CN116286888A - 一种黑莓黄酮醇合成酶基因RuFLS2及其应用 - Google Patents
一种黑莓黄酮醇合成酶基因RuFLS2及其应用 Download PDFInfo
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- CN116286888A CN116286888A CN202210814093.6A CN202210814093A CN116286888A CN 116286888 A CN116286888 A CN 116286888A CN 202210814093 A CN202210814093 A CN 202210814093A CN 116286888 A CN116286888 A CN 116286888A
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- flavonol
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Abstract
本发明公开了一种黑莓黄酮醇合成酶基因RuFLS2及其应用,属于基因工程技术领域。本申请成功鉴定且分离了RuFLS2基因,其ORF长996bp,编码331个氨基酸组成的蛋白。时空表达分析显示RuFLS2在黑莓果实中表达量较高,在果实发育至红紫色时表达量达到峰值。异源过量表达烟草发现黄酮醇类物质柚皮素‑7‑O‑葡萄糖苷、矢车菊素、芹菜素‑7‑葡萄糖苷、山奈酚‑3‑O‑芸香糖苷、紫云英苷与槲皮素等合成含量增加,表明RuFLS2是调控黑莓黄酮醇类物质合成的关键酶基因,且能提高转基因植物黄酮醇类合成量,在植物药物制备技术领域具有广大应用前景。
Description
技术领域
本发明属于基因工程技术领域,更具体地说,涉及一种黑莓黄酮醇合成酶基因RuFLS2及其应用。
背景技术
黑莓(Rubus spp.)是蔷薇科(Rasaceae)悬钩子属(Rubus)灌木,为多年生小浆果果树。它原产于北美,在欧美地区有较长的栽培历史,1986年,黑莓由江苏省中国科学院植物研究所首次引入中国并推广,其具有抗旱、长势旺和繁殖容易等优点。其果实富含糖类、维生素C、维生素E、多酚和黄酮等营养物质,具有极高的营养及药用价值,包括抗菌、抗炎、抗氧化和抗衰老等功效,广泛应用于医疗和保健领域具有良好的应用前景。
黄酮类化合物是植物次生代谢产物中的一类多酚类化合物,具有典型的C6-C3-C6碳骨架。黄酮类化合物可分为黄酮类、异黄酮类、二氢黄酮类、黄酮醇类、黄烷酮类黄烷醇类以及花青素类等6个亚类,其中黄酮醇多个位点可发生修饰,包括酰基化、羟基化,糖基化等,这些修饰可以共存,造成黄酮醇结构的多样性,所以具有多种生物活性,如抗氧化、抗病毒、抗癌等。此外还具有抵御紫外线的辐射伤害、增强植物根系抵抗干旱胁迫的能力,在植物生长发育中担任重要的生物功能,是植物自身参与抵御外界生物和非生物胁迫的重要物质之一。
黄酮醇合成酶(fiavonol synthase,FLS)是黄酮类化合物生物合成途径中的关键酶之一,催化底物二氢黄酮醇生成槲皮素、山奈酚等黄酮醇类化合物,在黄酮类化合物生物合成途径中起重要作用。二氢黄酮醇4-还原酶(dihydroflavonol 4-reductase,DFR)与FLS共同竞争底物二氢黄酮醇合成花色素,对植物中花青素和黄酮醇含量的积累有重要影响,DFR参与的代谢过程合成有颜色的花青素,FLS催化生成黄酮醇是无色的,因此,FLS的表达对植物颜色的变化具有重要影响。FLS基因对于植物器官呈现白色或者黄色有着关键的作用,葡萄风信子(Muscari aucheri)FLS基因的过表达使花色呈现白色。FLS在植物中常常是由多拷贝基因编码的,在不同植物中拷贝数不同,其表达水平在植物的不同组织部位、代谢时期和生长发育阶段具有显著的时空差异性,影响植物体内黄酮醇的含量。目前,FLS基因已在多种植物中进行了鉴定和功能研究,在某些植物中FLS基因功能相似,如虎眼万年青(Ornithogalum caudatum)的OcFLS1和OcFLS2都能催化二氢黄酮醇生成黄酮醇,且均具有黄烷酮-3-羟化酶(flavanone 3-hydroxylase,F3H)活性,而某些植物中多个FLS功能不同,拟南芥(Arabidopsis thaliana)中FLS1参与黄酮的生物合成,FLS3则催化生成黄连素(avonols)。因此,尽管FLS参与黄酮醇的合成途径已被研究,但其在黑莓中的表达模式和具体的调控机制仍待进一步研究验证。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一种黑莓黄酮醇合成酶基因RuFLS2。本发明所要解决的另一技术问题在于提供所述黑莓黄酮醇合成酶基因RuFLS2的具体应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
