Functional characterization of Delta9 and omega9 desaturase genes in Mortierella alpina 1S-4 and its derivative mutants.
Functional characterization of Delta9 and omega9 desaturase genes in Mortierella alpina 1S-4 and its derivative mutants.
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发表时间：
2006
影响因子：
5
通讯作者：
T. Abe;E. Sakuradani;T. Asano;H. Kanamaru;S. Shimizu
中科院分区：
文献类型：
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作者：
T. Abe;E. Sakuradani;T. Asano;H. Kanamaru;S. Shimizu
高山被孢霉1 S-4 Delta 9去饱和酶（Delta 9 I）基因的克隆和特性分析已被报道。在这项研究中，两个基因编码Delta 9去饱和酶同源物分离出这种真菌。一种是Delta 9去饱和酶（Delta 9 II），其与Delta 9 I具有86%的氨基酸序列相似性。涉及编码基因在曲霉中表达的功能分析显示，Delta 9 II表现出Delta 9去饱和酶活性，18：0转化为18：1 Delta 9。然而，与Delta 9 I不同的是，Delta 9 II的累积量很低，为16：1Delta 9。另一个同源物是ω 9去饱和酶（ω 9），其分别与Delta 9 I和Delta 9 II具有56%和58%的氨基酸序列相似性。在用Aspergilluslavis进行的功能分析中，发现ω 9不能将18：0转化为18：1Delta 9，而是分别将24：0和26：0转化为24：1 omega 9和26：1 omega 9。另一方面，Delta 9去饱和缺陷型突变体的特征在于18：0的积累，来自M. alpina 1 S-4进行诱变，并对Delta 9去饱和酶基因的突变位点进行了鉴定。假设Delta 9 I基因上的突变分别在突变体（HR 222、T4和ST 56）中引起氨基酸替换（W136 Stop、G265 D和W360 Stop）。在这些突变体中，Delta 9 II和omega 9基因上没有突变位点。实时荧光定量PCR（RTQ-PCR）分析结果表明：（1）在培养的第5天，突变株的Delta 9 Ⅰ基因的转录水平远高于野生株;（2）在培养的第4天，突变株的Delta 9 Ⅱ基因的转录水平与野生株的Delta 9 Ⅰ基因的转录水平相同，而野生株的Delta 9 II基因转录水平较低，和（3）突变体和野生株中ω 9基因的转录水平均较低，即，与野生株的Delta 9 II基因一样低。在这些Delta 9去饱和缺陷突变体中，Delta 9 II可能在Delta 9去饱和中起重要作用。
Cloning and characterization of the Delta9 desaturase (Delta9I) gene of a fungus, Mortierella alpina 1S-4, was previously reported. In this study, two genes encoding Delta9 desaturase homologs were isolated from this fungus. One is a Delta9 desaturase (Delta9II) that exhibits 86% amino acid sequence similarity to Delta9I. Functional analysis involving expression of the encoding gene in Aspergillus oryzae revealed that Delta9II exhibits Delta9 desaturase activity, 18:0 being converted to 18:1Delta9. However, unlike Delta9I, the Delta9II transformant accumulated a low amount of 16:1Delta9. The other homolog is a omega9 desaturase (omega9) that exhibits 56 and 58% amino acid sequence similarity to Delta9I and Delta9II, respectively. On functional analysis with the Aspergillus transformant, it was found that omega9 does not convert 18:0 to 18:1Delta9, but converts 24:0 and 26:0 to 24:1omega9 and 26:1omega9, respectively. On the other hand, Delta9 desaturation-defective mutants characterized by accumulation of 18:0 were derived from M. alpina 1S-4 with a chemical mutagen, and the mutated sites of the Delta9 desaturase genes were identified. The mutation on the Delta9I gene was assumed to cause an amino acid replacement (W136Stop, G265D, and W360Stop) in the mutants (HR222, T4, and ST56), respectively. In these mutants, there was no mutated site on the Delta9II and omega9 genes. Real-time quantitative PCR (RTQ-PCR) analysis revealed that (1) the transcriptional level of the Delta9I gene in HR222 and T4 was much higher than that in the wild strain until the fifth day of the cultivation periods, (2) the Delta9II gene of the mutants was transcribed until the fourth day at the same level as the Delta9I gene of the wild strain, whereas the Delta9II gene of the wild strain was transcribed at a lower level, and (3) the transcriptional level of the omega9 gene in both the mutants and the wild strain was low, i.e., as low as that of the Delta9II gene of the wild strain. In these Delta9 desaturation-defective mutants, Delta9II is likely to play an important role in Delta9 desaturation.