ANALYSIS OF 1, 3-DIOLEOYL-2-PALMITOYLGLYCEROL OF CHLAMYDOMONAS REINHARDTII BY ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM TRIPLE QUADRUPOLE MASS SPECTROMETRY
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摘要: 研究建立了基于超高效液相色谱串联三重四级杆质谱技术的1,3-二油酸-2-棕榈酸甘油三酯(1,3-Dioleoyl-2-palmitoylglycerol, OPO)分析方法。在正离子模式下, 以[876>577]为定量离子对, 通过多反应监测模式(Multiple Reaction Monitoring, MRM)扫描, 采用内标法对OPO进行定量分析。对缺氮胁迫条件下一株野生型莱茵衣藻(Chlamydomonas reinhardtii)cc-5325及其半乳糖基水解酶基因敲降突变体M08的OPO进行了定量分析, 研究结果发现莱茵衣藻中OPO在缺氮胁迫期间显著积累, 且不同遗传背景的莱茵衣藻OPO积累量不同。在缺氮第1至第3天, M08的OPO含量相较于cc-5325分别提高了3.70、3.04和2.74倍, M08的OPO产量相较于cc-5325分别提高了1.13、1.53和1.33倍, 说明通过遗传改造莱茵衣藻的某些脂质代谢途径相关基因可以增加其OPO含量, 因此莱茵衣藻具有改成为商业化油脂新食品原料的潜在应用价值。研究建立的功能脂质OPO的定量分析方法对进一步研究不同遗传改造的莱茵衣藻中OPO结构脂的检测提供了技术支持和分析基础。
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关键词:
- 1,3-二油酸-2-棕榈酸甘油三酯 /
- 超高效液相色谱串联三重四级杆质谱 /
- 缺氮胁迫 /
- 积累 /
- 莱茵衣藻
Abstract: In human breast milk, over 50% energy is in the form of triacylglycerol (TAG), which is dominated by a functional lipid, 1,3-dioleoyl-2-palmitoyl-glycerol (OPO). A number of studies have shown that OPO possesses particular stereospecific arrangement of acyl chains which is essential for the absorption of fatty acids in infant gut. However, most of the commercial OPOs in the domestic market rely on imports currently. The contradiction between price and quality has restricted the wide application of OPO in the infant fomula. Chlamydomonas reinhardtii is a model organism for investigating lipid metabolism, and numerous studies suggested that C. reinhardtii is able to accumulate OPO under stress conditions such as nitrogen deficiency. This study established a method to quantify 1,3-dioleoyl-2-palmitoyl-glycerol OPO by employing ultra-high performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry. The OPO content was quantified with the internal standard method through multi-reaction monitoring mode (MRM) under positive mode, and the selected ion-pair was [876>577]. The OPO production was investigated in the wild-type C. reinhardtii strain cc-5325 and the galactosyl hydrolase gene mutant strain M08. The results showed that the content of OPO in C. reinhardtii accumulated greatly under nitrogen deprivation with great difference in the two strains. Compared to cc-5325, from day1 to day3, nitrogen deprivation increased the content of OPO of M08 by 3.70, 3.04 and 2.74 times, and it increased the yield of OPO of M08 by 1.13, 1.53 and 1.33 times, respectively. These data suggest that modifying lipid metabolic pathway can increase the production of OPO in C. reinhardtii. Therefore, C. reinhardtii has potential as a new food ingredient for commercial oils. This study provide technical support and basis to increase OPO production in different genetically engineered C. reinhardtii. -
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图 1 OPO子离子扫描质谱图
Figure 1. The product-ion scanning spectrum of the [M + NH4]+ ion of OPO
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图 4 MRM检测模式下特征离子扫描鉴定OPO
A—D. 缺氮0—3d
Figure 4. Identification of OPO by characteristic ion scanning detected by MRM
A—D. Day 0—3 of nitrogen deficiency
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图 5 莱茵衣藻野生型cc-5325和Crgh突变株M08的性状表征
A. 正常培养条件下的Crgh基因转录水平; B. 缺氮培养时的细胞干重; C. 缺氮培养时的细胞密度
Figure 5. Characterization of wild-type cc-5325 and Crgh mutant strain M08
A. The expression level of Crgh gene under normal culture conditions; B. The dry weight under nitrogen deprived condition; C. The cell density under nitrogen deprived condition
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图 6 缺氮培养时莱茵衣藻野生型cc-5325和Crgh突变株M08的OPO积累(*P<0.05)
A. OPO含量; B. OPO产量
Figure 6. The accumulation of OPO in wild-type cc-5325 and Crgh mutant strain M08 under nitrogen deprivation (*P<0.05)
A. The content of OPO (% dry weight); B. The yield of OPO
表 1 UPLC梯度洗脱条件
Table 1 Gradient elution conditions of UPLC
时间
Time (min)流动相A
Mobile phase A (%)流动相B
Mobile phase B (%)0.0 80.0 20.0 1.0 80.0 20.0 35.0 20.0 80.0 40.0 20.0 80.0 45.0 80.0 20.0 50.0 80.0 20.0 
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