目的 研究茉莉酸甲酯(methyl jasmonate,MeJA)、硫酸铈铵(ammonium cerous sulfate,ACS)、花生四烯酸(arachidonic acid,AA)、光和促进剂(photosynthetic induction factor,PIF)、光合抑制剂[3-(3,4-dichlorophenyl)-1,1-dimethylurea,DCMU]和不同光质对三角褐指藻3-羟基-3-甲基戊二酸单酰辅酶A还原酶(3-hydroxy-3-methylglutarate monoacyl-CoA reductase,HMGR)基因PtHMGR表达的影响,研究不同外源因素处理下PtHMGR基因表达与岩藻黄素含量之间的关系。方法 通过转录组测序获得PtHMGR基因cDNA序列,并对其进行生物信息学分析,有机溶剂提取法测定MeJA、ACS、AA、PIF、DCMU和不同光质处理下三角褐指藻岩藻黄素含量,荧光定量多聚酶链式反应(polymerase chain reaction,PCR)检测PtHMGR基因表达。结果 PtHMGR基因全长2 161 bp,其ORF长1 914 bp,编码637个氨基酸。PtHMGR蛋白分别含有2个烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADP)和3-羟基-3-甲基戊二酸单酰辅酶A(3-hydroxy-3-methyl glutaryl coenzyme A reductase,HMG-CoA)结合基序,催化结构域包含L-domain、S-domain和N-domain,N端存在3个跨膜螺旋区。系统进化树分析结果表明,动物和植物HMGR明显分为2支,三角褐指藻与假微型海莲藻(Thalassiosira pseudonana)HMGR蛋白的亲缘关系最近,同处于水生植物一支。荧光定量PCR分析结果表明,12.5 mg·L-1 AA、0.4 mg·L-1ACS、0.05 μg·L-1 PIF、0.4 mg·L-1 DCMU和紫光处理下PtHMGR基因表达水平最高。相关性分析结果表明,ACS和不同光质处理下岩藻黄素含量与PtHMGR基因表达呈正相关。结论 PtHMGR基因是参与ACS和不同光质调控三角褐指藻岩藻黄素合成的关键基因之一,PtHMGR与三角褐指藻岩藻黄素的合成与积累具有密切关系。
Abstract
OBJECTIVE To study the effects of methyl jasmonate (MeJA), arachidonic acid(AA), ammonium cerous sulfate(ACS), photosynthetic induction factor(PIF), photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) and different light quality on expression of 3-hydroxy-3-methylglutarate monoacyl-coA reductase(HMGR) gene in Phaeodactylum tricornutum and study the relationship between the expression of PtHMGR gene and fucoxanthin content in P.tricornutum treated by different exogenous factors. METHODS The cDNA of PtHMGR gene in P.tricornutum was obtained by transcriptome sequencing, and bioinformatics analysis was conducted. An organic solvent extraction method was used to determine fucoxanthin content in P.tricornutum and PtHMGR gene expression was detected by real-time fluorescence quantitative PCR. RESULTS A 2 161 bp PtHMGR gene was cloned, containing a complete open reading frame of 1 914 bp, encoding 637 amino acids. P.tricornutum PtHMGR protein contains two NADP and HMG-COA binding motifs respectively. It has a typical structure with a catalytic regionincluding L, N and S domains, and there are three transmembrane helical regions at the N-terminal. The phylogenetic tree analysis showed that PtHMGR proteins are divided into two branches, animal HMGRs group and plant HMGRs group. The P.tricornutum and Thalassiosira pseudonana HMGR are closely related, and both belong to the branch of aquatic plants. The analysis of PtHMGR gene transcription results showed that 12.5 mg·L-1 AA, 0.4 mg·L-1 ACS, 0.05 μg·L-1 PIF, 0.4 mg·L-1 DCMU, and purple light promoted the expression of P.tricornutum PtHMGR gene. Correlation analysis results showed that fucoxanthin content was positively correlated with PtHMGR gene expression under ACS and different light quality treatments. CONCLUSION PtHMGR gene is one of the key genes involved in the regulation of fucoxanthin synthesis in P.tricornutum under ACS and different light quality treatment, and HMGR is closely related to the synthesis and accumulation of fucoxanthin.
关键词
三角褐指藻 /
岩藻黄素 /
3-羟基-3-甲基戊二酸单酰辅酶A还原酶 /
克隆 /
诱导表达
{{custom_keyword}} /
Key words
Phaeodactylum tricornutum /
fucoxanthin /
3-hydroxy-3-methylglutarate monoacyl-CoA reductase /
gene cloning /
induced expression
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] PENG J, YUAN J P, WU C F, et al. Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms: metabolism and bioactivities relevant to human health[J]. Mar Drugs, 2011, 9(10): 1806-1828.
