Departamento de Ecología, Center for Advanced Studies in Ecology and Biodiversity, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 6513677, Santiago, Chile.
Mar Biotechnol (NY). 2011 Jun;13(3):544-56. doi: 10.1007/s10126-010-9325-8. Epub 2010 Oct 9.
In order to identify genes/proteins involved in copper tolerance, the marine alga Ulva compressa was cultivated with 10 μM copper for 3 days. The activities of antioxidant enzymes ascorbate peroxidase (AP), peroxiredoxin (PRX), thioredoxin (TRX), and glutathione-S-transferase (GST) and the level of lipoperoxides were determined in the alga cultivated with and without copper addition. Antioxidant enzyme activities and lipoperoxides level increased in response to copper excess, indicating that the alga was under oxidative stress. A cDNA library was prepared using U. compressa cultivated with 10 μM copper for 3 days. A total of 3 × 10(4) clones were isolated and 480 clones were sequenced, resulting in 235 non-redundant ESTs, of which 104 encode proteins with known functions. Among them, we identified proteins involved in (1) antioxidant metabolism such as AP, PRX, TRX, GST, and metalothionein (MET), (2) signal transduction, such as calmodulin (CAM), (3) calcium-dependent protein kinase (CDPK) and nucleoside diphosphate kinase (NDK), (4) gene expression, (5) protein synthesis and degradation, and (6) chloroplast and mitochondria electron transport chains. Half of the identified proteins are potentially localized in organelles. The relative level of 18 genes, including those coding for AP, PRX, TRX, GST, MET, CAM, CDPK, and NDK were determined by quantitative RT-PCR in the alga cultivated with 10 μM copper for 0 to 7 days. Transcript levels increased in response to copper stress and most of them reached a maximum at days 3 and 5. Thus, the selected genes are induced by copper stress and they are probably involved in copper acclimation and tolerance.
为了鉴定耐铜基因/蛋白，将海洋绿藻 U. compressa 在 10 μM 铜下培养 3 天。在添加和不添加铜的情况下，测定抗氧化酶抗坏血酸过氧化物酶（AP）、过氧化物酶（PRX）、硫氧还蛋白（TRX）和谷胱甘肽-S-转移酶（GST）的活性以及脂过氧化物的水平。抗氧化酶活性和脂过氧化物水平的增加表明藻类受到了氧化应激。使用在 10 μM 铜下培养 3 天的 U. compressa 制备 cDNA 文库。共分离了 3×104 个克隆，对 480 个克隆进行测序，得到 235 个非冗余 EST，其中 104 个编码具有已知功能的蛋白质。其中，我们鉴定了参与（1）抗氧化代谢的蛋白质，如 AP、PRX、TRX、GST 和金属硫蛋白（MET），（2）信号转导，如钙调蛋白（CAM），（3）钙依赖蛋白激酶（CDPK）和核苷二磷酸激酶（NDK），（4）基因表达，（5）蛋白质合成和降解，以及（6）叶绿体和线粒体电子传递链的蛋白质。鉴定出的蛋白质中有一半可能定位于细胞器中。在 10 μM 铜下培养 0 至 7 天的藻类中，通过定量 RT-PCR 测定了包括编码 AP、PRX、TRX、GST、MET、CAM、CDPK 和 NDK 的 18 个基因的相对水平。在铜胁迫下，转录水平增加，大多数基因在第 3 和第 5 天达到最大值。因此，所选基因受铜胁迫诱导，可能参与铜适应和耐受。
