆子均赋予大肠杆菌卡那抗性，但抗性的强度不同，卡那霉素抗性水平最高表现为3500μg／mL，最低表现为1000μg／mL。这些结果充分说明了不同的启动子片段对卡那抗性基因的启动表达能力不同。这些结果与启动GFP报告基因表达的结果相吻合，9个亚克隆片段启动子的功能得到最终确认。
A 5' flanking region DNA encoding alkaline lipase from Penicillium expansumPF898 with about 2000 bp was specially amplified by LA-PCR, then sequenced andsubmitted into the GenBank data (GenBank accession NO. DQ677520). The sequencecontained a novel promoter region with TATA box、CAAT box and GC box-like elementsin the identical positions shared by the eucaryote promoter sequence regions incomparison with the sequences in the GenBank.
The amplified sub-cloned products of 5' flanking region were ligated into the vectorwhich contained the green fluorescence protein (GFP) gene as the reporter gene toconstruct the expression plasmids. The recombined plasmids were transfered toEscherichia.coil TOP10. Many Escherichia.coil cells with the recombined plasmid couldemit fluorescence. The result indicated that the 5' flanking region had the promoter genesegment, which could promote GFP gene expression in Escherichia.coil cells.
The sub-cloned products of 5' flanking region were ligated into the promoter-probevector pSUPV4 with kanamycin-resistant gene (Kan~r) as the reporter gene to construct therecombined plasmids, which were transfered to Escherichia.coil DH5a to determine thepromoter function of the sub-cloned PCR produces. Escherichia.coils with therecombinant Kan~r gene promoted by the above fragments showed the different resistancesto Kanamycin in the range of 1000-3500μg/mL. The result was similar to the analysisresult of promoter function using GFP as the reporter gene, showing that the promoterfunction was sured.
引文
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