一种黑莓黄酮醇合成酶基因RuFLS2,其核苷酸序列如SEQ ID NO.1所示。
所述的黑莓黄酮醇合成酶基因RuFLS2的表达蛋白,其氨基酸序列如SEQ ID NO.2所示。
含有所述的黑莓黄酮醇合成酶基因RuFLS2的载体。
所述的含有黑莓黄酮醇合成酶基因RuFLS2的载体,是植物表达载体。
进一步地,所述的植物表达载体是PB1121-RuFLS2。
所述的黑莓黄酮醇合成酶基因RuFLS2在提高植物中黄酮醇类物质中的应用。
进一步地,所述的黑莓黄酮醇合成酶基因RuFLS2在提高植物中黄酮醇类物质中的应用,包括以下步骤:
1)构建黑莓黄酮醇合成酶基因RuFLS2的载体;
2)将所构建的黑莓黄酮醇合成酶基因RuFLS2的载体转化到植物或植物细胞中;
3)培育筛选得到黄酮醇类物质含量提高的转基因植物。
所述的应用中,所述的黄酮醇类物质为柚皮素-7-O-葡萄糖苷、矢车菊素、芹菜素-7-葡萄糖苷、山奈酚-3-O-芸香糖苷、紫云英苷、槲皮素中的一种或几种。
相比于现有技术,本发明的有益效果为:
本申请成功鉴定且分离了RuFLS2基因,其ORF长996bp,编码331个氨基酸组成的疏水酸性蛋白,属于2OG-Fe(II)_Oxygenase超家族。时空表达分析显示RuFLS2基因在黑莓果实中表达量较高,在果实发育至红紫色时表达量达到峰值。异源过量表达烟草发现黄酮醇类物质,如柚皮素-7-O-葡萄糖苷(naringenin-7-O-glucoside)、矢车菊素(cyanidin)、芹菜素-7-葡萄糖苷(apigenin 7-glucoside)、山奈酚-3-O-芸香糖苷(kaempferol-3-O-rutinoside)、紫云英苷(astragalin)、槲皮素(quercitrin)等的合成含量增加,且RuFLS2的高表达提高了转基因烟草中NtF3H、NtFLS、NtDFR、NtANS基因的表达水平,说明RuFLS2是调控黑莓黄酮醇类物质合成的关键酶基因,在黄酮醇类物质的合成中有重要作用,有望应用于培育获得具有高含量黄酮类化物和酚类化合物的转基因植物,在植物药物制备技术领域具有广大应用前景。
附图说明
图1为黑莓“Chester”果实不同发育阶段的表型图;
图2为RuFLS蛋白亲水性和结构域的预测图:(a)RuFLS1的亲水性;(b)RuFLS2的亲水性;(c)RuFLS1的结构域;(d)RuFLS2的结构域;
图3为RuFLSs中的保守域或基序图;
图4为RuFLS2基因在黑莓不同组织(a)和不同果实发育阶段的表达模式(b)图,不同字母表示有显著差异(p<0.05);
图5为WT和RuFLS2过表达烟草中类黄酮生物合成途径基因的表达模式图,其中:(a)RuFLS2;(b)NtFLS;(c)NtF3H;(d)NtDFR;(e)NtANS;(f)NtLAR;
图6为黑莓黄酮类化合物的生物合成途径图;
图7为对照组和FLS组烟草的差异表达代谢物(DEM)分析图:(a)正、负离子模式下的DEM数量;(b):不同途径DEM的KEGG富集分析图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1:黑莓黄酮醇合成酶基因的克隆与分析
一、实验材料
实验材料为黑莓‘Chester’品种,来自南京市溧水区白马试验基地(119°11′E,31°36N)。该地区属于亚热带季风气候区,年平均温度15.6℃,年降雨量1031.9mm,平均日照50%,其中,6-8月平均温度27.1℃,降雨量435.6mm,土壤为酸性粘土,pH5.52,含有机质18.67g/kg,全氮1.25g/kg,速效磷4.83mg/kg,速效钾94.21mg/kg。分别采摘黑莓的根、茎、叶、花、结果枝和果实作为不同组织部位表达分析的植物材料。此外,根据果实呈现颜色区分果实发育阶段的采样时期,分别采摘青色果、青红色果、红色果、红紫色果和紫色果(图1)。所有实验均生物学重复至少3次,采集之后液氮保存,带回实验室后-80℃冰箱贮存备用。
