海南龙血树类黄酮3′-羟化酶基因(DcF3′H)的克隆与表达分析
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摘要
类黄酮3′-羟化酶(Flavonoid 3′-hydroxylase,F3′H)是植物黄酮类化合物骨架修饰途径中的关键酶之一,在形成结构多样化的黄酮类化合物中起重要作用。在前期已获得的龙血树转录组数据基础上克隆了1个海南龙血树F3′H基因,命名为DcF3′H。DcF3′H含有的开放阅读框长1 533 bp,编码510个氨基酸。推导的DcF3′H分子量为58 ku,等电点p I为6.59。DcF3′H含有细胞色素P450的保守结构域和CYP基序,与甘蔗、玉米、猕猴桃、小麦、烟草、黄瓜和大豆等植物的F3′H同源性分别为76%、75%、77%、74%、75%、76%和75%。进化树分析表明,DcF3′H与中国水仙亲缘关系较近,与苜蓿和鹰嘴豆的亲缘关系较远。荧光定量表达结果显示:DcF3′H在根、茎、叶、花和果等组织中均有表达,其中在花中表达量最高,根中表达量最低;无机盐诱导剂处理能显著提高DcF3'H的表达量,处理3 d时DcF3′H的表达量提高了5.58倍。此结果将为进一步研究海南龙血树类黄酮3′-羟化酶基因的功能奠定基础。
Flavonoid 3′-hydroxylase is a key enzym e in flavonoid biosynthesis in many plants. In this study, a gene encoding flavonoid 3′-hydroxylase, designated as DcF3′ H, was cloned from Dracaena cambodiana using the result of RNA-seq. DcF3′ H contains an open reading frame of 1 533 bp, encoding a polypeptide of 510 amino acids with a molecular weight of 58 ku and an isoelectric point of 6.59. The deduced amino acid sequences of DcF3′H contained p450 conserved domains and CYP motif and showed high identities of 76%, 75%, 77%, 74%,75%, 76% and 75% to those of the F3′ H from Saccharum officinarum, Zea mays, Actinidia chinensis, Triticum urartu, Nicotiana sylvestris, Cucumis sativus and Glycine max. The phylogenetic analyses revealed that DcF3′ H was the closest to that from Narcissus tazetta and far form Medicago truncatula and Cicer arietinum. DcF3′H was expressed at different levels with the highest transcription in the flowers and lowest in the roots. The transcription of DcF3′H was induced by salt, the expression level increased 5.58-fold after 3 days treatment by inorganic salt.This study lays a foundation for studying the gene expression pattern and regulating mechanisms of DcF3′ H in flavonoid biosynthesis in D. cambodiana.
Flavonoid 3′-hydroxylase is a key enzym e in flavonoid biosynthesis in many plants. In this study, a gene encoding flavonoid 3′-hydroxylase, designated as DcF3′ H, was cloned from Dracaena cambodiana using the result of RNA-seq. DcF3′ H contains an open reading frame of 1 533 bp, encoding a polypeptide of 510 amino acids with a molecular weight of 58 ku and an isoelectric point of 6.59. The deduced amino acid sequences of DcF3′H contained p450 conserved domains and CYP motif and showed high identities of 76%, 75%, 77%, 74%,75%, 76% and 75% to those of the F3′ H from Saccharum officinarum, Zea mays, Actinidia chinensis, Triticum urartu, Nicotiana sylvestris, Cucumis sativus and Glycine max. The phylogenetic analyses revealed that DcF3′ H was the closest to that from Narcissus tazetta and far form Medicago truncatula and Cicer arietinum. DcF3′H was expressed at different levels with the highest transcription in the flowers and lowest in the roots. The transcription of DcF3′H was induced by salt, the expression level increased 5.58-fold after 3 days treatment by inorganic salt.This study lays a foundation for studying the gene expression pattern and regulating mechanisms of DcF3′ H in flavonoid biosynthesis in D. cambodiana.
引文
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[29]黄文坤,程红梅,郭建英,等.紫茎泽兰类黄酮3′-羟化酶基因的克隆、序列分析和原核表达[J].植物生理学通讯,2007,43(5):821-826.
[30]Iwashina T,Githiri S M,Benitez E R,et al.Analysis of fl avonoids in fl ower petals of soybean near-isogenic lines for flower and pubescence color genes[J].J Hered,2007,98(3):250-257.
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[32]Zhang F,Gonzalez A,Zhao M,et al.A network of redundant b HLH proteins functions in all TTG1-dependent pathways of Arabidopsis[J].Development,2003,130(20):4 859-4 869.
[33]Zhang B,Hu Z,Zhang Y,et al.A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale(Brassica Oleracea var.acephala f.tricolor)[J].Plant Cell Rep,2012,31(2):281-289.
[2]王晓丹,滕玉芳,郝吉福,等.不同来源血竭的研究进展[J].中成药,2013,35(8):1 752-1 756.
[3]Gupta D,Bleakley B,Gupta R K.Dragon′s blood:botany,chemistry and therapeutic uses[J].J Ethnopharmaco,2008,115(3):361-380.
[4]Pearson J,Prendergast H D V.Daemonorops,Dracaena and other dragon′s blood[J].Economic Bot,2001,55(4):474-477.
[5]Fan J Y,Yi T,Sze-To C M,et al.A systematic review of the botanical,phytochemical and pharmacological profile of Dracaena cochinchinensis,a plant source of the ethnomedicine“Dragon′s Blood”[J].Molecules,2014,19:10 650-10 669.
