异源表达海苏特氏菌Argonaute蛋白基因提高酿酒酵母脂肪酸含量研究
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摘要
Argonaute蛋白作为一类高度保守的蛋白,是小RNA调控代谢机制的重要参与者,是RNA干扰所必须的,然而此蛋白在酿酒酵母中却未被发现。为初步探究海苏特氏菌Argonaute蛋白基因对脂肪酸含量的影响,以海苏特氏菌全基因组为模板,克隆并表达了海苏特氏菌Argonaute蛋白基因的CDS区。生物信息学分析发现该基因具有基因沉默调节因子SIR2结构域。将海苏特氏菌Argonaute蛋白基因连接至载体p ZMG中获得重组载体p ZMG-Jm-Argonaute并转化至酿酒酵母BY4742中,GC-MS检测显示,重组菌株总脂肪酸含量相对于野生菌株显著提高了6. 4%,其中不饱和脂肪酸C161含量显著提高了8. 3%,结果表明Jm-Argonaute蛋白基因能够提高酿酒酵母BY4742不饱和脂肪酸C161的含量,从而为深入研究该基因在菌体内对脂肪酸代谢的调节机制提供参考。
As a highly conserved protein,Argonaute protein is an important participant in the regulation of the metabolism of small RNA and is necessary for RNA interference,however,this protein was not found in Saccharomyces cerevisiae. In order to investigate the influence of Argonaute protein gene on the content of fatty acids,the whole genome of Joostella marina was used as a template to clone and verify the CDS region of the Argonaute protein gene. Bioinformatics analysis had found that the gene has a gene silencing regulator SIR2 domain. The recombinant plasmid p ZMGJm-Argonaute was constructed by ligating the Argonaute protein gene of Joostella marina to the vector p ZMG and transformed into S. cerevisiae BY4742. The total fatty acid content of the recombinant strain was significantly increased by6. 4% as compared with the wild strain,and the content of unsaturated fatty acid C161 was significantly increased by 8. 3%. The results showed that the Jm-Argonaute protein gene can mainly increase the C161 content of unsaturated fatty acids in S. cerevisiae BY4742,which laid the foundation for the further study of the regulation mechanism of fatty acid metabolism in this strain.
As a highly conserved protein,Argonaute protein is an important participant in the regulation of the metabolism of small RNA and is necessary for RNA interference,however,this protein was not found in Saccharomyces cerevisiae. In order to investigate the influence of Argonaute protein gene on the content of fatty acids,the whole genome of Joostella marina was used as a template to clone and verify the CDS region of the Argonaute protein gene. Bioinformatics analysis had found that the gene has a gene silencing regulator SIR2 domain. The recombinant plasmid p ZMGJm-Argonaute was constructed by ligating the Argonaute protein gene of Joostella marina to the vector p ZMG and transformed into S. cerevisiae BY4742. The total fatty acid content of the recombinant strain was significantly increased by6. 4% as compared with the wild strain,and the content of unsaturated fatty acid C161 was significantly increased by 8. 3%. The results showed that the Jm-Argonaute protein gene can mainly increase the C161 content of unsaturated fatty acids in S. cerevisiae BY4742,which laid the foundation for the further study of the regulation mechanism of fatty acid metabolism in this strain.
引文
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[3] Kaifa Wei,Lingjuan Wu,Yanhui Chen,et al. Argonaute protein as a linker to command center of physiological processes[J].Chinese Journal of Cancer Research,2013,25(4):430-441.
[4] Huang V,Li LC. Demystifying the nuclear function of Argonaute proteins[J]. Rna Biology,2014,11(1):18-24.
[5] Boland A,Huntzinger E,Schmidt S,et al. Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein[J]. Proceedings of the National Academy of Sciences of the United States of America,2011,108(26):10466-10471.
[6] Hutvagner G,Simard MJ. Argonaute proteins:key players in RNA silencing[J]. Nat Rev Mol Cell Biol,2008,9(9):22-32.
[7] Quévillon HM,Zeitler DM,Hauptmann J,et al. Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing in vivo[J]. Embo Journal,2017:e201696386.
[8] Till S,Lejeune E,Thermann R,et al. A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain[J]. Nature Structural&Molecular Biology,2007,14(10):897-903.
[9] H9ck J,Meister G. The Argonaute protein family[J]. Genome Biology,2008,9(2):1-8.
[10]温剑.真菌miRNA的计算机预测及鉴定[D].南昌:南昌大学,2008.
[11] Drinnenberg IA,Weinberg DE,Xie KT,et al. RNAi in budding yeast[J]. Science,2009,326(5952):544-550.
[12] Moore KJ,Rayner KJ,Suárez Y,et al. The Role of MicroRNAs in Cholesterol Efflux and Hepatic Lipid Metabolism[J]. Annual Review of Nutrition,2011,31(1):49-63.
[13]李超,杜志游,陈集双.解读AGO蛋白结构及其功能[J].中国生物化学与分子生物学报,2009,25(11):969-976.
[14] Corinna Giorgi,Carlo Cogoni,Caterina Catalanotto. From transcription to translation:new insights in the structure and function of Argonaute protein[J]. BioMolecular Concepts,2012,3(6):545-559.
[15] Poulsen C,Vaucheret H,Brodersen P. Lessons on RNA silencing mechanisms in plants from eukaryotic argonaute structures[J]. Plant Cell,2013,25(1):22-37.
[16] Leber C,Da SN. Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids[J]. Biotechnology&Bioengineering,2013,111(2):347-358.
[17] Mcburney MW,Yang X,Jardine K,et al. The absence of SIR2alpha protein has no effect on global gene silencing in mouse embryonic stem cells[J]. Molecular Cancer Research,2003,1(5):402-409.
[18] Leber C,Polson B,Fernandez-Moya R,et al. Over production and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae[J]. Metabolic Engineering,2015,28:54-62.
[19] Tang X,Lee J,Chen WN. Engineering the fatty acid metabolic pathway in Saccharomyces cerevisiae,for advanced biofuel production[J]. Metabolic Engineering Communications,2015,2(C):58-66.
[20] Chen LW,Zhang JH,Chen WN. Engineering the Saccharomyces cerevisiaeβ-oxidation pathway to increase medium chain fatty acid production as potential biofuel[J]. Plos One,2014,9(1):1-10.
[21] Chen Z,Qiu H,Ma L,et al. miR-30e-5p and miR-15a Synergistically Regulate Fatty Acid Metabolism in Goat Mammary Epithelial Cells via LRP6 and YAP1[J]. International Journal of Molecular Sciences,2016,17(11):1909.
[22] Vergnes B,Gazanion E,Grentzinger T. Functional divergence of SIR2 orthologs between trypanosomatid parasites[J]. Molecular&Biochemical Parasitology,2016,207(2):96-101.