CN114574462B - 花斑形成与着色的关键糖基转移酶及其编码基因与应用 - Google Patents
花斑形成与着色的关键糖基转移酶及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了花斑形成与着色的关键糖基转移酶及其编码基因与应用。本发明所提供的糖基转移酶是如下a)或b)的蛋白质:a)由序列表中序列1所示的氨基酸序列组成的蛋白质;b)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有花斑形成与着色的糖基转移酶活性的由a)衍生的蛋白质。本发明所提供的糖基转移酶PhUGT78A22,它能精确地催化糖基化花青素在斑或非斑的转化,从而导致‘和谐’中花瓣斑和非斑花青苷的差异。本发明为花青苷生物合成和积累的分子调控提供了一个新的视角,为提高观赏价值的花色育种提供新的思路,同时也为深入了解花青苷生物合成和积累的分子调控机制提供参考。
Description
技术领域
本发明涉及生物技术领域,特别地涉及花斑形成与着色的关键糖基转移酶PhUGT78A22及其编码基因与应用。
背景技术
花的着色是自然界中最美丽的表现之一,通过吸引动物传粉者和参与对生物和非生物胁迫的反应,与进化和随后的花的多样化有关,这造成了今天观察到的被子植物多样性(Mekapogu M,Vasamsetti BMK,Kwon O-K,et al(2020)Anthocyanins in FloralColors:Biosynthesis and Regulation in Chrysanthemum Flowers.Int J Mol Sci 21:6537.https://doi.org/10.3390/ijms21186537)。花朵中不同寻常的颜色排列主要依赖于四种色素群:叶绿素、类胡萝卜素、类黄酮和甜菜素(Narbona E,del Valle JC,WhittallJB(2021)Painting the green canvas:how pigments produce flower colours.TheBiochemist 43:6–12.https://doi.org/10.1042/bio_2021_137)。最多样化的色素是黄酮类化合物,尤其是花青素,众所周知,花青素能赋予花朵鲜艳的橙色、粉色、红色、紫色和蓝色(-Ovando A,Pacheco-Hernández Ma de L,Páez-Hernández MaE,et al(2009)Chemical studies of anthocyanins:A review.Food Chem 113:859–871.https://doi.org/10.1016/j.foodchem.2008.09.001),其生物合成途径是目前植物中研究最广泛的途径之一。在花青素生物合成途径(ABP)早期,3个丙二酰-CoA分子和1个4-coumarpyl-CoA分子经查尔酮合成酶(CHS)缩合生成查尔酮,之后查尔酮异构酶(CHI)、黄烷酮3-羟化酶(F3H)、类黄酮3'-羟化酶(F3'H)和类黄酮3'5'-羟化酶(F3'5'H)将查尔酮转化为二氢黄酮醇。此外,在ABP后期,花青素由二氢黄酮醇还原酶(DFR)和花青苷元合酶(ANS)两种酶生成,并被UDP类黄酮糖基转移酶(UFGT)糖基化和/或类黄酮甲基转移酶(FOMT)甲基化。上述ABP酶基因主要受一个组织特异性表达的调控复合物控制:MYB(M)-basic helix-loop-helix(bHLH)-WD40(MBW)蛋白复合物(Gu Z,Zhu J,Hao Q,et al(2019)A Novel R2R3-MYBTranscription Factor Contributes to Petal Blotch Formation by RegulatingOrgan-Specific Expression of PsCHS in Tree Peony(Paeonia suffruticosa).PlantCell Physiol 60:599–611.https://doi.org/10.1093/pcp/pcy232)。
芍药属,芍药科的唯一一个属,包括三个组:牡丹组、北美芍药组、芍药组(Hao Q,Liu Z-A,Shu Q-Y,et al(2008)Studies on Paeonia cultivars and hybridsidentification based on SRAP analysis:Studies on Paeonia based on SRAPanalysis.Hereditas 145:38–47.https://doi.org/10.1111/j.0018-0661.2008.2013.x)。芍药属植物是世界著名的观赏植物,原产于中国,具有悠久的栽培选育历史(Zhou S-L,Zou X-H,Zhou Z-Q,et al(2014)Multiple species of wild treepeonies gave rise to the‘king of flowers‘,Paeonia suffruticosa Andrews.Proc RSoc B 281:20141687.https://doi.org/10.1098/rspb.2014.1687),牡丹组中,两种野生物种即紫斑牡丹和滇牡丹的花带有花瓣斑,因为花瓣斑是显性遗传性状,也可以在其后代中看到(Shi Q,Li L,Zhang X,et al(2017)Biochemical and ComparativeTranscriptomic Analyses Identify Candidate Genes Related to VariegationFormation in Paeonia rockii.Molecules 22:1364.https://doi.org/10.3390/molecules22081364)。花斑在每个花瓣基部表现出不同的颜色和大小,这为其品种提供了独特的观赏价值。在此之前,我们比较了35个牡丹组品种花瓣中斑部分和非斑部分的花青苷组成,结果表明,花青苷含量最多的是矢车菊素糖苷:矢车菊素-3-O-葡萄糖苷(Cy3G)和矢车菊素-3,5-O-双葡萄糖苷(Cy3G5G),而白色、无斑点的花瓣部分未检测到花青苷(ZhangJ,Wang L,Shu Q,et al(2007)Comparison of anthocyanins in non-blotches andblotches of the petals of Xibei tree peony.Scientia Horticulturae 114:104–111.https://doi.org/10.1016/j.scienta.2007.05.009)。通过对牡丹品种‘Jinrong‘花中紫色斑部分和白色无斑部分花瓣转录组的比较,表明花瓣斑的形成可能与PsCHS、PsF3’H、PsDFR和PsANS转录水平较高有关(Zhang Y,Cheng Y,Ya H,et al(2015)Transcriptomesequencing of purple petal spot region in tree peony reveals differentiallyexpressed anthocyanin structural genes.Front Plant Sci 6:.https://doi.org/10.3389/fpls.2015.00964)。