低温和外源不饱和脂肪酸对高山被孢霉脂肪酸脱氢酶基因表达的影响

Effects of low temperature and exogenous unsaturated fatty acids on fatty acid desaturase gene expression of Mortierella alpina ATCC 16266

摘要:

摘要:【目的】在转录水平上研究低温和外源不饱和脂肪酸对于高山被孢霉脂肪酸脱氢酶基因的表达调控机制。【方法】通过实时定量PCR 技术和启动子报告基因融合载体的方法，研究低温和外源不饱和脂肪酸对于高山被孢霉3 种脂肪酸脱氢酶基因表达随时间进程的影响。【结果】实时定量PCR 的结果表明:低温对于3种脂肪酸脱氢酶基因的转录具有激活作用，外源不饱和脂肪酸对基因转录起抑制作用，而且这两种作用都是快速响应的，随时间延长逐渐减弱并消失。脂肪酸组成测定结果证明了基因转录水平变化与对应产物变化之间没有相关性。低温能够在短时间内诱导pFAD6启动子活性增加，并随时间延长而持续增强;外源不饱和脂肪酸对pFAD6 启动子活性起抑制作用，其不饱和度和浓度越高，抑制作用越强，而且抑制作用是快速且持续的。【结论】低温和外源不饱和脂肪酸除了在转录水平上调控高山被孢霉脂肪酸脱氢酶基因表达发生变化之外，可能主要在转录后水平上介导了胞内脂肪酸组成的变化。而且，脂肪酸脱氢酶基因的表达可能受到胞内脂肪酸组成变化的反馈调节作用。本文首次在转录水平上对高山被孢霉脂肪酸脱氢酶基因的表达调控机制进行了探索，为深入了解脂肪酸脱氢酶基因表达及多不饱和脂肪酸合成对外界信号的应答机制提供了有用信息，也对应用微生物发酵和转基因技术生产不饱和脂肪酸具有指导意义。

Abstract:

Abstract:［Objective］In order to investigate the regulatory mechanisms of Mortierella alpina desaturase genes by low temperature and exogenous unsaturated fatty acids (UFAs) at the transcriptional level．［Methods］We performed timecourse studies of fatty acid desaturase gene expression by real-time PCR and determined fatty acid desaturase gene promoter activity using promoter-reporter constructs． ［Results］Relative expression results in real-time PCR showed that the mRNA levels of three fatty acid desaturase genes (Fad6，Fad12 and Fad3) were rapidly and transiently enhanced after 1 h of shifting to low temperature，in contrast，high concentration of exogenous oleic acid (OA) which was a monounsaturated fatty acid containing a D9 double bond suppressed the transcription of these genes and the transcriptional response appeared to be rapid and transient． Also，there was no absolute correlation between mRNA abundance and production of corresponding fatty acids． The pFAD6 promoter activity was induced by low temperature in a time-dependent manner and reduced in a dose- and time-dependent manner by addition of UFAs to the media，and α-linolenic acid (ALA) containing three double bonds appeared to have a more effective inhibition than linoleic acid (LA) and OA．［Conclusion］These results indicate that there may be post-transcriptional control and other modes of regulation of UFAs synthesis in M． alpina when facing different stimuli such as low temperature and exogenous unsaturated fatty acids besides the regulation in the transcription of fatty acid desaturase genes at the initial stage． Also，there may be an unknown endproduct (changes in fatty acid compositions) feedback regulation in the transcription of fatty acid desaturase genes to maintain cellular UFAs' homeostasis． In a word，we assessed mechanisms of transcriptional regulation in M． alpina fatty acid desaturase gene expression for the first time and we wish to make it possible to obtain a better understanding of the mechanisms and get some theoretical knowledge to offer some guidance to the industrial production of UFAs by transgenic technology and microbial fermentation technology．