|
|
Effects of Different Exogenous Elicitors on lcyb Gene Transcription and Fucoxanthin Content in Phaeodactylum tricornutum |
|
|
Abstract Lycopene beta-cyclase (LCYB), which catalyzes the formation of β-carotene and its oxides, is an enzyme act as branching point of the plant carotenoid synthesis pathway. This study investigated the effect of elicitors on the expression of lcyb gene and content of fucoidin in Phaeodactylum tricornutum. Firstly, the full-length cDNA of lcyb had been obtained from P. tricornutum, and bioinformatics analysis was carried out. Then, aethyl jasmonate (MeJA), aacetylsalicylic acid (ASA),arachidonic acid (AA) and ammonium cerous sulfate (ACS) were used to induce the expression of Ptlcyb gene and the content of fucoxanthin in P. tricornutum. The results showed that the lcyb gene of Phaeodactylum tricornutum obtained by de novo sequencing was same as the sequence on NCBI(GenBank No. XM002176576). The full length of lcyb gene was comprised by 2 060 bp, contanting a 1 980 bp open reading fragment(ORF), which encoded a polypeptide of 659 amio acids. Sequence analysis showed that P. tricornutum LCYB had a dinucleotides-biding site at the N-terminus and a Predicted TM helix at the C-terminus. In addition, signal peptide and the presence of chloroplast peptide han been found, which further proved that LCYB was located in the thylakoid membrane. Phylogenetic tree demostrated that diatom such as P. tricornutum coexisted in an evolutionary branch, which was closely related. Upon the observation of Ptlcyb regulation expression, the expression level of Ptlcyb had significant rise when the concentration was 50 μmol/L MeJA, 10 mg/L ASA, 0.1 mg /L AA and 0. 4 mg/L ACS. At the same time, the content of fucoxanthin in P. tricornutum was significantly higher than that in the control group (P <0.01). The variation trend of fucoxanthin content and Ptlcyb expression were basically the same, which showed that Ptlcyb played an important role in the synthesis of fucoxanthin. This study provide theoretical guidance for the regulation of fucoxanthin synthesis and also provide a reference for further improving the content of fucoxanthin by means of metabolic engineering in P. tricornutum.
|
Received: 24 March 2017
Published: 10 December 2017
|
|
|
|
陈选阳, 袁照年, 张招娟等. 2007. 甘薯番茄红素β-环化酶基因的克隆与转化烟草的研究[J]. 作物学报. 33(10): 1724-1728. (Chen X Y, Yuan Z N, Zhang Z J, et al. 2007. Cloning of lyc-b Gene from Sweetpotato (Ipomoea batatas L.) and Transferring the Gene into Tobacco[J]. Acta Agronomica Sinica, 33(10): 1724-1728.)段永波, 鲁放, 崔婷婷等. 2017. 非生物诱导子茉莉酸甲酯和水杨酸对半夏悬浮细胞中生物碱代谢的影响[J]. 中国中医药信息杂志, 24(1):87-90. (Duan Y B, Lu F, Cui T T, et al. 2017. Effects of Abiotic Elicitors MeJA and SA on Alkaloids Accumulation and Related Enzymes Metabolism in Pinellia ternata Suspension Cell Cultures[J]. Chinese Journal of Information on Traditional Chinese Medicine, 24(1): 87-90. )郭春芳. 2006. 影响植物细胞生长发育的质外体信号分子[J]. 福建教育学院学报. 7(1): 111-115. (Guo C F. 2006. Apoplast Signal Molecules that Affect the Growth of the Plant's Cells[J]. Journal of Fujian Education Institute, 7(1): 111-115. )韩娟, 杨艳, 祝传书等. 2012. 茉莉酸甲酯处理雷公藤悬浮细胞的基因差异表达分析[J]. 西北植物学报, 32(12): 2398-2404. (Han J, Yang Y, Zhu C S, et al. 2012. Analysis of Differentially Expressed Genes in the Cell Culture of Tripterugium willfordii Hook.f. With Elictor MeJA[J]. 32(12): 2398-2404.)李婷, 施春雷, 淦志兵等. 2009. 原壳小球藻番茄红素ε环化酶基因的克隆和分析[J]. 微生物学报. 49(9): 1180-118. (Li T, Shi C L, Gan Z B, et al. 2009. Cloning and analysis of the gene encoding lycopene epsilon cyclase in Chlorella protothecoides CS-41[J]. Acta microbiologica Sinica, 49(9): 1180-118. )刘浩, 杭雨晴, 朱帅旗等. 2016. 