另外,对紫斑牡丹、凤丹及其F1杂种花瓣斑的转录组分析表明,CHS、DFR和ANS可能参与了牡丹花斑的色素积累(Shi Q,Li L,Zhang X,et al(2017)Biochemical and Comparative Transcriptomic Analyses Identify Candidate GenesRelated to Variegation Formation in Paeonia rockii.Molecules 22:1364.https://doi.org/10.3390/molecules22081364)。在我们之前的研究中,我们发现了花青素-O-甲基转移酶(AOMT)负责将矢车菊素糖苷甲基化转化为芍药花素糖苷,并在牡丹的紫色着色中发挥重要作用(Du H,Wu J,Ji K-X,et al(2015)Methylation mediated by ananthocyanin,O-methyltransferase,is involved in purple flower coloration inPaeonia.J Exp Bot 66:6563–6577.https://doi.org/10.1093/jxb/erv365)。牡丹中花青素以糖苷化形式存在,糖基化在牡丹花色形成过程中发挥着重要作用,但糖基转移酶的基因功能却被忽略了。此外,我们在最近的研究中还报道了‘青海湖银波‘品种中PsCHS的转录调控所导致花瓣斑颜色形成的分子机制(Gu Z,Zhu J,Hao Q,et al(2019)A Novel R2R3-MYB Transcription Factor Contributes to Petal Blotch Formation by RegulatingOrgan-Specific Expression of PsCHS in Tree Peony(Paeonia suffruticosa).PlantCell Physiol 60:599–611.https://doi.org/10.1093/pcp/pcy232)。然而,上述对花斑转录组的研究仅局限于彩色斑对白色非斑花瓣部分(背景)的研究。有必要进一步研究彩色背景下带斑点的彩色花瓣,以揭示同一花瓣中色素图案的差异。
芍药属杂交品种提供了优良的观赏性状,自1948年Toichi Itoh获得第一个杂交品种以来,开启了杂交育种的新纪元,其后代以Itoh(伊藤)命名以纪念。在那之后,以‘杂交之父‘桑德斯为代表的育种家培育出了‘初到‘、‘雨中歌唱‘、‘鲜红天堂‘、‘柔辉‘、‘希拉里‘、‘金丝雀光辉‘、‘茱莉亚罗斯‘等著名品种,其中包括著名的伊藤杂交品种。在中国,我们有幸在牡丹组和芍药组之间发现了一个杂交品种,命名为‘和谐‘(Tong N,Peng L,Liu Z,et al(2021)Comparative transcriptomic analysis of genes involved in stemlignin biosynthesis in woody and herbaceous Paeonia species.Physiol Plant173:961–977.https://doi.org/10.1111/ppl.13495)。其表型结合了灌木牡丹和草本芍药的性状,特别是其带暗紫色斑点的紫色花朵具有较高的观赏价值。在之前的研究中,我们在―和谐”花瓣中发现了一个ring domain-containing蛋白(PhRINGH2),它与PhCHS发生物理互作,是PhCHS泛素化和降解所必需的,这表明在‘和谐‘类黄酮合成途径中转录后水平的调控是存在的,这为芍药属植物类黄酮合成调控提供了理论依据(Gu Z,Men S,Zhu J,etal(2019)Chalcone synthase is ubiquitinated and degraded via interactions witha RING-H2 protein in petals of Paeonia‘He Xie.‘J Exp Bot 70:4749–4762.https://doi.org/10.1093/jxb/erz245)。
糖基转移酶(Glycosyltransferase,GT)是普遍存在于生物体中能够通过合成糖苷键将糖基连接到特定受体的一大类酶,其糖基供体主要是尿苷二磷酸(uridinediphosphate,UDP)-糖,故这类酶又被称为尿苷二磷酸糖基转移酶(UGT)。目前在拟南芥、水稻、葡萄、月季、茶树、人参和苜蓿等植物中报道的UGTs分别有123、184、82、230、257、250和272个(Yonekura-Sakakibara K,Hanada K(2011)An evolutionary view of functionaldiversity in family 1 glycosyltransferases:Functional evolution in family1glycosyltransferases.Plant J 66:182–193.https://doi.org/10.1111/j.1365-313X.2011.04493.x;Wilson AE,Tian L(2019)Phylogenomic analysis of UDP-dependent glycosyltransferases provides insights into the evolutionarylandscape of glycosylation in plant metabolism.Plant J 100:1273–1288.https://doi.org/10.1111/tpj.14514)。鉴于糖基转移酶是一个大家族,如何精准找到关键酶基因,并通过分子生物学及植物化学等手段验证功能是迫切需要解决的技术难题。
发明内容
有鉴于此,本发明的主要目的是提供花斑形成与着色的关键糖基转移酶及其编码基因。
本发明的技术方案如下:
本发明提供了一种蛋白,是如下a)或b)的蛋白质:
a)由序列表中序列1所示的氨基酸序列组成的蛋白质;
b)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有花斑形成与着色的糖基转移酶活性的由a)衍生的蛋白质。
所述花斑形成与着色的糖基转移酶活性是催化单糖基化花青素向双糖基化花青素的转化。
所述催化单糖基化花青素向双糖基化花青素的转化为催化Cy3G转化为Cy3G5G,和/或催化Pn3G转化为Pn3G5G。
所述蛋白的编码基因也属于本发明的保护范围。
所述编码基因为如下1)或2)或3)或4)所示∶
1)其核苷酸序列是序列表中序列2所示DNA分子;
2)其核苷酸序列是序列表中序列3所示DNA分子;
3)在严格条件下与1)或2)限定的DNA分子杂交的DNA分子;
