米根霉AS 3.819基因启动子片段的克隆及功能鉴定

从L-乳酸生产菌米根霉（Rhizopus oryzae）菌株AS 3.819基因组DNA中分别扩增得到了乳酸脱氢酶基因（ldhA）、丙酮酸脱氢酶基因（pdcA）、淀粉糖化酶基因（amyA）以及磷酸甘油酸酯激酶基因（pgk1）的启动子片段，并构建启动子探针载体pUKMR，以β-内酰胺酶基因（bla）为报告基因在大肠杆菌JM109中对这些启动子片段进行筛选及启动活性检测。结果表明：4 种启动子片段成功启动报告基因表达；在非底物诱导情况下，ldhA和pgk1启动子启动活性较强；在有合适碳源底物诱导情况下pdcA和amyA启动子拥有更高的启动活性；ldhA基因的启动子启动活性随着启动子片段长度的增加有一定提高，而在长度为500 bp以上时，其启动活性变化不明显。本研究为Rhizopus oryzae提供了一种快速简便的启动子捕获分离及启动活性检测方法。

Cloning and Functional Characterization of Gene Promoters from Rhizopus oryzae AS 3.819

In this paper, promoter fragments of four genes, lactate dehydrogenase A (ldhA), pyruvate decarboxylase A (pdcA),
glucoamylase A (amyA) and phosphoglycerate kinase 1 (pgk1), from genome DNA of Rhizopus oryzae strain AS 3.819,
were amplified and screened in Escherichia coli strain JM109 by using promoter probe vector pUKMR containing β-lactamase
gene (bla) as its reporter gene. The results suggested that the four promoter fragments all possessed the ability to drive the
expression of the β-lactamase gene. The promoters of ldhA and pgk1 genes possessed high promoting strength even though
no inducing substrate was present, while the strength of pdcA and amyA promoters could be enhanced when the suitable
carbon source was adopted. The strength of ldhA promoter was enhanced as the fragment length increased until it reached
500 bp. This research provides a quick and easy method to isolate and detect gene promoters form Rhizopus oryzae.