氮元素对三角褐指藻岩藻黄素和油脂合成关键酶基因表达与代谢合成的影响
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摘要
为阐明氮元素对微藻次生代谢积累和调控的影响,以三角褐指藻为试验材料,研究不同氮浓度[896(CK)、448、112、28和0μmol·L~(-1)]处理对三角褐指藻细胞生长、岩藻黄素含量、油脂含量以及叶绿素a含量的影响,并对岩藻黄素-叶绿素蛋白复合体基因(FCPb)和酰基-酰基载体蛋白去饱和酶基因(FAB2)的表达进行实时荧光定量PCR分析。结果表明,氮限制极显著抑制了三角褐指藻细胞的生长和岩藻黄素的合成,但促进了油脂的合成。当氮浓度为112μmol·L~(-1)时,三角褐指藻岩藻黄素含量最低(0. 084 mg·g~(-1)DW),而油脂含量最高,较CK提高了1. 36倍。叶绿素a与岩藻黄素含量变化趋势一致。相关性分析表明,氮限制条件下,三角褐指藻岩藻黄素含量与油脂含量显著相关(R~2=0. 998 8)。实时荧光定量PCR分析表明,氮限制抑制了三角褐指藻中FCPb的表达,促进了FAB2的表达。综上,氮限制通过调控三角褐指藻岩藻黄素、油脂生物合成途径相关基因的表达影响了岩藻黄素和油脂的积累。本研究为进一步探究岩藻黄素与脂类物质代谢合成的关联性提供了一定的理论参考。
To demonstrate the effects of nitrogen on secondary metabolic accumulation and regulation of microalgae. The effect of different nitrogen concentrations on the growth, fucoxanthin, lipid and chlorophyll a of Phaeodactylum tricornutum was studied. The expression of fucoxanthin-chlorophyll protein complex gene( FCPb) and acyl-ACP desaturase gene( FAB2) were examined by real-time quantitive PCR( RT-qPCR). The results showed that nitrogen limitation reduced the growth of P. tricornutum cells and inhibited the synthesis of fucoxanthin in P. tricornutum,while it promoted the synthesis of lipid. When the nitrogen concentration was reached to 112 μmol·L~(-1),the fucoxanthin content was the lowest( only 0. 084 mg·g~(-1) DW~(-1)),while the lipid content was the highest and it was 1. 37 times higher than that of the control group. The chlorophyll a content was consistent with the change rhythm of fucoxanthin content.Correlation analysis showed that under nitrogen limitation,the fucoxanthin content of P. tricornutum was significantly correlated with the relative content of lipid( R~2= 0. 998 8). Nitrogen limitation inhibited the expression of FCPb in P.tricornutum,and promoted the expression of FAB2 in P. tricornutum. These findings indicate that nitrogen limitation affects fucoxanthin and total lipid accumulation by regulating the expression of fucoxanthin and lipid biosynthesis pathway-related genes in P. tricornutum. The results provide a theoretical reference for further exploring the correlation between fucoxanthin and lipid metabolism and synthesis.
To demonstrate the effects of nitrogen on secondary metabolic accumulation and regulation of microalgae. The effect of different nitrogen concentrations on the growth, fucoxanthin, lipid and chlorophyll a of Phaeodactylum tricornutum was studied. The expression of fucoxanthin-chlorophyll protein complex gene( FCPb) and acyl-ACP desaturase gene( FAB2) were examined by real-time quantitive PCR( RT-qPCR). The results showed that nitrogen limitation reduced the growth of P. tricornutum cells and inhibited the synthesis of fucoxanthin in P. tricornutum,while it promoted the synthesis of lipid. When the nitrogen concentration was reached to 112 μmol·L~(-1),the fucoxanthin content was the lowest( only 0. 084 mg·g~(-1) DW~(-1)),while the lipid content was the highest and it was 1. 37 times higher than that of the control group. The chlorophyll a content was consistent with the change rhythm of fucoxanthin content.Correlation analysis showed that under nitrogen limitation,the fucoxanthin content of P. tricornutum was significantly correlated with the relative content of lipid( R~2= 0. 998 8). Nitrogen limitation inhibited the expression of FCPb in P.tricornutum,and promoted the expression of FAB2 in P. tricornutum. These findings indicate that nitrogen limitation affects fucoxanthin and total lipid accumulation by regulating the expression of fucoxanthin and lipid biosynthesis pathway-related genes in P. tricornutum. The results provide a theoretical reference for further exploring the correlation between fucoxanthin and lipid metabolism and synthesis.
