氮饥饿引起缺刻缘绿藻积累三酰甘油的机理研究

In the oleaginous microalga Myrmecia incisa Reisigl H4301, the content of triacylglycerols (TAG) significantly increased being accompanied with the degradation of membrane lipid and the inhibition of de novo biosynthesis of fatty acid under nitrogen starvation. In addition, diacylglycerol acyltransferase (DGAT), phospholipid:diacylglycerol acyltransferase (PDAT) and galactolipase coding genes were annotated in transcriptome of M. incisa, which suggested that TAG might accumulated at the expense of membrane lipids when M. incisa was cultured under nitrogen starvation. However, the accurate pathway of TAG biosynthesis in this microalga is unknown. Hence, to gain more insights into the molecular mechanism of TAG accumulation induced by nitrogen starvation in M. incisa, this proposal is planning to utilize the techniques of lipidomics combined with the techniques of molecular biology. Firstly, ultra performance lipid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) and multivariate data analysis methods are applied to identify and quantify the species of changed lipids from M. incisa under nitrogen starvation. Secondly, rapid amplification of cDNA ends (RACE) technique is applied to clone the full-length cDNAs of DGAT, PDAT and galactolipase coding gene. Subsequently, the subcellular localization of DGAT, PDAT and galactolipase are determined, followed by the functional analysis of these enzyme coding genes. Finally, the transcript quantification of DGAT, PDAT and galactolipase coding gene from M. incisa under nitrogen starvation are performed to elucidate the TAG biosynthesis pathway of this microalga with the results from lipidomics analysis. The fulfillment of this project will not only enable us to understand the biological significance in microalgae that the intracellular TAG accumulate under nitrogen starvation but degrade to help reconstruct chloroplastic membrane under the following suitable environment, but also lay a foundation for gene-manipulation of biofuel produced microalgae.

在氮饥饿条件下，缺刻缘绿藻细胞的三酰甘油（TAG）含量随着膜脂含量和脂肪酸从头合成能力的下降而显著上升，以及在该藻转录组文库中存在二酰甘油酰基转移酶（DGAT）、磷脂:二酰甘油酰基转移酶（PDAT）和半乳糖脂酶等基因信息，都提示在此条件下该藻TAG主要由膜脂转变而来。但这个过程究竟如何，尚不清楚。为此，本项目首先利用脂代谢组学分析手段对该藻在氮饥饿胁迫前后的变化脂质分子种类进行定性和定量研究；然后在DGAT、PDAT和半乳糖脂酶等基因序列克隆的基础上，鉴定其功能并确定其编码酶的亚细胞定位；最后利用实时荧光定量PCR技术分析氮饥饿胁迫前后这些基因转录量的变化，并结合变化的脂质综合探讨氮饥饿引起缺刻缘绿藻累积TAG的分子机理。本项目研究任务的完成不仅有利于了解藻细胞在逆境条件下积累TAG而在环境适宜时提供TAG以重建细胞的生物学意义，也为如何培养高TAG含量的微藻以生产生物柴油提供理论指导。

在氮饥饿条件下培养缺刻缘绿藻，其细胞的三酰甘油（TAG）含量随着膜脂含量和脂肪酸从头合成能力的下降而显著上升，以及在该藻转录组文库中存在二酰甘油酰基转移酶（DGAT）、磷脂:二酰甘油酰基转移酶（PDAT）和半乳糖脂酶等基因信息，都提示该藻TAG在氮饥饿胁迫下的合成主要是通过消耗膜脂来实现的。但这个过程究竟如何，尚不清楚。为此，本项目研究氮饥饿（4 d）/氮恢复（4 d和8 d）培养下缺刻缘绿藻细胞中脂类及其中脂肪酸组成的变化，发现TAG含量在氮饥饿胁迫下显著上升，而在氮恢复培养下逐渐下降；DGDG含量的变化趋势与TAG相反；MGDG的含量则始终呈现上升趋势；花生四烯酸（ArA）主要存在于TAG中。克隆得到1条DGAT1基因（命名为MiDGAT1）和2条DGAT2（分别命名为MiDGAT2A和MiDGAT2B）基因cDNA全长序列，通过在TAG代谢缺陷的酿酒酵母突变株中进行表达，证实了它们的二酰甘油酰基转移酶功能。同时，通过实时荧光定量PCR检测发现MiDGAT1和MiDGAT2A转录水平受氮饥饿影响上调较明显的，表明在氮饥饿培养条件下，MiDGAT1和MiDGAT2A在缺刻缘绿藻TAG合成过程中起主要作用。对克隆得到的PDAT基因（命名为MiPDAT）进行亚细胞定位和功能分析结果表明，该质膜型的PDAT能够合成TAG且偏好以磷脂酰胆碱（PC）为作用底物。在氮饥饿胁迫下MiPDAT转录水平逐渐增加。利用超高效液相色谱-四极杆-时间飞行-质谱技术对氮饥饿/氮恢复培养下藻细胞的总脂样品进行分离并收集脂代谢组学数据，并进行定性和定量分析。通过比较发现在氮饥饿胁迫条件下培养缺刻缘绿藻细胞4 d，细胞中PC、DGDG含量下降，TAG含量上升。从分子种类来看，上升的TAG中除含有ArA的分子种类，还有较多原核型的分子种类。综合上述研究结果我们推测原核型的TAG主要是经由叶绿体中MiDGAT1催化的从头合成，真核型的TAG主要是经由DGAT2A催化和由DGDG和PC通过PDAT催化转向而来的。本项目研究任务的顺利完成不仅有利于了解藻细胞在逆境条件下积累TAG而在环境适宜时提供TAG以重建细胞的生物学意义，也为如何培养高TAG含量的微藻以生产生物柴油提供理论指导。