Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source
Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source
复制标题
DOI：
10.1016/j.jbiotec.2003.08.006
复制
发表时间：
2003-12-19
影响因子：
4.1
通讯作者：
Tauch, A
中科院分区：
文献类型：
--
作者：
Hüser, AT;Becker, A;Tauch, A
开发了一种DNA微阵列来分析氨基酸产生菌谷氨酸棒杆菌的全局基因表达。PCR产物占预测值的93.4%。制备谷氨酸杆菌基因，并将其以四倍量点样到3-氨基丙基三甲氧基硅烷包被的载玻片上。C.通过与荧光标记的cDNA探针的共杂交证实了谷氨酸DNA微阵列。技术方差分析表明，C。以不同强度检测到的导致比值大于1.52或小于-1.52的谷氨酸杆菌基因可以被认为是差异表达的，置信水平大于95%。在一个验证的例子中，我们测量了在C.以乙酸盐和丙酸盐为碳源，使用乙酸盐生长的谷氨酸棒杆菌培养物作为参考。在95%的置信区间，117个基因显示在丙酸盐的存在下增加的转录水平，而43个基因显示出与乙酸盐生长的培养物相比降低的表达。全局表达谱分析证实了prpD 2B 2C 2基因簇的诱导，已知该基因簇是通过2-甲基柠檬酸循环进行丙酸盐降解所必需的。除了许多功能未知的基因外，旁系同源的prpD 1B 1C 1基因簇以及fasI-B（编码脂肪酸合酶IB）、dtsR 1和dtsR 2（酰基辅酶A羧化酶的组分）、gluABCD（谷氨酸转运系统）、putP（脯氨酸转运系统）和pyc（丙酮酸羧化酶）的表达水平显著增加。这些基因的差异表达，证实了实时逆转录（RT）PCR检测。(C)2003 Elsevier B. V.保留所有权利。
A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays. (C) 2003 Elsevier B.V. All rights reserved.