4)与1)或2)或3)限定的DNA分子具有90%以上的同源性的DNA分子。
将该基因命名为PhUGT78A22,其核苷酸序列如序列表中序列2所示,其中第122位到1492位为编码区,其编码基因含有1371个核苷酸(序列表中序列3),将其编码的蛋白命名为PhUGT78A22,PhUGT78A22蛋白含有456个氨基酸(序列表中序列1)。
含有权利要求所述编码基因的表达盒、重组表达载体、转基因细胞系或重组微生物也属于本发明的保护范围。
PhUGT78A22蛋白在作为花斑形成与着色的糖基转移酶中的应用也属于本发明的保护范围。
所述花斑形成与着色的糖基转移酶催化单糖基化花青素向双糖基化花青素的转化。
所述催化单糖基化花青素向双糖基化花青素的转化为催化Cy3G转化为Cy3G5G,和/或催化Pn3G转化为Pn3G5G。
所述的蛋白、所述编码基因在催化Cy3G和/或Pn3G向双糖基化花青素的转化中的应用也属于本发明的保护范围。
为了进一步探究紫色花瓣中深紫色斑的形成,本发明比较了花瓣斑部分和非斑部分的花青苷成分,然后将花瓣斑部分和非斑部分分离出来进行转录组分析和比较,发现了一个关键的UDP-糖基转移酶(UGT)PhUGT78A22,它能精确地催化糖基化花青素在斑或非斑的转化,从而导致‘和谐‘中花瓣斑和非斑花青苷的差异。本发明为花青素生物合成和积累的分子调控提供了一个新的视角,为提高观赏价值的花色育种提供新的思路,同时也为深入了解花青素生物合成和积累的分子调控机制提供参考。
附图说明
为了说明而非限制的目的,现在将根据本发明的优选实施例、特别是参考附图来描述本发明,其中:
图1为‘和谐‘不同发育期花瓣斑(B)和非斑(NB)表型。1级:花蕾未开放,花瓣浅黄绿色没有花斑;2级花蕾未开放,红色花斑在花瓣基部出现,非斑部分由浅黄绿色转为浅粉色;3级花蕾部分开放,斑部分扩大、颜色加深,非斑部分完全转变为粉色并加深颜色;4级花朵完全开放,斑部分面积达到最大。标尺1cm。
图2为‘和谐‘花朵发育过程中斑和非斑花青苷积累模式分析;n=3。
图3为‘和谐‘4级花瓣斑花青苷(525nm)的UPLC色谱图。
图4为‘和谐‘4级花瓣非斑花青苷(525nm)的UPLC色谱图。
图5为6个UAGTs在‘和谐‘不同发育期花瓣斑和非斑表达模式;n=3。
图6为c99617_g1蛋白包含PSPG基序。
图7为c99617_g1与拟南芥UGTs的进化关系分析。
图8为PhUGT78A22的烟草叶片亚细胞定位,标尺25μm。
图9为GST-PhUGT78A22对Cy3G的催化反应;GST作为负对照。
图10为GST-PhUGT78A22对Pn3G的催化反应;GST作为负对照。
图11为PhUGT78A22沉默对‘和谐‘芽鳞花青苷的影响;A,PhUGT78A22沉默会改变芽鳞的颜色,标尺2mm,*,P<0.05;B,对照和PhUGT78A22沉默芽鳞中PhUGT78A22的表达量n=3,**,P<0.01;C,对照和PhUGT78A22沉默芽鳞中Lab值,n=6;D,PhUGT78A22沉默改变了芽鳞中花青苷的积累,n=3,**,P<0.01。
具体实施方式
实施例1、克隆花斑形成与着色的关键糖基转移酶PhUGT78A22的编码基因
植物材料:
本发明以中国科学院植物研究所北京植物园(北纬39°59′,东经116°12′,海拔70m)栽培的牡丹芍药远缘杂交种‘和谐‘(P.‘He Xie‘)(郝青,刘政安*,舒庆艳,王亮生,陈富飞.2008.中国首例芍药牡丹远缘杂交种的发现及鉴定.园艺学报,35(6):853-858)为研究对象。花瓣分为斑和非斑两部分,分别在4个发育阶段采集:第一阶段花蕾未开放,花瓣浅黄绿色没有花斑(1级);第二阶段花蕾未开放,红色花斑在花瓣基部出现,非斑部分由浅黄绿色转为浅粉色(2级);第三阶段花蕾部分开放,斑部分扩大、颜色加深,非斑部分完全转变为粉色并加深颜色(3级);第四阶段花朵完全开放,斑部分面积达到最大(4级,图1)。芽鳞从‘和谐‘花芽中分离出来。
方法:
超高效液相色谱-二极管阵列检测器(UPLC-DAD)分析:
从每个发育阶段的花瓣斑和非斑部分或芽鳞样本中提取花青素进行分析,试验方法参考Gu等(Gu Z,Zhu J,Hao Q,et al(2019)ANovel R2R3-MYB Transcription FactorContributes to Petal Blotch Formation by Regulating Organ-Specific Expressionof PsCHS in Tree Peony(Paeonia suffruticosa).Plant Cell Physiol 60:599–611.https://doi.org/10.1093/pcp/pcy232)。取0.2g新鲜样品,用1mL 0.2%甲酸/甲醇(v/v)溶液超声破碎20min,避光静置2h,提取液在12000rpm下离心5min,收集上清。重复以上步骤,直到花青素全部被提取。收集的上清液经0.22μm膜过滤,-20℃保存。
采用UPLC-DAD(ACQUITY-Class,Waters,Massachusetts,USA)检测花青苷的成分和含量。分析柱为ACQUITY />HSS T3 1.8μm柱(Waters,Massachusetts,USA)。四种标品均购自Solarbio(Beijing,China),分别为Cy3G、Cy3G5G、芍药花素-3-O-葡萄糖苷(Pn3G)、芍药花素-3,5-O-双葡萄糖苷(Pn3G5G)。其中,以Cy3G为标准曲线,通过线性回归定量分析花青苷含量。
转录组测序和分析:
提取RNA后,用1.0%琼脂糖凝胶分析评价总RNA的完整性,然后用K5500显微分光光度计(Kaiao,Beijing,China)检测其质量,最后用2100 RNA Nano 6000试验设备(Agilent Technologies,CA,USA)检测RNA完整性。文库的构建和RNA-seq分析由安诺优达基因技术公司(Beijing,China)使用Illumina平台(CA,USA)进行。然后对原始数据进行处理,通过删除被污染的、低质量和大于5%N的数据来获得干净的reads。随后,使用Trinity(vision 20140717)完成转录组组装。
使用RPKM方法计算unigenes的表达水平。用DESeq检测差异表达基因(DEGs),|log2Ratio|≥1且q<0.05的基因为差异表达。此外,利用Gene Ontology(GO)和KyotoEncyclopedia of Genes and Genomes(KEGG)数据库对DEGs进行注释和分类。
荧光定量PCR(RT-qPCR)分析:
采用E.Z.N.A.Plant RNAKit(Omega Bio-Tek,GA,USA)提取‘和谐‘花瓣斑部分和非斑部分的总RNA。然后,用HiScript II逆转录酶试剂盒(Vazyme,Nanjing,China)获得用于RT-qPCR的cDNA模板。RT-qPCR反应采用StepONE Real-Time PCR系统(AppliedBiosystems,Carlsbad,USA)和2×M5 HiPer Realtime PCR Super mix(Mei5bio,Beijing,China)进行。PoTUB作内参。RT-qPCR中使用的引物见表1。