[2] MERESSE S, FODIL M, FLEURY F, et al. Fucoxanthin, a marine-derived carotenoid from brown seaweeds and microalgae: a promising bioactive compound for cancer therapy[J]. Int J Mol Sci, 2020, 21(23): 9273-9300.
[3] SATOMI Y. Antitumor and cancer-preventative function of fucoxanthin: a marine carotenoid[J]. Anticancer Res, 2017, 37(4): 1557-1562.
[4] TAKATANI N, KONO Y, BEPPU F, et al. Fucoxanthin inhibits hepatic oxidative stress, inflammation, and fibrosis in diet-induced nonalcoholic steatohepatitis model mice[J]. Biochem Biophys Res Commun, 2020, 528(2): 305-310.
[5] MIYASHITA K, HOSOKAWA M. Fucoxanthin in the management of obesity and its related disorders[J]. J Funct Foods, 2017, 36: 195-202. Doi:10.1016/j.jff.2017.07.009.
[6] LIN J J, HUANG L, YU J, et al. Fucoxanthin, a marine carotenoid, reverses scopolamine-induced cognitive impairments in mice and inhibits acetylcholinesterase in vitro[J]. Mar Drugs, 2016, 14(4): 67-84.
[7] XIANG S Y, LIU F F, LIN J J, et al. Fucoxanthin inhibits β-amyloid assembly and attenuates β-amyloid oligomer-induced cognitive impairments[J]. J Agric Food Chem, 2017, 65(20): 4092-4102.
[8] LIN J J, YU J, ZHAO J Y, et al. Fucoxanthin, a marine carotenoid, attenuates β-amyloid oligomer-induced neurotoxicity possibly via regulating the PI3K/Akt and the ERK pathways in SH-SY5Y cells[J]. Oxid Med Cell Longev, 2017, 2017: 1-10.Doi:10.1155/2017/6792543.
[9] MOHAMADNIA S, TAVAKOLI O, FARAMARZI M A. Enhancing production of fucoxanthin by the optimization of culture media of the microalga Tisochrysis lutea[J]. Aquaculture, 2020, 533: 736074. Doi:10.1016/j.aquaculture.2020.736074.
[10] ZHANG N N, LUO L, CHEN Z, et al. Biosynthesis pathway of fucoxanthin and expression levels of key genes for fucoxanthin synthesis in response to high irradiance in Phaeodactylum tricornutum[J]. Chin J Oil Crop Sci(中国油料作物学报), 2017, 39(1): 128-136.
[11] ZHANG W, LIANG C W. Research progress of plant isoprenoids biosynthetic pathway[J]. Shandong Chem Ind(山东化工), 2014, 43(5): 57-58.
[12] LI Q, LI B, GUO S X. Cloning and expression analysis of hmgr gene in Dendrobium nobile in response to mycorrhizal fungal inoculation[J]. Chin Pharm J(中国药学杂志), 2017, 52(22): 1976-1982.
[13] WANG H, WEI J S, YANG J F, et al. Overexpression of HMGR and DXR from Amomum villosum Lour. affects the biosynthesis of terpenoids in Tobacco[J]. Mod Tradit Chin Med Mater Med World Sci Technol (世界科学技术-中医药现代化), 2014, 16(7): 1513-1527.
[14] ZHU C S, CHEN M M, LIU Y,et al. Effect of over-expressing TwHMGR and TwDXR on the biosynthesis of terpenoids in Tripterygium wilfordi[J]. J Agric Biotechnol(农业生物技术学报), 2018, 26(6): 940-948.
[15] ZHANG B. Study on regulations of hmgr and dxr genes on bios ynthesis of terpenoids in Tripterygium wilfordii HOOK. F. using RNAi[D]. Xianyang: Northwest A&F University, 2015.
[16] CHAN Y P, SHEN T, QIU F, et al. Molecular cloning and expression analysis of HMG-CoA(3-hydroxy-3-methylglutaryl-CoA) reductase gene familyupon MeJA and mechanical injury treatments in Artemisia annua L.[J]. J Southwest Univ Nat Sci Ed(西南大学学报自然科学版), 2017, 39(12): 44-51.
[17] LIAO P. Cloning of genes involved in tanshinones biosynthesis and its metabolic regulation[D]. Shanghai: Shanghai Normal University, 2010.
