Molecular cloning and characterization of a cDNA for low-temperature inducible cytosolic glucose 6-phosphate dehydrogenase gene from Chlorella vulgaris and expression of the gene in Saccharomyces cerevisiae
Molecular cloning and characterization of a cDNA for low-temperature inducible cytosolic glucose 6-phosphate dehydrogenase gene from Chlorella vulgaris and expression of the gene in Saccharomyces cerevisiae
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DOI：
10.1016/j.plantsci.2006.12.004
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发表时间：
2007-03-01
期刊：
影响因子：
5.2
通讯作者：
Iio, Masayoshi
中科院分区：
文献类型：
--
作者：
Honjoh, Kenichi;Machida, Takeshi;Iio, Masayoshi
从小球藻耐寒菌株C-27的Poly(A)(+)RNA构建的cDNA文库中克隆了葡萄糖6-磷酸脱氢酶基因(E.C.1.1.1.49)。该克隆命名为Cvcg6pdh。Cvcg6pdh全长1845个，编码521个氨基酸。Cvcg6pdh的氨基酸序列与其他高等植物的胞质G6PDH具有同源性，而不是与叶绿体G6PDH的同源性。对Cvcg6pdh基因转录本的Northern杂交分析表明，该基因曾下调过一次，12小时硬化后诱导表达。活性检测还表明，其中一个G6PDH亚型的表达模式与该基因的转录本相似。基因组DNA的Southern杂交分析表明，Cvcg6pdh似乎至少与G6PDH基因的一个拷贝杂交。用聚合酶链式反应扩增该克隆的编码区，并用pTG887表达载体导入酿酒酵母中。转化酵母中G6PDH的活性提高到对照菌株的8.7倍。冻融后，转化pTG887/Cvcg6pdh的酵母细胞活力显著高于pTG887的对照酵母细胞。(C)2006爱思唯尔爱尔兰有限公司。保留所有权利。
A cDNA clone corresponding to the gene for glucose 6-phosphate dehydrogenase (E.C.1.1.1.49) was isolated from a cDNA library constructed from poly(A)(+)RNA from Chlorella vulgaris C-27, a frost hardy strain. The cDNA clone was designated as Cvcg6pdh. The length of Cvcg6pdh was 1845 by and the clone coded for 521 amino acids. The deduced amino acid sequence of Cvcg6pdh showed sequence homology to cytosolic G6PDHs from other higher plants rather than chloroplastic ones. Northern blot analysis of a transcript of Cvcg6pdh gene showed that the gene was down-regulated once and then induced after 12-h hardening. Activity staining also showed that the expression pattern of one G6PDH isoform was similar to that of the transcript of the gene. Southern blot analysis of genomic DNA showed that Cvcg6pdh seems to hybridize with, at least, one copy of g6pdh genes. The coding region of the clone was amplified by PCR and the product was introduced into Saccharomyces cerevisiae by using a pTG887 expression vector. The activity of G6PDH in transformed yeast was enhanced up to 8.7 times that of the control strain. Furthermore, after freezing-thawing, the viability of the yeast transformed with pTG887/Cvcg6pdh was significantly higher than that of the control yeast cells carrying pTG887. (c) 2006 Elsevier Ireland Ltd. All rights reserved.