表1 RT-qPCR中使用的引物
系统进化分析:
系统发育分析方法参考(Ross J,Li Y,Lim E-K,Bowles DJ(2001)Higher plantglycosyltransferases.Genome Biol.2:3004.1–3004.6 http://genomebiology.com/2001/2/2/reviews/3004.1;Yin Q,Shen G,Chang Z,et al(2016)Involvement of threeputative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis in Lotus japonicus seeds.J Exp Bot 2016 68:597–612.https://doi.org/10.1093/jxb/erw420)。所有拟南芥UGT均来源于Carbohydrate-Active Enzymes(CAZymes)数据库(http://www.cazy.org/Home.html)。利用MEGA软件(版本X)和Evolview(http://www.evolgenius.info/evolview.html),使用Neighbor-Joining法构建c99617_g1和拟南芥UGTs的系统发育树。其中,所有包含空白和缺失数据的位置已被删除,共分析了123个氨基酸序列。
PhUGT78A22的亚细胞定位:
利用Super启动子构建Super promoter::PhUGT78A22-GFP质粒,与核定位蛋白质粒Super promoter::NF-YA4-mCherry共同注射烟草叶片,异源表达(Chen J,Li Y,Li Y,etal(2021)AUXIN RESPONSE FACTOR 18–HISTONE DEACETYLASE 6module regulates floralorgan identity in rose(Rosa hybrida).Plant Physiol 186:1074–1087.https://doi.org/10.1093/plphys/kiab130)。侵染3天后用共聚焦显微镜(Leica TCS SP5,Germany)观察GFP或mCherry荧光。亚细胞定位所用的引物序列见表2。
表2亚细胞定位所用的引物序列
同源建模与分子对接:
PhUGT78A22的同源性模型采用SWISS-MODEL(https://swissmodel.expasy.org/)同源建模系统进行计算,并以葡萄(Vitis vinifera)花青素3-O-葡萄糖基转移酶[ProteinData Bank(PDB)编码为22c1x]为模板。使用Molecular Operating Environment(MOE)Dock软件进行PhUGT78A22的分子对接,其中,UDP-葡萄糖作为糖供体,矢车菊素(Cy)、Cy3G、芍药花素(Pn)和Pn3G作为糖受体。UDP-葡萄糖、Cy、Cy3G、Pn和Pn3G的二维(2D)结构从PubChem(https://pubchem.ncbi.nlm.nih.gov/)获得,并在MOE中以能量最小化的形式转化为三维(3D)结构。最优的分子对接模式由PyMOL(www.pymol.org)可视化展示。
PhUGT78A22的体外酶活分析:
体外酶活检测参考Yin等(Yin Q,Shen G,Chang Z,et al(2016)Involvement ofthree putative glucosyltransferases from the UGT72 family in flavonolglucoside/rhamnoside biosynthesis in Lotus japonicus seeds.EXBOTJerw420.https://doi.org/10.1093/jxb/erw420)之前的方法,将PhUGT78A22的编码区序列构建到pGEX4T-2(Chen J,Li Y,Li Y,et al(2021)AUXIN RESPONSE FACTOR 18–HISTONEDEACETYLASE 6module regulates floral organ identity in rose(Rosa hybrida).Plant Physiol 186:1074–1087.https://doi.org/10.1093/plphys/kiab130)中,并在大肠杆菌菌株Rosetta(购自北京庄盟国际生物基因科技有限公司,北京,中国)中表达。加入isopropylthio-β-D-galactoside(IPTG,0.3mM)诱导蛋白,并于16℃,120rpm孵育24h。GST-PhUGT78A22蛋白用Glutathione Sepharose 4B(GE Healthcare,PA,USA)纯化。酶活试验以UDP-葡萄糖(Solarbio,Beijing,China)作为糖供体,以Cy、Cy3G、Pn和Pn3G(Solarbio,China)作为糖受体底物。PhUGT78A22酶活试验的反应液由100mM Tris-HCl(pH 7.0)、0.1mM底物、2mM UDP-葡萄糖和3–10μg PhUGT78A22蛋白组成,反应体积为50μL。37℃孵育1小时后,加入0.2%甲酸/甲醇(v/v)溶液终止反应,然后采用UPLC-DAD对产物进行分析。酶活分析中使用的引物见表3。
表3体外酶活分析中使用的引物
病毒诱导的基因沉默(VIGS):
VIGS试验参考Chen等(Chen J,Li Y,Y,et al(2021)AUXIN RESPONSE FACTOR 18–HISTONE DEACETYLASE 6module regulates floral organ identity in rose(Rosahybrida).Plant Physiol 186:1074–1087.https://doi.org/10.1093/plphys/kiab130)的方法。利用PhUGT78A22基因特异性片段(327bp)构建载体pTRV2-PhUGT78A22。分别将pTRV1、pTRV2(Chen J,Li Y,Y,et al(2021)AUXIN RESPONSE FACTOR 18–HISTONEDEACETYLASE 6 module regulates floral organ identity in rose(Rosa hybrida).Plant Physiol 186:1074–1087.https://doi.org/10.1093/plphys/kiab130)和pTRV2-PhUGT78A22转化根癌农杆菌GV3101(购自北京庄盟国际生物基因科技有限公司,北京,中国),并在Luria-Bertani(LB)培养基(含50mg/L利福平和50mg/L卡那霉素)中培养,集菌后重悬于侵染液中(10mM MgCl2、200mM乙酰丁香酮和10mM MES,pH 5.6)。将含有pTRV1的菌液与pTRV2-PhUGT78A22或阴性对照pTRV2按1:1(v/v)的比例混合,并置于室温黑暗处4-6h。接着,取大小和颜色基本一致的的‘和谐‘芽鳞,浸于含pTRV1+pTRV2或pTRV1+pTRV2-PhUGT78A22的侵染液中,-0.9atm真空处理2次,每次5min。侵染后的芽鳞用蒸馏水冲洗,置于Murashige和Skoog培养基(Solarbio,Beijing,China)上8℃黑暗环境下孵育3天后,检测沉默和对照组芽鳞的表型、PhUGT78A22的表达和花青苷含量。VIGS中使用的引物见表4。