葡萄糖对三角褐指藻生长、岩藻黄素含量及相关基因表达的影响[J]. 中国药学杂志. 51(14): 1230-1234. (Liu H, Hang Y Q, Zhu S Q, et al. 2016. Effects of Glucose on the Growth of Phaeodactylum tricornutum, Fucoxanthin Content and Related Gene Expression[J]. Chinese Pharmaceutical Journal, 51(14): 1230-1234.)牧文, 刘文哲. 2007. 伤害及诱导子对喜树幼苗中喜树碱含量的影响[J]. 西北植物学报, 27(5):977-982. (Mu W, Liu W Z. 2007. Effects of Mechanical Wounding and Elicitors on Camptothecin Content in Camptotheca acuminata Seedlings[J]. Acta Botanica Boreali-Occidentalia Sinica, 27(5): 977-982.)苗晓燕, 朱维红, 张筱梅等. 2016. 水杨酸、茉莉酸甲酯及超声波对北沙参愈伤组织生长及产香豆素的影响[J]. 食品科学, 37(9):181-185. (Miao X Y, Zhu W H, Zhang X M, et al. 2016. Effects of Salicylic Acid, Methyl Jasmonate and Ultrasound on the Production of Cumarins and Growth of Radix Glehniae Callus[J]. Food Science, 37(9):181-185.)邵磊, 辛现良, 耿美玉. 2005. 昆布多糖药理作用的研究进展[J]. 中国海洋药物. 24(2): 57-60. (Shao L, Xin X L, Geng M Y. 2005. Research progress in the pharmacological effect of polysaccharides from Laminaria japonica[J]. Chinese Journal of Marine Drugs, 24(2): 57-60. )陶俊, 刘兴满. 2006. 枇杷番茄红素β环化酶基因片段的克隆和序列分析[J]. 苏州科技学院学报(社会科学). 23(4) :43-46. (Tao J, Liu X M. 2006. Molecular Cloning and Sequence Analysis of lycopene β- cyclase Gene Fragment from Loquat[J]. Journal of University of Science and Technology of Suzhou: Social Science, 23(4): 43-46. )汪曙晖, 薛长湖. 2010. 岩藻黄素的结构、性质和功能[J]. 食品工业科技, 31(6):408-410. (Wang S H, Xue C H. 2010. Chenical strucyure, propertiees and bioacticities of fucoxanthin[J]. Science and Technology of Food Industry, 31(6): 408-410.)王丽丽, 李惠咏, 龚一富等. 2010. 花生四烯酸对雨生红球藻细胞生长和虾青素含量的影响[J]. 水产科学. 29(3): 142-146. (Wang L L, Li H Y, Gong Y F, et al. 2010. The Effects of Arachidonic Acid (AA) on the Cell Growth and Astaxanthin Content in Alge Haematococcus pluvialis[J]. Fisheries Science, 29(3): 142-146. )王鑫威, 王丽丽, 龚一富等. 2011. 茉莉酸甲酯对雨生红球藻虾青素含量和dxs基因表达的影响[J]. 水产学报. 35(12): 1822-1828. (Wang X W, Wang L L, Gong Y F, et al. 2011. The effects of methyl jasmonate(MeJA) on the astaxanthin production and dxs gene expression of Haematococcus pluvialis[J]. Journal of Fisheries of China, 35(12): 1822-1828.)严小军, 范晓, 娄清香等. 2001. 海藻中类胡萝卜素的提取及含量测定[J]. 海洋科学集刊, 43(00): 113-119. (Yan X J, Fan X, Lou Q X, et al. 2001. Isolation and quantification of carotenoids from marine algae[J]. Studia Marina Sinica, 43(00): 113-119.)晏琼, 胡宗定, 吴建勇. 2006. 生物和非生物诱导子对丹参毛状根培养生产丹参酮的影响[J]. 中草药, 37(2): 262-265. (Yan Q, Hu Z D, Wu J Y. 2006. Influence of biotic and abiotic elicitors on production of tanshinones in Salviam iltiorrhiza hairy root culture[J]. Chinese Traditional and Herbal Drugs, 37(2): 262-265.)晏露, 姜建国, 刘冠楠等. 2010. 盐藻番茄红素β-环化酶全长cDNA的克隆及结构分析[J]. 现代食品科技. 26(3): 218-221. (Yan L, Jiang J G, Liu G N, et al. 2010. Cloning and Structure Analysis of the Full Length cDNA of Lycopeneβ-Cyclase from Dunaliella salina[J]. Modern Food Science & Technology, 26(3): 218-221.)张坚, 马琳, 李兴林等. 2016. 茉莉酸甲酯对杠柳不定根中重要次生代谢产物诱导及生理机制的研究[J]. 中国科技论文在线. (Zhang J, Ma l, li X L, et al. 2016. Study on MeJA Inducing Effect to Important Secondary Metabolites and on Plant Physiological Mechanism[J]. www.paper.edu.cn)章丽, 龚一富, 刘晓丹等. 2013. 盐生杜氏藻β-胡萝卜素羟化酶基因(chyb)的克隆及表达分析[J]. 农业生物技术学报, 21(8):920-930. (Zhang L, Gong Y F, Liu X D, et al. 2013. Cloning and Expression Analysis of β-carotene Hydroxylase Gene (chyb) from Dunaliella salina[J]. Chinese journal of agricultural biotechnolog, 21(8):920-930.)余桂兰, 胡晗华, 李秀波等. 2007. 海洋富油微拟球藻nannochloropsis gaditana ccap849/5的转化及外源基因整合方式[J]. 热带海洋学报. 33(2): 72-77. (Yu G L, Hu H H, Li X B, et al. 2007. Transformation of marine oleaginous alga Nannochloropsis gaditana CCAP849/5 and heterologous gene integration[J]. Journal of Tropical Oceanography, 33(2): 72-77.)