引文
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[18]蔡卓平,段舜山,朱红惠.三角褐指藻在多波长下藻液吸光度与细胞密度的关系研究[J].广东药学院学报,2012,28(6):608-611
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[24]Ghazaryan A,Akhtar P,Garab G,Lambrev P H,Büchel C.Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes[J].Biochimica et Biophysica Acta,2016,1857(9):1373-1379
[25]杨莹,张娇娇,卜宁,董彩虹.氮源对蛹虫草生长及类胡萝卜素产生的影响[J].微生物学通报,2019,46(1):130-138
[26]郭丽丽,郭帅,董林林,沈亮,李西文,徐江.无公害人参氮肥精细化栽培关键技术研究[J].中国中药杂志,2018,43(7):1427-1433
[27]赵佩佩,顾文辉,伍松翠,解修俊,赵宇鹏,黄爱优.氮限制有利于三角褐指藻脂质积累[J].科学通报,2015(23):2196-2208
[28]Breuer G,Lamers P P,Martens D E,Draaisma R B,Wijffels R H.The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains[J].Bioresour Technology,2012,124(6):217-226
[29]Caron D A,Sanders R W,Lim E L,MarraséC,Amaral L A,Whitney S,Aoki R B,Porters K G.Light-dependent phagotrophy in the freshwater mixotrophic chrysophyte Dinobryon cylindricum[J].Microbial Ecology,1993,25(1):93-111
[30]Carvalho W F,Granéli E.Contribution of phagotrophy versus autotrophy to Prymnesium parvum growth under nitrogen and phosphorus sufficiency and deficiency[J].Harmful Algae,2010,9(1):105-115
[31]Nomura M,Kamogawa H,Susanto E,Kawagoe C,Yasui H,Saga N,Hosokawa M,Miyashita K.Seasonal variations of total lipids,fatty acid composition,and fucoxanthin contents of Sargassum horneri,(Turner)and Cystoseira hakodatensis,(Yendo)from the northern seashore of Japan[J].Journal of Applied Phycology,2013,25(4):1159-1169
[32]吴琼芳.不同氮素浓度下产油普通小球藻的光合生理及生化特征研究[D].广州:暨南大学,2016:27-33
[33]Matsumoto M,Hosokawa M,Matsukawa N,Hagio M,Shinoki A,Nishimukai M.Suppressive effects of the marine carotenoids,fucoxanthin and fucoxanthinol on triglyceride absorption in lymph duct-cannulated rats[J].European Journal of Nutrition,2010,49(4):243-249
[34]Tsukui T,Baba N,Hosokawa M,Sashima T,Miyashita K.Enhancement of hepatic docosahexaenoic acid and arachidonic acid contents in C57BL/6J mice by dietary fucoxanthin[J].Fisheries Science,2009,75(1):261-263
[35]李小梅,夏建荣.氮磷营养限制影响三角褐指藻光合无机碳利用和碳酸酐酶活性[J].水生生物学报,2013,37(3):405-412
[36]Juergens M T,Deshpande R R,Lucker B F,Park J J,Wang H,Gargouri M.The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii[J].Plant Physiology,2015,167(2):558-573
[37]Osuka A,Kume T,Haggquist G W,Jávorfi T,Lima J C,Melo E.Photophysical characteristics of two model antenna systems:Afucoxanthin-pyropheoporbide dyad and its peridinin analogue[J].Chemical Physics Letters,1999,313(3/4):499-504
[38]Zhang L,Li L,He M,Liu J.The role of photorespiration during H2photoproduction in Chlorella protothecoides under nitrogen limitation[J].Plant Cell Reports,2016,35(1):1-4
[39]Longworth J,Wu D,Hueteortega M,Vaidyanathan S.Proteome response of Phaeodactylum tricornutum,during lipid accumulation induced by nitrogen depletion[J].Algal Research,2016,18(8):213-224
[40]Tasseva G,Virville J D D,Cantrel C,Moreau F,Zachowski A.Changes in the endoplasmic reticulum lipid properties in response to low temperature in Brassica napus[J].Plant Physiology and Biochemistry(Paris),2004,42(10):811-822