表4 VIGS中使用的引物
颜色测量(CIELab系统):
颜色分析参考(Gonnet J-F(1998)Colour effects of co-pigmentation ofanthocyanins revisited—1.A colorimetric definition using the CIELABscale.Food Chem 63:409–415.https://doi.org/10.1016/S0308-8146(98)00053-3;DuH,Wu J,Ji K-X,et al(2015)Methylation mediated by an anthocyanin,O-methyltransferase,is involved in purple flower coloration in Paeonia.J ExpBot 66:6563–6577.https://doi.org/10.1093/jxb/erv365)。颜色用L*、a*和b*值表示。L*是从黑(0)到白(100),代表亮度。a*表示红色(正数)到绿色(负数)。b*从黄色(正)到蓝色(负)。颜色测量使用分光光度计NF555(Nippon Denshoku,Tokyo,Japan)。
统计学分析:
使用GraphPad Prism(版本8)进行统计分析。所有实验数据均采用双侧t分布检验(two-sided Student‘s t-test)。
结果:
1.―和谐”花瓣在着色和开放过程中存在差异糖基化花青素
本发明以―和谐”花瓣为研究对象,研究了花瓣发育的4个主要阶段:第一阶段,花蕾未开放,花瓣呈淡黄绿色,无斑(1级);第二阶段,花蕾未开放,花瓣基部出现红色斑,无斑部分开始由浅黄绿色变为浅粉色(2级);第三阶段,花蕾部分开放,斑面积增长,颜色加深,非斑部分完全变为粉红色(3级);第四阶段,花朵完全开放,斑面积达到最高(4级,图1)。为研究花瓣发育过程中和非斑区色素的积累规律,我们测定了花瓣中花青苷的组成和含量。将斑和非斑部分分离进行UPLC-DAD分析,结果表明,随着花瓣颜色的加深,花瓣总花青苷含量逐渐增加,在4级达到最高水平(4.17mg/g FW)。在2级后,斑的花青苷含量的部分总是高于非斑,在斑和非斑中,花青苷含量在2级分别为0.91mg/g FW和0.37mg/g FW,在3级为1.32mg/g FW和0.51mg/g FW,在4级为2.73mg/g FW和1.43mg/g FW(图2)。斑检测到4种花色苷,分别为Cy3G、Cy3G5G、Pn3G和Pn3G5G。而在非斑仅检测到Cy3G5G和Pn3G5G两种组分(图2、图3和图4)。这些结果表明,花青素糖基化在斑和非斑是不同的。
2.‘和谐‘花瓣花青素生物合成的转录组和unigenes分析。
为了确定在斑和非斑花青素生物合成的关键基因,利用Illumina测序平台对‘和谐‘花瓣的2~4级斑和非斑混合样本进行转录组分析。共对6个样本(每组3个生物重复)进行测序,得到4056-4587万个碱基分布合理、平均质量位置合适的Clean reads,且Q30基率高于93.44%,这表明序列质量较好可供进一步分析。过滤低质量reads后得到127,287个unigenes,平均长度为630bp,从转录组中得到的数据比对率为89.07%。
通过比较‘和谐‘花瓣的斑和非斑转录组数据,检测到2,433个差异表达基因,其中包括1,494个上调基因和939个下调基因。花青素的生物合成在花瓣颜色的决定中起着关键作用,同时根据花瓣斑与非斑色素组成差异,重点关注了糖基转移酶基因,UDP花青素3-O-葡糖基转移酶(UA3GT)和UDP花青素5-O-葡糖基转移酶(UA5GT)。涉及花青素糖基化的UAGT表达水平的变化较为复杂的。3个UAGTs(c132695_g1、c126517_g1和c9624_g1)在斑和非斑之间差异不大,但斑中有9个UAGTs(c95107_g1、c128309_g1、c8473_g1、c99617_g1、c44319_g1、c51545_g1、c44249_g1、c93571_g1和c96046_g2)下调和5个UAGTs(c97667_g1、c51545_g2、c93787_g2、c95696_g1和c95696_g2)表达上调。
3.‘和谐‘花瓣中参与花青苷合成的PhUGTs表达模式及序列分析
为了进一步研究‘和谐‘花青素糖基化的差异,对log2差异倍数绝对值大于0.5的UAGTs(c95107_g1、c128309_g1、c8473_g1、c99617_g1、c95696_g1和c95696_g2)进行了RT-qPCR检测。与其他UAGTs基因不同,c99617_g1在‘和谐‘花瓣发育过程表达量很高。在斑出现在花瓣基部(2级)后,c99617_g1在非斑中的表达水平显著高于斑中(图5),这与之前的猜想相吻合:双糖基化更多地出现在非斑而不是斑中。c99617_g1的氨基酸序列分析表明,其C端结构域附近含有一个PSPG(plant secondary product glycosyltransferase)基序(331-374aa)(图6)。在c99617_g1中也观察到PSPG基序中最后一个谷氨酰胺(Q)残基,它被认为对作为糖供体的UDP-葡萄糖具有特异性。系统发育分析显示c99617_g1接近拟南芥UGT超家族中的F组(图7)。我们还从UGT命名委员会(https://prime.vetmed.wsu.edu/resources/udp-glucuronsyltransferase-homepage)获得了c99617_g1的名称,结合进化关系分析和UGT命名委员会命名结果,将c99617_g1命名为PhUGT78A22。
该基因PhUGT78A22核苷酸序列如序列表中序列2所示,其中第122位到1492位为编码区,其编码基因含有1371个核苷酸(序列表中序列3),将其编码的蛋白命名为PhUGT78A22,PhUGT78A22蛋白含有456个氨基酸(序列表中序列1)。
花青苷是在内质网的外表面合成的。亚细胞定位结果表明,其主要在细胞质及细胞核中表达(图8)。
4.PhUGT78A22的建模、对接及底物特异性分析