邹宁, 李艳, 孙东红. 2005. 几种有经济价值的微藻及其应用[J]. 鲁东大学学报(自然科学版). 21(1): 59-63. (Zhou N, Li Y, Sun D H. 2005. Several species of economic microalage and their applications[J]. Ludong University Journal (Natural Science Edition), 21(1): 59-63.)朱长甫, 陈 星, 王英典. 2004. 植物类胡萝卜素生物合成及其相关基因在基因工程中的应用[J].植物生理与分子生物学学报. 30(6):609-618. (Zhu C F, Chen X, Wang Y D. 2004. Carotenoid Biosynthesis in Plants and Application of Its Relative Genes in Gene Engineering[J]. Journal of Plant Physiology and Molecular Biology, 30(6): 609-618.)朱帅旗, 龚一富, 刘浩等. 2014. 硫酸铈铵对三角褐指藻岩藻黄素含量的影响及转录差异研究[J]. 中国稀土学报. 32(6): 750-757. (Zhu S Q, Gong Y F, Liu H, et al. 2014. Effects of Ammonium Cerous Sulfate on Fucoxanthin Content in PhaeodactylumTricornutum and Research of Transcriptional Differences[J]. Journal of The Chinese Rare Earth Society, 32(6): 750-757.)朱跃辉, 姜建国. 2005. 类胡萝卜素代谢途径中相关番茄红素环化酶的功能[J]. 中国食品添加剂. 5: 40-43. (Zhu Y H, Jiang J G. 2005. Functi ons of related lycopene cyclases in the metabolic pathway of carotenoids]J]. China Food Additives, 5: 40-43.)Aoki K, Yano K, Suzuki A, et al. 2010. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics.[J]. BMC Genomics, 11(1): 210.Bendsten J D, Nielsen H, Heijne G V, et al. 2004. Prediction of signal peptides: SignalP3.0[J]. Journal of Biochemistry, 340: 783-795 .Bramley P, Teulieres C, Blain I, et al. (1992). Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5[J]. Plant Journal, 2(4): 343-349.Bowler C, Allen A E, Badger J H, et al. 2008. The Phaeodactylum genome reveals the evolutionary history of diatom genomes[J]. Nature, 456(7219): 239-44.Cunningham F X, Gantt E. 1994. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.[J]. Plant Cell, 6(8): 1107.Dharmapuri S, Rosati C, Pallara P, et al. 2002. Metabolic engineering of xanthophyll content in tomato fruits[J]. Febs Letters, 519(1): 30-34.Diretto G, Tavazza R, Welsch R, et al. 2006. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase[J]. BMC Plant Biology, 6(1): 13.Emanuelesson O, Nielsen H. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites[J]. Protein Science, 8(5): 978-984.Gasteiger E, Hoogland C, Gattiker A, et al. 2005. Protein identification and analysis tools on the ExPAsy server[M]. Swiss, Humana Press, pp: 571-607.Geourjon C, Deleage G. 1995. 1SOPMA: Significant improvement in protein secondary structure prediction by consensus prediction from multiple alinments[J]. Computer Applications in the Biosciences: CABISO, 1(6): 681-684.Gong Y F, Liao Z H, Guo B H, et al. 2006. 7Molecular cloning and expression profile analysis of Ginkgo biloba DXS gene encoding 1-deoxy-D-xylulose 5-phosphate synthase, the first committed enzyme of the 2-C-methyl-D-erythrito l,4-phosphate pathway[J]. Planta Medica, 2: 329-335.Gundlach H, Muller M J, Kutchan T M, et al. 1992. Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures[J]. Proceedings of the National Academy of Sciences of the United States of America, 89: 2389-2393.Guex N, Peitsch M C. 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling[J]. Electrophoresis, 18: 2714-2723.Haugan J A, Aakermann T, Liaaen J S. 1992. Isolation of fucoxanthin and peridinin[J]. Methods in Enzymology, 213: 231-245.Higgins D, Thompson J, Gibson T, et al. 1999. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gapenalties and weight matrix choice[J]. Nucleic Acids Research, 22: 4673-4680.Hosokawa M, Miyashita T, Nishikawa S, et al. 2010. Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK-A y mice[J]. Archives of Biochemistry and Biophysics, 504(1): 17-25.Jr C F, Pogson B, Sun Z, et al. 1996. Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation.[J]. The Plant cell, 8(9):1613-26.Kim S M, Jung Y J, Kwon O N, et al. 2012. A potential commercial source of fucoxanthin extracted from the microalga Phaeodactylum tricornutum.[J]. Applied Biochemistry and Biotechnology, 166(7): 1843-1855.Kumar S, Nei M, Dudley J, et al. 2008. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences[J]. Briefings in Bioinformatics, 9(4): 299-306.Maeda H, Hosokawa M, Sashima T, et al. 2007. Dietary combination of fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and decreases blood glucose in obese/diabetic KK-A y mice[J]. Journal of Agricultural and Food Chemistry, 55(19): 7701-7706.Mount D W. 2003. Bioinformatics[M]. Higher Education Press, Beijing, pp: 736-767.Osborne B, Geider R. 1986. Effect of nitrate limitation on photosynthesis of the diatom Phaeodactylum tricornutum Bohlin (Bacillarophyceae)[J]. Plant Cell & Environment, 9(8): 617-625.Phillip D M, Young A J. 2006. Preferential inhibition of the lycopene epsilon-cyclase by the substituted triethylamine compound MPTA in higher plants.[J]. Journal of Plant Physiology, 163(4): 383-391.Ronen G, Cohen M, Zamir D, et al. 1997. Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant Delta.[J]. Plant Journal, 17(4): 341–351.Ruizsola M A, Rodríguezvillalón A, Rodríguezconcepción M. 2014. Light-sensitive Phytochrome-Interacting Factors (PIFs) are not required to regulate phytoene synthase gene expression in the root.[J]. Plant Signaling & Behavior, 9(8): 353-372.Sachindra N M, Airanthi M K W A, Hosokawa M, et al. 2010. Radical scavenging and singlet oxygen quenching activity of extracts from Indian seaweeds[J]. Journal of Food Science & Technology, 47(1):94-99.Server O T E. 2008. Protein Analysis Tools on the ExPASy Server 571 52 Protein Identification and Analysis Tools[J]. Methods in Molecular Biology, 112(112): 531-52.Shiratori K, Ohgami K, Ilieva I, et al. 2005. Effects of fucoxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo.[J]. Experimental Eye Research, 81(4):422-428.Singh M, Brutnell T P. 2003. Activator Mutagenesis of the Pink scutellum1/viviparous7 Locus of Maize[J]. Plant Cell, 15(4): 874-884.Tian L, Magallanes-Lundback M, Musetti V, et al. 2003. Functional Analysis of β - and ε - Ring Carotenoid Hydroxylases in Arabidopsis[J]. Plant Cell, 15(6): 1320.Verpoorte R, Heijden R V D, Hoge J H C, et al. 1994. Plant cell biotechnology for the production of secondary metabolites[J]. Pure & Applied Chemistry, 66(10-11): 2307-2310.Waegemann K and Soll J, 1996. Phosphorylation of the transit sequence of choloroplast precursor proteins[J]. The Journal of Biological Chemistry, 271 (11) : 6545~6554.Wasternack C, Parthier B. 1997. Jasmonate-signalled plant gene expression[J]. Trends in Plant Science, 2(8): 302-307.Wierstra, I., Kloppstech, K. 2000. Differential effects of methyl jasmonate on the expression of the early light-inducible proteins and other light-regulated genes in barley[J]. Plant Physiol, 124(2): 833–844.Woo M N, Jeon S M, Kim H J, et al. 2010. Fucoxanthin supplementation improves plasma and hepatic lipid metabolism and blood glucose concentration in high-fat fed C57BL/6N mice[J]. Chemico-Biological Interactions, 186(3): 316-322.Yao P, Yu Z G, Deng C M. 2006. Pigment signatures of some diatoms isolated from China seas[J]. Acta Oceanologica Sinica, 25(1):108-118.Zhao J, Zhu W, Hu Q. 2001. Selection of fungal elicitors to increase indole alkaloid accumulation in Catharanthus roseus suspension cell culture[J]. Enzyme & Microbial Technology, 28(7–8): 666-672. |
|
|
|