甘油激酶基因对三角褐指藻理化性质的影响
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摘要
为了探索甘油激酶(glycerol kinase,GK)基因对三角褐指藻(Phaeodactylum tricornutum)生长的影响,以GK基因过表达藻株、干扰藻株和野生型藻株为研究对象,测定了其生长情况、GK基因转录水平、GK活性、脂质含量和金藻昆布多糖等参数.结果显示:转基因藻株的比生长速率与野生型藻株无显著差异;过表达藻株GKOE1-8、GKOE7-1的转录水平显著上调,GK活性增强了3.55倍和2.58倍,中性油脂含量增加64.95%和18.49%,金藻昆布多糖含量增加50.89%和42.11%;而干扰藻株GKRNAi3-1、GKRNAi4-1的转录水平则显著下调,伴随着GK活性降低52.34%和6.64%,中性油脂含量减少26.88%和16.98%,金藻昆布多糖含量减少16.99%和10.62%.研究结果表明:在三角褐指藻中,过表达甘油激酶基因可以促进中性油脂和金藻昆布多糖等生物活性物质的积累,这为通过基因工程的手段获得产油率高的转基因藻株提供了参考.
In order to know the mechanism of glycerol kinase in Phaeodactylum tricornutum,we studied its growth,transcriptional level,enzyme activity,lipid content and chrysolaminarin content.Results showed that the growth rate of transgenic types was not significantly different from that of the control,but the transcriptional level of GK during GKOE1-8 and GKOE7-1 was significantly up-regulated.The activity of GK increased 3.55 times and 2.58 times compared with that of the control,respectively.Neutral lipid content increased by 64.95% and 18.49%.Chrysolaminarin content increased by 50.89% and 42.11%.While the transcriptional level of GKRNAi3-1 and GKRNAi4-1 were significantly down-regulated,the decrease of GK activity was 52.34% and 6.64%,respectively.The content of neutral lipid was reduced by 26.88% and 16.98%,and chrysolaminarin content decreased by 16.99% and 10.62%.The results revealed that overexpression of GK could promote the accumulation of bioactive compounds,such as neutral lipid and chrysolaminarin,and thus provide some reference for obtaining transgenic algae with high lipid through genetic engineering.
In order to know the mechanism of glycerol kinase in Phaeodactylum tricornutum,we studied its growth,transcriptional level,enzyme activity,lipid content and chrysolaminarin content.Results showed that the growth rate of transgenic types was not significantly different from that of the control,but the transcriptional level of GK during GKOE1-8 and GKOE7-1 was significantly up-regulated.The activity of GK increased 3.55 times and 2.58 times compared with that of the control,respectively.Neutral lipid content increased by 64.95% and 18.49%.Chrysolaminarin content increased by 50.89% and 42.11%.While the transcriptional level of GKRNAi3-1 and GKRNAi4-1 were significantly down-regulated,the decrease of GK activity was 52.34% and 6.64%,respectively.The content of neutral lipid was reduced by 26.88% and 16.98%,and chrysolaminarin content decreased by 16.99% and 10.62%.The results revealed that overexpression of GK could promote the accumulation of bioactive compounds,such as neutral lipid and chrysolaminarin,and thus provide some reference for obtaining transgenic algae with high lipid through genetic engineering.
引文
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[12] Lin H Y,Yen S C,Kuo P C,et al. Alkaline phosphatase promoter as an efficient driving element for exogenic recombinant in the marine diatom Phaeodactylum tricornutum[J]. Algal Research,2017,23:59-65.
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[16] Osborne D R,Voogt P. Análisis de los nutrientes de los alimentos[J]. Reproduction Nutrition Developmen,1985,43(5):136-153.
[2] Nagle N,Lemke P. Production of methyl ester fuel from microalgae[J]. Applied Biochemistry Biotechnolgy,1990,24(1):355-361.
[3]陈波，黄龙，郭婷，等.三角褐指藻甘油兼养条件下甘油激酶的功能研究[J].四川大学学报：自然科学版，2015,52(1):144-148.
[4] Bowler C,Allen A,Badger J,et al. The Phaeodactylum genome reveals the evolutionary history of diatom genomes[J]. Nature,2008,456(7219):239-244.
[5] Zwaig N,Kistler W S,Lin E C. Glycerol kinase,the pacemaker for the dissimilation of glycerol in Escherichia coli[J]. Journal of Bacteriology,1970,102(3):753-759.
[6] Hayashi S L,Lin E C. Purification and properties of glycerol kinase from Escherichia coli[J]. Journal of Biological Chemistry,1967,242(5):1030-1035.
[7] Lin E C. Glycerol dissimilation and its regulation in bacteria[J]. Annual Review of Microbiology,1976,30(1):535-538.
[8] Muto M,Tanaka M,Liang Y,et al. Enhancement of glycerol metabolism in the oleaginous marine diatom Fistulifera solaris JPCC DA0580 to improve triacylglycerol productivity[J]. Biotechnology for Biofuels,2015,8(1):4.
[9] Yu K O,Ju J,Ramzi A B,et al. Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate[J].Biotechnology&Bioengineering,2013,110(1):343-347.
[10]樊改丽，赵敖，陈理彬，等.稻瘟病菌中甘油激酶基因在碳源代谢途径中的功能分析[J].基因组学与应用生物学，2016,35(10):2716-2723.
[11]张文源，高保燕，李爱芬，等.不同培养条件对三角褐指藻生长及其生物活性成分积累的影响[J].海洋科学，2016,40(5):57-65.
[12] Lin H Y,Yen S C,Kuo P C,et al. Alkaline phosphatase promoter as an efficient driving element for exogenic recombinant in the marine diatom Phaeodactylum tricornutum[J]. Algal Research,2017,23:59-65.
[13] Trinder P. Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen[J]. Journal of Clinical Pathology,1969,22(2):158-161.
[14] Liu Z Y,Wang G C,Zhou B C. Effect of iron on growth and lipid accumulation in Chlorella vulgaris[J]. Bioresource Technology,2008,99(11):4717-4722.
[15] Granum E,Myklestad S M. A simple combined method for determination ofβ-1,3-glucan and cell wall polysaccharides in diatoms[J]. Hydrobiologia,2002,477(1/2/3):155-161.
[16] Osborne D R,Voogt P. Análisis de los nutrientes de los alimentos[J]. Reproduction Nutrition Developmen,1985,43(5):136-153.