[41]Gundermann K,Büchel C.The fluorescence yield of the trimeric fucoxanthin-chlorophyll-protein FCPa in the diatom Cyclotella meneghinianais dependent on the amount of bound diatoxanthin[J].Photosynthesis Research,2008,95(2/3):229-235
[2]Mikami N,Hosokawa M,Miyashita K,Miyashita K,Sohma H,Ito Y M,Kokai Y.Reduction of HbA1c levels by fucoxanthin-enriched akamoku oil possibly involves the thrifty allele of uncoupling protein1(UCP1):A randomised controlled trial in normal-weight and obese Japanese adults[J].Journal of Nutritional Science,2017,6(1):1-9
[3]Ana S,Diana P.Overview on the antihypertensive and anti-obesity effects of secondary metabolites from seaweeds[J].Marine Drugs,2018,16(7):e237
[4]Heo S J,Jeon Y J.Protective effect of fucoxanthin isolated from Sargassum siliquastrum on UV-B induced cell damage[J].Journal of Photochemistry and Photobiology B:Biology,2009,95(2):101-107
[5]Zeng J,Zhang Y,Ruan J,Ruan J,Yang Z,Wang C,Hong Z,Zuo Z.Protective effects of fucoxanthin and fucoxanthinol against tributyltin-induced oxidative stress in HepG2 cells[J].Environmental Science and Pollution Research,2018,25(6):5582-5589
[6]Lin J J,Yu J,Zhao J Y,Zhang K,Zheng J C,Wang J L,Huang CH,Zhang J R,Yan X J.Fucoxanthin,a marine carotenoid,attenuatesβ-amyloid oligomer-induced neurotoxicity possibly via regulating the PI3K/Akt and the ERK pathways in SH-SY5Y cells[J].Oxidative Medicine and Cellular Longevity,2017,10(25):1-10
[7]Terasaki M,Iida T,Kikuchi F,Tamura K,Endo T,Kuramitsu Y,Tanaka T,Maeda H,Miyashita K,Mutoh M.Fucoxanthin potentiates anoikis in colon mucosa and prevents carcinogenesis in AOM/DSS model mice[J].Journal of Nutritional Biochemistry,2019,64(6):198-205
[8]管悦琳,龚一富,朱帅旗,俞凯,王何瑜,严小军.三角褐指藻crtiso基因克隆与表达调控研究[J].核农学报,2018,32(11):2098-2106
[9]徐才哲,王文达,艾鹏飞,靳占忠,桑敏,匡廷云.三角褐指藻岩藻黄素-叶绿素蛋白复合体的分离纯化和功能研究[J].水生生物学报,2016,40(1):109-115
[10]刘澈,刘文君.紫外线对原水中几种微藻的作用效果研究[J].给水排水,2011,37(11):115-119
[11]夏金兰,万民熙,王润民,刘鹏,李丽,黄斌,邱冠周.微藻生物柴油的现状与进展[J].中国生物工程杂志,2009,29(7):118-126
[12]郭雪洁.衣藻FAB2基因分离和功能鉴定以及不同胁迫条件下表达模式研究[D].青岛:中国科学院研究生院(海洋研究所),2011:19-25
[13]Bowler C,Allen A E,Badger J H,et al.The Phaeodactylum genome reveals the evolutionary history of diatom genomes[J].Nature,2008,456(7219):239-244
[14]韦剑锋,韦冬萍,陈超君,蓝立斌,熊建文,梁和.施氮水平对甘蔗氮素吸收与利用的影响[J].核农学报,2012,26(3):609-614
[15]Yang M,Meng Y,Chu Y,Fan Y,Cao X,Xue S,Chi Z.Triacylglycerol accumulates exclusively outside the chloroplast in short-term nitrogen-deprived Chlamydomonas reinhardtii[J].Biochimica et Biophysica Acta,2018,1863(12):1478-1487
[16]郑彩云,卢伟婷,王艳,张崇禧.不同氮浓度对紫球藻生长及藻胆蛋白和叶绿素a含量变化的影响[J].中国酿造,2013,32(6):133-135
[17]宋雨晴,靳翠丽,胡文峰,封克,周晓见.氮源对盐藻生长及细胞物质组成的影响[J].生态环境学报,2017,26(2):268-274
[18]蔡卓平,段舜山,朱红惠.三角褐指藻在多波长下藻液吸光度与细胞密度的关系研究[J].广东药学院学报,2012,28(6):608-611
[19]闫相勇,刘翼翔,吴永沛,卢珍华,郭彩华.海带岩藻黄素的提取及纯化工艺研究[J].中国食品学报,2014,14(3):115-121