为了确定PhUGT78A22特异性的分子基础,首先利用SWISS-MODEL构建PhUGT78A22的结构模型。PhUGT78A22结构与模板UGT结构基本一致(PDB编码:22c1x),三维结构重叠的均方根偏差(RMSD)值为两者具有相同的α螺旋和β折叠区域。PhUGT78A22与22c1x的氨基酸序列相似度为66.37%,对PhUGT78A22与底物UDP-葡萄糖之间潜在相互作用预测结果表明,PhUGT78A22的9个氨基酸(Thr19、Gly278、Thr279、Ser305、Trp331、Ala332、His349、Glu357和Asp373)与糖供体UDP-葡萄糖相互作用。PhUGT78A22与UDP-葡萄糖的对接得分为-10.06kCal/mol(表5)。为了研究PhUGT78A22在‘和谐‘花瓣花青苷形成中的作用,我们将PhUGT78A22与UDP-葡萄糖以及底物Cy、Cy3G、Pn和Pn3G分别进行对接。PhUGT78A22的4个氨基酸(His20,Phe118,Glu186和Gly372)与底物Cy相互作用;5个氨基酸(Gln81,His147,Glu186,Ala280和Phe371)与底物Cy3G相互作用;4个氨基酸(Phe118,Glu186,Gly372和Asp373)与底物Pn相互作用;PhUGT78A22的7个氨基酸(Phe118,Glu186,Thr279,Ala280,Phe371,Asp373和Gln374)与底物Pn3G相互作用。此外,PhUGT78A22+UDP-葡萄糖与Cy、Cy3G、Pn和Pn3G的对接得分分别为-6.66、-8.39、-6.53和-9.08kCal/mol(表5)。结合能量越低,结合亲和力越高。结果表明,Cy3G和Pn3G对PhUGT78A22+UDP-glucose的结合能力预测更强。
表5 PhUGT78A22与配体对接结果
为了进一步分析PhUGT78A22的体外功能,将PhUGT78A22在大肠杆菌中表达,并通过酶活检测对融合GST标签的PhUGT78A22蛋白进行评价。UDP-葡萄糖和两种花青苷(Cy3G和Pn3G)分别作为糖的供体和底物。以葡萄糖为糖供体,PhUGT78A22表现出对Cy3G和Pn3G的催化活性。UPLC-DAD对酶促产物的分析表明,重组PhUGT78A22蛋白催化Cy3G转化为Cy3G5G(图9),Pn3G转化为Pn3G5G(图10)。结果表明,PhUGT78A22催化了Cy3G和Pn3G葡萄糖向糖基化花青素的转移。
5.PhUGT78A22基因的沉默改变了'和谐'中Cy3G5G和Pn3G5G的生物合成。
为了进一步验证PhUGT78A22的体内活性,我们利用VIGS技术,用烟草脆裂病毒在‘和谐‘芽鳞上沉默PhUGT78A22。‘和谐‘芽鳞花青苷成分与花斑部分相同,PhUGT78A22的沉默改变了芽鳞的颜色(图11中A)。RT-qPCR检测表明,PhUGT78A22在TRV-PhUGT78A22中表达量显著降低(图11中B)。对颜色进行检测发现,Ph UGT78A22沉默株系的a*值明显降低,向绿色转变,而b*和L*无显著变化(图11中C),这与表型结果一致。
为了进一步确认PhUGT78A22沉默后花青苷的变化,我们检测了TRV-PhUGT78A22株系中花青苷的成分和含量。PhUGT78A22沉默的芽鳞中,Cy3G5G和Pn3G5G显著降低,而Cy3G显著升高。此外,PhUGT78A22沉默的芽鳞中Pn3G也有所增加,但未观察到统计学差异(图11中D)。这表明,PhUGT78A22的沉默会减少Cy3G和Pn3G向双糖苷花青素的转化。
综上所述,我们提出了UDP-糖基转移酶PhUGT78A22在‘和谐‘花瓣斑形成时参与单糖基化花青素向双糖基化花青素的转化模型:在斑中,PhUGT78A22的表达量较低,只有部分Cy3G和Pn3G能够分别糖基化成Cy3G5G和Pn3G5G。在非斑中,PhUGT78A22的表达量较高,全部Cy3G和Pn3G都可以分别被糖基化为Cy3G5G和Pn3G5G。糖基化花青素成分和含量的差异解释了‘和谐‘花瓣中斑的形成和颜色差异。
上述具体实施方式,并不构成对本发明保护范围的限制。本领域技术人员应该明白的是,取决于设计要求和其他因素,可以发生各种各样的修改、组合、子组合和替代。任何在本发明的精神和原则之内所作的修改、等同替换和改进等,均应包含在本发明保护范围之内。
序列表
<110> 中国科学院植物研究所
<120> 花斑形成与着色的关键糖基转移酶及其编码基因与应用
<130> SPI22017
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 456
<212> PRT
<213> 牡丹芍药远缘杂交种'和谐' (P.'He Xie')
<400> 1
Met Thr Ser Met Thr Asn Glu Pro His Val Ala Val Phe Ala Phe Pro
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Phe Gly Thr His Ala Ala Pro Leu Leu Thr Ile Ile Arg Tyr Leu Ser
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Lys Ala Ala Pro Asn Val His Phe Ser Phe Phe Ser Thr Ala Glu Ser
35 40 45
Asn Thr Met Ile Phe Ser Asn Ser Asn Asn Asp Ala Val Lys Ala Tyr
50 55 60
Asn Val Ser Asp Gly Val Pro Asp Lys Tyr Val Phe Thr Gly Lys His
65 70 75 80
Gln Glu Lys Ile Asp Leu Phe Met Lys Ala Ala Pro Glu Asn Phe Arg
85 90 95
Lys Cys Met Glu Ala Ala Val Ala Glu Thr Gly Arg Lys Val Ser Cys
100 105 110
Leu Val Thr Asp Gly Phe Phe Trp Phe Ala Ala Glu Met Ala Glu Glu
115 120 125
Met Gly Val Pro Trp Val Pro Phe Trp Thr Ala Gly Pro Asn Ser Leu
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Ser Thr His Val Leu Thr Asp Phe Ile Arg Asp Lys Val Gly Ala Gly
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Gly Ile Glu Gly Arg Glu Glu Glu Pro Leu Ala Phe Ile Pro Gly Met
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Ser Lys Leu Arg Leu Arg Asp Leu Pro Glu Gly Ile Leu Val Gly Asn
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Leu Asn Ser Ile Phe Ser Thr Met Leu His Lys Met Gly Gln Met Leu
