Isolation of quizalofop-resistant mutants of Nannochloropsis oculata (Eustigmatophyceae) with high eicosapentaenoic acid following N-methyl-N-nitrosourea-induced random mutagenesis
Isolation of quizalofop-resistant mutants of Nannochloropsis oculata (Eustigmatophyceae) with high eicosapentaenoic acid following N-methyl-N-nitrosourea-induced random mutagenesis
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DOI：
10.1023/b:japh.0000044826.70360.8e
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发表时间：
2004-03-01
影响因子：
3.3
通讯作者：
Fujita, Y
中科院分区：
文献类型：
--
作者：
Chaturvedi, R;Uppalapati, SR;Fujita, Y
在喹禾灵（一种已知的乙酰辅酶A羧化酶（ACCase）活性抑制剂）的选择压力下，对微拟球藻（Nannochloropsis oculata）进行N-甲基-N-亚硝基脲诱导的诱变，目的是产生具有改变的脂肪酸代谢的遗传上易处理的突变体。两个突变体，QUIZ 1和QUIZ 2，具有稳定的抗喹禾灵分离和部分特征。突变株的生长特性和形态与出发菌株基本一致。然而，在突变体中观察到耐热性。对喹禾灵的抗性增强表明存在ACCase的除草剂抗性同种型。ACCase活性的体外测定表明，野生菌株中的ACCase比突变菌株对喹禾灵更敏感。脂肪酸的气相色谱分析显示，突变株富含多不饱和脂肪酸（n-3 PUFAs），以及总脂肪酸含量，这是伴随着三酰甘油（TAG），其次是亚油酸（18：2），花生四烯酸（20：4 n-6）和EPA（20：5 n-3）的增加。这些结果表明，体内和体外突变菌株中底物池（丙二酰辅酶A）增加（由于ACCase的比活性增加）可能导致TAG蓄积增加。随机诱变被证明是一个很好的工具，操纵PUFAs和EPA在微拟球藻。开发的菌株将是有用的，在了解脂肪酸代谢的遗传和生物化学方法，也为他们直接用于海水养殖。
Nannochloropsis oculata was subjected to N-methyl-N-nitrosourea-induced mutagenesis under the selection pressure of quizalofop, a known inhibitor of acetyl-CoA carboxylase (ACCase) activity with the objective of generating genetically tractable mutants with altered fatty acid metabolism. Two mutants, QUIZ1 and QUIZ2, with stable resistance to quizalofop were isolated and partially characterized. The growth properties and morphology of the mutants appeared identical with the parent strain. However thermo-tolerance was observed in the mutants. Enhanced resistance to quizalofop suggested the presence of herbicide resistant isoforms of ACCase. In vitro assays for ACCase activity showed that ACCase in the wild strains was much more sensitive to quizalofop than the mutant strains. Gas chromatographic analysis of fatty acids revealed that the mutant strains were rich in polyunsaturated fatty acids (n-3PUFAs), as well as total fatty acid contents; this was accompanied by a concomitant increase in triacylglycerol (TAG) followed by linoleic acid (18: 2), arachidonic acid (20:4 n-6) and EPA (20:5 n-3). These results suggest that an increased substrate pool (malonyl-CoA) (due to increased specific activity of ACCase) in the mutant strains in vivo and in vitro may have led to the increased TAG accumulation. Random mutagenesis was shown to be a good tool to manipulate PUFAs and EPA in Nannochloropsis. The strains developed will be useful in understanding fatty acid metabolism using genetic and biochemical approaches and also for their direct use in mariculture.