[20]张敬键,吕雪娟,李爱芬,万凌琳,吴洪,尹顺吉.微藻细胞油脂含量的快速检测方法[J].中国生物工程杂志,2012,32(1):64-72
[21]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408
[22]李涛,许瑾,吴华莲,王铭,向文洲.不同氮浓度对一株产油绿球藻生长、脂类积累及脂肪酸分布的影响[J].生物技术通报,2018,34(5):154-162
[23]LundgrenⅤM,Glibert P M,Granéli E,Vidyarathna N K,Fiori E,Ou L.Metabolic and physiological changes in Prymnesium parvum when grown under,and grazing on prey of variable nitrogen:Phosphorus stoichiometry[J].Harmful Algae,2016,55(5):1-12
[24]Ghazaryan A,Akhtar P,Garab G,Lambrev P H,Büchel C.Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes[J].Biochimica et Biophysica Acta,2016,1857(9):1373-1379
[25]杨莹,张娇娇,卜宁,董彩虹.氮源对蛹虫草生长及类胡萝卜素产生的影响[J].微生物学通报,2019,46(1):130-138
[26]郭丽丽,郭帅,董林林,沈亮,李西文,徐江.无公害人参氮肥精细化栽培关键技术研究[J].中国中药杂志,2018,43(7):1427-1433
[27]赵佩佩,顾文辉,伍松翠,解修俊,赵宇鹏,黄爱优.氮限制有利于三角褐指藻脂质积累[J].科学通报,2015(23):2196-2208
[28]Breuer G,Lamers P P,Martens D E,Draaisma R B,Wijffels R H.The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains[J].Bioresour Technology,2012,124(6):217-226
[29]Caron D A,Sanders R W,Lim E L,MarraséC,Amaral L A,Whitney S,Aoki R B,Porters K G.Light-dependent phagotrophy in the freshwater mixotrophic chrysophyte Dinobryon cylindricum[J].Microbial Ecology,1993,25(1):93-111
[30]Carvalho W F,Granéli E.Contribution of phagotrophy versus autotrophy to Prymnesium parvum growth under nitrogen and phosphorus sufficiency and deficiency[J].Harmful Algae,2010,9(1):105-115
[31]Nomura M,Kamogawa H,Susanto E,Kawagoe C,Yasui H,Saga N,Hosokawa M,Miyashita K.Seasonal variations of total lipids,fatty acid composition,and fucoxanthin contents of Sargassum horneri,(Turner)and Cystoseira hakodatensis,(Yendo)from the northern seashore of Japan[J].Journal of Applied Phycology,2013,25(4):1159-1169
[32]吴琼芳.不同氮素浓度下产油普通小球藻的光合生理及生化特征研究[D].广州:暨南大学,2016:27-33
[33]Matsumoto M,Hosokawa M,Matsukawa N,Hagio M,Shinoki A,Nishimukai M.Suppressive effects of the marine carotenoids,fucoxanthin and fucoxanthinol on triglyceride absorption in lymph duct-cannulated rats[J].European Journal of Nutrition,2010,49(4):243-249
[34]Tsukui T,Baba N,Hosokawa M,Sashima T,Miyashita K.Enhancement of hepatic docosahexaenoic acid and arachidonic acid contents in C57BL/6J mice by dietary fucoxanthin[J].Fisheries Science,2009,75(1):261-263
[35]李小梅,夏建荣.氮磷营养限制影响三角褐指藻光合无机碳利用和碳酸酐酶活性[J].水生生物学报,2013,37(3):405-412
[36]Juergens M T,Deshpande R R,Lucker B F,Park J J,Wang H,Gargouri M.The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii[J].Plant Physiology,2015,167(2):558-573
[37]Osuka A,Kume T,Haggquist G W,Jávorfi T,Lima J C,Melo E.Photophysical characteristics of two model antenna systems:Afucoxanthin-pyropheoporbide dyad and its peridinin analogue[J].Chemical Physics Letters,1999,313(3/4):499-504
[38]Zhang L,Li L,He M,Liu J.The role of photorespiration during H2photoproduction in Chlorella protothecoides under nitrogen limitation[J].Plant Cell Reports,2016,35(1):1-4
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