Sugimoto Manabu, Saiki Yuji, Zhang Demin, Kawai Fusako
Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama, Japan.
FEMS Microbiol Lett. 2004 Jan 15;230(1):137-42. doi: 10.1016/S0378-1097(03)00886-3.
cDNAs expressed preferentially in an Al-tolerant microorganism were isolated by subtraction hybridization with cDNAs of Al-sensitive Penicillium chrysogenum IFO4626 as driver cDNA and cDNAs of the Al-tolerant mutant derived from the wild cells by UV irradiation as tester cDNA. Northern blot analysis revealed that mRNA levels of six genes were increased significantly in the Al-tolerant mutant after exposure to Al stress when compared with the wild cells. Two genes accumulated in both the presence and absence of Al stress and four genes were induced by Al stress in the Al-tolerant mutant. cDNA fragments were amplified by rapid amplification of cDNA ends and sequenced to obtain full-length cDNAs of the six genes. Two genes were novel or predicted ones and the others showed significant homology to known genes, ADP/ATP translocase, enolase, cysteine synthase, and glucoamylase, which are induced by environmental stresses in prokaryotic and eukaryotic cells. These enzyme activities increased in the Al-tolerant mutant when compared to those in the wild cells, showing that not only the levels of gene expression but also the levels of enzyme activities increased in the Al-tolerant mutant.
通过与作为驱动cDNA的铝敏感产黄青霉IFO4626的cDNA以及通过紫外线照射从野生细胞衍生的铝耐受突变体的cDNA进行消减杂交，分离出在耐铝微生物中优先表达的cDNA。Northern印迹分析表明，与野生细胞相比，耐铝突变体在暴露于铝胁迫后，六个基因的mRNA水平显著增加。在耐铝突变体中，两个基因在有铝胁迫和无铝胁迫时均积累，四个基因由铝胁迫诱导。通过cDNA末端快速扩增扩增cDNA片段并测序，以获得这六个基因的全长cDNA。两个基因是新基因或预测基因，其他基因与已知基因ADP/ATP转位酶、烯醇化酶、半胱氨酸合酶和糖化酶具有显著同源性，这些基因在原核和真核细胞中受环境胁迫诱导。与野生细胞相比，耐铝突变体中的这些酶活性增加，表明耐铝突变体中不仅基因表达水平增加，酶活性水平也增加。