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Pro Gln Ala Thr Ala Val Phe Ile Asn Ser Phe Glu Glu Leu Asp Pro
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Thr Leu Thr Asn Asp Leu Asn Ser Lys Phe Lys Lys Phe Leu Asn Ile
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Gly Pro Phe Asn Leu Leu Ser Pro Leu Pro Pro Pro Ser Thr Pro Asp
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Ala Asn Asn Cys Leu Ser Trp Leu Asn Asp Gln Lys Ala Glu Ser Val
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Ala Tyr Ile Ser Phe Gly Thr Ala Ala Thr Pro Pro Pro Thr Glu Ile
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Leu Ala Ile Ala Glu Ala Leu Glu Ala Ser Gly Val Ala Phe Leu Trp
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Ser Leu Lys Asp His Leu Asn Val His Leu Pro Lys Gly Phe Leu Asp
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Lys Thr Arg Ala Cys Gly Met Val Val Pro Trp Ala Pro Gln Leu Gln
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Ile Leu Ala His Gly Ala Val Gly Val Phe Val Thr His Cys Gly Trp
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Asn Ser Val Leu Glu Ser Ile Gly Gly Gly Val Pro Met Ile Cys Arg
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Pro Phe Phe Gly Asp Gln Lys Leu Asn Ala Cys Met Val Glu Asp Val
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Trp Glu Ile Gly Val Lys Ile Asp Gly Gly Val Phe Thr Lys Asn Gly
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Leu Ile Ser Ser Leu Asp Leu Val Leu Ser Gln Glu Lys Gly Lys Lys
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Met Arg Gly Glu Ile Arg Gly Leu Lys Gly Leu Ala Glu Lys Ala Val
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Gly Pro Gln Gly Ser Ser Thr Glu Asn Leu Lys Thr Leu Leu Ser Leu
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Val Ser Arg His Lys Asp Ile Ala
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<213> 牡丹芍药远缘杂交种'和谐' (P.'He Xie')
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atttgcctcg gggttatcac gtgcttcact tgctcacaac ctactcacaa acctaccacc 60
gcatatttct cacaaatata accaaccaca gctccactca tttctattac cttctccaga 120
aatgacgtcg atgactaacg agccacacgt ggcggtcttc gccttcccct tcggcacaca 180
tgccgctccc ctccttacca tcatccgcta cctttcgaaa gccgccccaa acgttcactt 240
ctcattcttt agcaccgccg aatccaatac catgatattc tccaactcca acaacgacgc 300
cgtaaaggcc tataatgttt ctgatggcgt gccggataag tacgtgttca ccgggaagca 360
ccaggagaaa atcgaccttt tcatgaaagc ggcgccggag aattttcgga agtgtatgga 420
ggcggcggtg gcggagacgg gaaggaaggt gagctgttta gtgacggatg gattcttttg 480
gtttgcggca gagatggctg aggagatggg ggtgccttgg gtgccgtttt ggactgctgg 540
gcctaactca ctttccactc atgtcctcac tgatttcatc cgagacaagg ttggagctgg 600
aggtattgaa gggagagaag aagaaccact tgcattcatt ccaggaatgt ccaaattacg 660
cctccgggac ttgccggaag gaatcctcgt cggaaacttg aactccatct tctcaaccat 720
gctacacaaa atgggacaaa tgctgccaca agcaaccgcg gtttttatca actccttcga 780
agaattagat cctactttaa caaatgactt aaactccaag ttcaaaaaat tcctaaacat 840
tggccctttt aatctattat caccgttacc gccaccatct acgcctgatg caaataattg 900
cctgtcatgg ctcaatgacc aaaaggctga atccgtcgcg tacatcagct ttggcacggc 960
