Dolui Achintya Kumar, Latha Mahadev, Vijayaraj Panneerselvam
1Department of Lipid Science, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka 570020 India.
2Academy of Scientific and Innovative Research, Ghaziabad, Uttar Pradesh 201002 India.
3 Biotech. 2020 Jan;10(1):30. doi: 10.1007/s13205-019-2016-x. Epub 2020 Jan 6.
Hydrolysis of phospholipid monolayer by phospholipases is an important event in the mobilization of stored lipids for seed germination. However, the identification and functional characterization of cereal phospholipases, especially during rice germination, are limited. In the present study, we have identified and characterized a phospholipase gene expressed during germination. The full-length coding region of was cloned into pRSETA as well as pYES2/NTC vector. The recombinant protein was successfully expressed in both and The recombinant protein was purified to homogeneity by affinity chromatography, and it was further confirmed by MS/MS analysis. In vitro lipase assay and lipidome analysis using high-resolution mass spectrometry showed phosphatidylcholine (PC) specific phospholipase B activity. The results revealed that protein encoded by gene prefers to hydrolyze PCs with C28, C32, and C34 containing unsaturated fatty acids. Collectively, the present study describes the identification and characterization of a phospholipase B, which hydrolyze PC, a major component of phospholipid monolayer covering storage lipid, as an initial event during rice seed germination.
磷脂酶对磷脂单层的水解作用是种子萌发过程中储存脂质动员的一个重要事件。然而,谷物磷脂酶的鉴定和功能表征,尤其是在水稻萌发过程中的相关研究还很有限。在本研究中,我们鉴定并表征了一个在萌发过程中表达的磷脂酶基因。该基因的全长编码区被克隆到pRSETA以及pYES2/NTC载体中。重组蛋白在大肠杆菌和毕赤酵母中均成功表达。通过亲和层析将重组蛋白纯化至同质,并用串联质谱分析进一步确认。体外脂肪酶测定和使用高分辨率质谱的脂质组分析显示该蛋白具有磷脂酰胆碱(PC)特异性磷脂酶B活性。结果表明,该基因编码的蛋白更倾向于水解含有C28、C32和C34不饱和脂肪酸的PC。总体而言,本研究描述了一种磷脂酶B的鉴定和表征,它水解PC(覆盖储存脂质的磷脂单层的主要成分),是水稻种子萌发过程中的一个初始事件。