[18] FAN Y F. Discovery of sesquiterpenes inducer and cloning, geneexpression of terpenoids synthase from Aquilaria sinensis[D]. Guangzhou: Guangdong Pharmaceutical University, 2017.
[19] ZHANG W J, CAO X Y, JIANG J H. Triterpene biosynthesis in Euphorbia pekinensis induced by methyl jasmonate[J]. Guihaia(广西植物), 2015, 35(4): 590-596.
[20] ZHU F. Cloning functional expression, and expressional regulation of the Ganoderma lucidum hydroxy methylglutaryl-coenzyme are ductase gene[D]. Nanjing: Nanjing Agricultural University, 2007.
[21] YU K, GONG Y F, ZHU S Q, et al. Effects of different exogenous elicitors on lcyb gene transcription and fucoxanthin content in Phaeodactylum tricornutum[J]. J Agric Biotechnol(农业生物技术学报), 2017, 25(12): 2017-2019.
[22] LIN R Y. Studies on the physiological and molecular mechanism of alkaloid synthesis of in vitro cultures in Dendrobium huoshanense[D]. Fuzhou: Fujian Agriculture and Forestry University, 2014.
[23] XU R J, GONG Y F, WEI F J, et al. Correlation analysis of photosynthetic physiological indexes and content of fucoxanthin in Phaeodactylum tricornutum under different light quality conditions[J]. Chin J Lasers(中国激光), 2020, 47(5): 471-479.
[24] LI S R, ZHENG X Y, GONG Y F, et al. Exploring the potential of photosynthetic induction factor for the commercial production of fucoxanthin in Phaeodactylum tricornutum[J]. Bioprocess Biosyst Eng, 2021, 44(3): 1769-1779.
[25] ZHENG X Y, GONG Y F, LI S R, et al. Effects of the photosynthesis inhibitor DCMU on fucoxanthin content, chlorophyll fluorescence characteristics and key genes of Phaeodactylum tricornutum[J]. J Nucl Agric Sci(核农学报), 2020, 34(8): 1705-1712.
[26] SONG Y Q, JIN C L, HU W F, et al. The effect of nitrogen supply on the growth and cell chemical composition of Dunaliella salina[J]. Ecol Environ Sci(生态环境学报), 2017, 26(2): 268-274.
[27] YIN H, YUAN L Y, YU K. Bioinformatics and expression differences of GPAT in Phaeodactylum tricornutum[J]. J Nucl Agric Sci(核农学报), 2019, 33(8): 1483-1489.
[28] TROTT O, OLSON A J. Software news and update autodockvina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[J]. J Comput Chem, 2010, 31(2): 455-461.
[29] ISTVAN ES, DEISENHOFER J. The structure of the catalytic portion of human HMG-CoA reductase[J]. Biochim Biophys Acta Mol Cell Biol Lipids, 2000, 1529(1-3): 9-18.
[30] FRIESEN J A, RODWELL V W. The 3-hydroxy-3-methylglutaryl coenzyme-A(HMG-CoA) reductases[J]. Genome Biol, 2004, 5(11): 248-255.
[31] DARABI M, MASOUDI-NEJAD A, NEMAT-ZADEH G. Bioinformatics study of the 3-hydroxy-3-methylglotaryl-coenzyme A reductase(HMGR) gene in Gramineae[J]. Mol Biol Rep, 2012, 39(9): 8925-8935.
[32] DARABI M, FARHADI-NEJAD H. Study of the 3-hydroxy-3-methylglotaryl-coenzyme A reductase(HMGR) protein in Rosaceae by bioinformatics tools[J]. Caryologia, 2013, 66(4): 351-359.
[33] ISTVAN ES, PALNITKAR M, BUCHANAN SK, et al. Crystal structure of the catalytic portion of human HMG-CoA reductase: insights into regulation of activity and catalysis[J]. Embo J, 2014, 19(5):819-830.
[34] BHAMBHANI S, LAKHWANI D, SHUKLA T, et al. Genes encoding members of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR) gene family from Azadirachta indica and correlation with azadirachtin biosynthesis[J]. Acta Physiol Plant, 2017, 39(2): 65-78.
[35] LIU W, ZHANG Z Q, LI W,et al. Genome-wide identification and comparative analysis of the 3-hydroxy-3-methylglutaryl coenzyme a reductase(HMGR) gene family in gossypium[J]. Molecules, 2018, 23(2): 193-211.