tgcgactcca cctccaaccg agattttagc tatagcagaa gcattagaag caagtggggt 1020
tgccttttta tggtcactta aggaccattt aaatgtgcat ttaccaaaag ggtttttaga 1080
caaaacaaga gcatgtggaa tggttgtgcc atgggctcct caattacaaa tactagcaca 1140
tggtgctgtt ggggtgtttg taacgcattg tggttggaac tctgtactag agagtatcgg 1200
aggtggagtg cctatgattt gtaggccatt cttcggcgat caaaagttga acgcgtgtat 1260
ggtggaggac gtgtgggaaa ttggtgtgaa aatcgatggt ggagtattta cgaagaatgg 1320
gttaataagt agtttggatc tagttttatc acaagaaaag ggaaagaaga tgagggggga 1380
gatcagaggg ttgaaaggac ttgctgaaaa agcggtcgga ccgcaaggga gttctactga 1440
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ttagtataga ttaaaggttt cagatggtca ttgggagagg ataagatcat ggtcgctctt 1620
taggttttga tgtggtgatc agtttgttac ttgtagtctt gtgataattt tagtaccctt 1680
ttgtattgtt ttattatggt aaattttttt cttatcatct tttgtatgct ttctctcttt 1740
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atgacgtcga tgactaacga gccacacgtg gcggtcttcg ccttcccctt cggcacacat 60
gccgctcccc tccttaccat catccgctac ctttcgaaag ccgccccaaa cgttcacttc 120
tcattcttta gcaccgccga atccaatacc atgatattct ccaactccaa caacgacgcc 180
gtaaaggcct ataatgtttc tgatggcgtg ccggataagt acgtgttcac cgggaagcac 240
caggagaaaa tcgacctttt catgaaagcg gcgccggaga attttcggaa gtgtatggag 300
gcggcggtgg cggagacggg aaggaaggtg agctgtttag tgacggatgg attcttttgg 360
tttgcggcag agatggctga ggagatgggg gtgccttggg tgccgttttg gactgctggg 420
cctaactcac tttccactca tgtcctcact gatttcatcc gagacaaggt tggagctgga 480
ggtattgaag ggagagaaga agaaccactt gcattcattc caggaatgtc caaattacgc 540
ctccgggact tgccggaagg aatcctcgtc ggaaacttga actccatctt ctcaaccatg 600
ctacacaaaa tgggacaaat gctgccacaa gcaaccgcgg tttttatcaa ctccttcgaa 660
gaattagatc ctactttaac aaatgactta aactccaagt tcaaaaaatt cctaaacatt 720
ggccctttta atctattatc accgttaccg ccaccatcta cgcctgatgc aaataattgc 780
ctgtcatggc tcaatgacca aaaggctgaa tccgtcgcgt acatcagctt tggcacggct 840
gcgactccac ctccaaccga gattttagct atagcagaag cattagaagc aagtggggtt 900
gcctttttat ggtcacttaa ggaccattta aatgtgcatt taccaaaagg gtttttagac 960
aaaacaagag catgtggaat ggttgtgcca tgggctcctc aattacaaat actagcacat 1020
ggtgctgttg gggtgtttgt aacgcattgt ggttggaact ctgtactaga gagtatcgga 1080
ggtggagtgc ctatgatttg taggccattc ttcggcgatc aaaagttgaa cgcgtgtatg 1140
gtggaggacg tgtgggaaat tggtgtgaaa atcgatggtg gagtatttac gaagaatggg 1200
ttaataagta gtttggatct agttttatca caagaaaagg gaaagaagat gaggggggag 1260
atcagagggt tgaaaggact tgctgaaaaa gcggtcggac cgcaagggag ttctactgag 1320
aatttgaaaa ctttgttgag tctagtatca aggcataagg atattgctta a 1371
Claims (9)
1.一种蛋白,所述蛋白的氨基酸序列如序列表中序列1所示。
2.权利要求1所述蛋白的编码基因,其特征在于:所述编码基因为如下1)或2)所示∶
1)其核苷酸序列是序列表中序列2所示DNA分子;
2)其核苷酸序列是序列表中序列3所示DNA分子。
3.含有权利要求2所述编码基因的表达盒、重组表达载体或重组微生物。
4.权利要求1所述的蛋白在作为牡丹芍药远缘杂交种‘和谐’(P.‘He Xie’)的花斑形成与着色的糖基转移酶中的应用。
5.根据权利要求4所述的应用,其特征在于:所述花斑形成与着色的糖基转移酶催化单糖基化花青素向双糖基化花青素的转化。
6.根据权利要求5所述的应用,其特征在于:所述催化单糖基化花青素向双糖基化花青素的转化为催化Cy3G转化为Cy3G5G。
7.根据权利要求5所述的应用,其特征在于:所述催化单糖基化花青素向双糖基化花青素的转化为催化Pn3G转化为Pn3G5G。
8.权利要求1所述的蛋白在催化Cy3G和/或Pn3G向双糖基化花青素的转化中的应用。
9.权利要求2所述的编码基因在催化Cy3G和/或Pn3G向双糖基化花青素的转化中的应用。
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