[6]Ying Luo,Hui Wang,You-xing Zhao,et al.Cytotoxic and antibacterial flavonoids from dragon′s blood of Dracaena cambodiana[J].Plant Medica,2011,77:2 053-2 056.
[7]Hui Wang,Jian Liu,Jiao Wu,et al.Flavonoids from Dracaena cambodiana[J].Chemistry of natural compounds,2011,47(4):624-626.
[8]Hao-fu Dai,Jian Liu,Jiao Wu,et al.Two new biflavonoids from the stem of Dracaena cambodiana[J].Chemistry of Natural Compounds,2012,48(3):376-378.
[9]Chen H Q,Zuo W J,Wang H,et al.Two new antimicrobial flavanes from dragon′s blood of Dracaena cambodiana[J].Asian Nat Product Res,2012,14(5):436-440.
[10]Wen-Li Mei,Ying Luo,Hui Wang,et al.Two new flavonoids from dragon′s blood of Dracaena cambodiana.[J].Bull.Korean Chem.Soc.2013,34(6):1 791-1 794.
[11]赵婷,杨玲玲,刘姮,等.血竭诱导及形成机制的研究进展[J].中草药,2010,41(4):5-8.
[12]梅文莉,戴好富,王辉.一种人工诱导血竭的方法:中国[P].ZL201310207182.5.2013-09-11.
[13]Chapple C.Molecular-genetic analysis of plant cytochrome P450-dependent monooxygenases[J].Ann Review Plant Biol,1998,49(1):311-343.
[14]侯杰,佟玲,崔国新,等.植物类黄酮3′-羟化酶基因的研究进展[J].植物生理学报,2011,47(7):641-647.
[15]蔡华,许雪峰,诸立新,等.滁菊类黄酮3-羟化酶基因的克隆及分子特性[J].食品科学,2012,33(17):145-149.
[16]Schlangen K,Miosic S,Topuz F,et al.Chalcone 3-hydroxylation is not a general property of flavonoid 3′-hydroxylase[J].Plant Sci,2009,177(2):97-102.
[17]Brugliera F,Barri-Rewell G,Holton T A,et al.Isolation and characterization of a flavonoid 3′-hydroxylase c DNA clone corresponding to the Ht1 locus of Petunia hybrida[J].Plant J,1999,19(4):441-451.
[18]Bogs J,Ebadi A,Mc David D,et al.Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development[J].Plant Physiol,2006,140(1):279-291.
[19]Jeong S T,Goto-Yamamoto N,Hashizume K,et al.Expression of the flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes and flavonoid composition in grape(Vitis vinifera)[J].Plant Sci,2006,170(1):61-69.
[20]Owens D K,Crosby K C,Runac J,et al.Biochemical and genetic characterization of Arabidopsis flavanone 3β-hydroxylase[J].Plant Physiol Biochem,2008,46(10):833-843.
[21]Nagamatsu A,Masuta C,Matsuura H,et al.Down-regulation of flavonoid 3′-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold m RNA level associated with pigmentation in pubescence[J].J Plant Physiol,2009,166(1):32-39.
[22]王佳媛,戴好富,李辉亮,等.海南龙血树查尔酮合酶基因(Dc CHS)的克隆及表达分析[J].热带作物学报,2014,35(8):1 539-1 545.
[23]蔡文伟,杨本鹏,张树珍,等.龙血树总RNA提取方法的研究[J].中国热带医学,2006,6(2):242-243.
[24]董美超,唐燕琼,莫饶,等.海南龙血树基因组DNA提取方法比较[J].中国农学通报,2010,26(11):28-31.
[25]胡利宗,张佳慧,王秋霞,等.植物F3′H基因的序列与进化分析[J].东北农业大学学报,2015,46(3):41-49.
[26]Yamazaki S1,Sato K,Suhara K,et al.Importance of the prolinerich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s[J].J Biochem,1993,114(5):652-657.
[27]Murakami K1,Mihara K,Omura T.The transmembrane region of microsomal cytochrome P450 identified as the endoplasmic reticulum retention signal[J].J Biochem,1994,116(1):164-75.
[28]Jayanand Boddua,Catherine Svabeka,Rajandeep Sekhon,et al.Expression of a putative flavonoid 3′-hydroxylase in sorghum mesocotyls synthesizing 3-deoxyanthocyanidin phytoalexins[J].Physiol Mol Plant Patho,2004,65(2):101-113.
[29]黄文坤,程红梅,郭建英,等.紫茎泽兰类黄酮3′-羟化酶基因的克隆、序列分析和原核表达[J].植物生理学通讯,2007,43(5):821-826.
[30]Iwashina T,Githiri S M,Benitez E R,et al.Analysis of fl avonoids in fl ower petals of soybean near-isogenic lines for flower and pubescence color genes[J].J Hered,2007,98(3):250-257.
[31]Kobayashi H,Suzuki S,Tanzawa F,et al.Low expressionof flavonoid 3′,5′-hydroxylase(F3′5′H)associated with cyanidinbased anthocyanins in grape leaf[J].Am J Enol Vitic,2009,60(3):362-367
[32]Zhang F,Gonzalez A,Zhao M,et al.A network of redundant b HLH proteins functions in all TTG1-dependent pathways of Arabidopsis[J].Development,2003,130(20):4 859-4 869.
[33]Zhang B,Hu Z,Zhang Y,et al.A putative functional MYB transcription factor induced by low temperature regulates anthocyanin biosynthesis in purple kale(Brassica Oleracea var.acephala f.tricolor)[J].Plant Cell Rep,2012,31(2):281-289.