[36] AKHTAR N, GUPTA P, SANGWAN N S, et al. Cloning and functional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania somnifera: an important medicinal plant[J]. Protoplasma, 2013, 250(2): 613-622.
[37] GUAN Y L, GONG Y F, ZHU S Q, et al. Cloning and expression analysis of carotenoid isomerase gene from Phaeodactylum tricornutum[J]. J Nucl Agric Sci(核农学报), 2018, 32(11): 2098-2106.
[38] XIA Y, GU W, CHAO J G, et al. Effects of exogenous calcium chloride on growth physiological indexes and leaf key enzyme gene expression of Atractylodes lancea(Thunb.) DC. under high temperature stress[J]. J South Agric(南方农业学报), 2020, 51(2): 267-274.
[39] MIAO Z Q, WEI Z J, YUAN Y J. Studies on the acting point of arachidonic acid in taxol biosynthetic pathway[J]. Chin Tradit Herb Drugs(中草药), 2000, 31(6): 20-23.
[40] XU Q M, YUAN Y J, CHENG J S, et al. Cell membrane permeability changes induced by Ce4+ in suspension cultures of taxus cuspidate[J]. Chin Rare Earths(稀土), 2004, 25(2): 50-53.
[41] LV D M, ZHANG T T, DENG S, et al. Functional analysis of the Malus domestica MdHMGR2 gene promoter in transgenic Arabidopsis thaliana [J]. Biol Plant, 2016, 60(4): 1-10.
[42] LV D M, ZHANG Y H. Isolation and functional analysis of apple MdHMGR1 and MdHMGR4 gene promoters in transgenic Arabidopsis thaliana[J]. Plant Cell Tissue Organ Cult, 2016, 129(1): 1-11.
[43] ZHU C S, LIU Y, PU S, et al. Cloning and expression analysis of 4 oxidosqualene cyclase genes in tripterygium wilfordii[J]. J Agric Biotechnol(农业生物技术学报), 2019, 27(2): 237-247.
[44] GUO S, LIU J, LU K H, et al. Influence of elicitors on effective constituents and expression of key metabolic enzyme genes in hairyroots of Atropa belladonna[J]. Chin Tradit Herb Drugs(中草药), 2018, 49(4): 897-902.
[45] LI Y M, ZHANG N, LI H, et al. Cloning and expression characteristics of a transcription factor gene RpMYB4 in Rheum palmatum L.[J]. Acta Pharm Sin(药学学报), 2021, 56(4): 1170-1177.
[46] WANG X W, WANG L L, GONG Y F,et al. The effects of methyl jasmonate(MeJA) on the astaxanthin production and dxs gene expression of Haematococcus pluvialis[J]. J Fish China(水产学报), 2011, 35(12): 1822-1828.
[47] XU R J, GONG Y F, CHEN W T, et al. Effects of LED monochromatic light quality of different colors on fucoxanthin content and expression levels of related genes in Phaeodactylum tricornutum[J]. Acta Opt Sin(光学学报), 2019, 39(9): 299-307.
[48] LU Q J, CHAO J G, GU W, et al. Effects of copper stress on accumulation of three medicinal compositions and expression of two keyenzyme genes in biosynthesis of Atractylodes lancea[J]. Chin Tradit Herb Drugs(中草药), 2019, 50(3): 710-715.
[49] MA F L, LI D M, LI Z, et al.GhHMGR gene function in ovule development of cotton(Gossypium hirsutum L.)[J]. Acta Agron Sin(作物学报), 2016, 42(2): 222-229.
[50] JOHNSON B M, DEBOSE-BOYD R A . Underlying mechanisms for sterol-induced ubiquitination and ER-associated degradation of HMG CoA reductase[J]. Semin Cell Dev Biol, 2017, 81: 121-128. Doi:10.1016/j.semcdb.2017.10.019.
[51] ZEIDABAD D D, JAVARAN M J, DEHGHANI H, et al. An investigation of the HMGR gene and IPI gene expression in black caraway(Bunium persicum)[J]. 3 Biotech, 2018, 8(9): 405. Doi:10.1007/s13205-018-1404-y.
[52] WANG J, LI J F, ZHANG T T, et al. Effect of endophytic fungi on expression of key enzyme genes in polysaccharide and alkaloid synthesis from Dendrobium officinale[J]. Chin Tradit Herb Drugs(中草药), 2019, 50(23): 5838-5846.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
宁波市社发重大项目资助(2017C510002)
{{custom_fund}}
PDF(2586 KB)