二、总RNA提取和cDNA反转
总RNA提取使用通用植物总RNA快速提取试剂盒(BioTeKe,Beijing,China),具体方法参照说明书进行,然后采用PrimeScript RT Master Mix(Perfect Real Time)试剂盒(TaKaRa,Dalian,China)对黑莓不同组织部位和果实发育阶段的总RNA反转录合成cDNA。以稀释至浓度约为300ng·μL-1的cDNA为模板进行后续的表达分析。
三、黑莓RuFLS基因的克隆
首先基于黑莓转录组数据中筛选的差异表达的RuFLS Unigene序列,然后通过BioXM2.6软件分析获得其开放阅读框(open reading frame,ORF)和三代全长转录本集进行校正,使用Oligo 6.0软件设计引物,利用高保真PCR酶PrimeSTAR Max DNA Polymerase(TaKaRa,Dalian,China)克隆其ORF序列。
50μL PCR反应体系:Primer Star Max 25μL、前后引物各1μL、cDNA模板1μL、ddH2O22μL。PCR反应程序:98℃3min;98℃10s、55℃5s、72℃15s,35个循环;72℃3min,4℃永久保温。
扩增产物按照pClone007 Blunt Vector Kit试剂盒(TSINGKE,Nanjing,China)要求与载体连接,转入大肠杆菌感受态中,选择摇床37℃250rpm培养6-8h后潜在的阳性菌液用2×T5 Super PCR Mix(Colony)(TSINGKE,Nanjing,China)做菌液PCR验证,将阳性菌液送至擎科生物技术有限公司进行一代Sanger测序验证。引物序列列于表1。
表1黑莓FLS2基因和转基因烟草等相关PCR引物信息
从黑莓不同发育阶段的转录组数据中(https://doi.org/10.1007/s00468-022-02291-3)筛选出3条可能编码FLSs蛋白的基因,其中一条未查询和验证到ORF的存在,另外2条序列经鉴定后分别命名为RuFLS1、RuFLS2。结构分析显示,RuFLS1、RuFLS2的ORF序列长度分别为1017bp和996bp(SEQ ID NO.1),编码由338和331个氨基酸(SEQ ID NO.2)组成的多肽,终止密码子分别为TAA和TAG。
四、生物信息学分析
使用DNAMAN软件对黑莓FLSs基因编码的蛋白序列进行分析,使用ExPASy软件(https://web.expasy.org)预测其物理、化学性质和蛋白质亲水性。利用MEME在线工具(https://meme-suite.org/meme/tools/meme)预测RuFLSs蛋白的motif,通过SMART在线工具(http://smart.embl-heidelberg.de/)和NCBI在线工具CDART(Conserved DomainArchitecture Retrieval Tool)(https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi#opennewwindow)对RuFLSs蛋白的结构域进行分析,包括跨膜域、功能域和保守结构域。此外,采用SOPMA在线工具分析其蛋白二级结构;利用SWISS-MODEL(https://swissmodel.expasy.org/)构建三维模型进行验证。使用MEGAX进行RuFLSs蛋白序列比对(相似度)并以邻接法(Neighbour-joining,NJ)自举检验1000次构建系统进化树。
利用ProtParam软件分析RuFLS1和RuFLS2蛋白的理化性质如表2所示,其蛋白分子量为39.03kDa、37.30kDa,理论等电点为5.72、5.44,带负电荷残基总数(Asp+Glu)为42、44,带正电荷残基总数(Arg+Lys)为32、33,不稳定指数为36.45、34.70,不稳定系数均小于40,均属于稳定蛋白,脂肪指数为81.36、88.91。
通过ProtScale在线软件预测RuFLS1、RuFLS2的总亲水性为-0.494、-0.341(图2a、b),推测其均为疏水酸性蛋白。
对RuFLS1、RuFLS2进行功能域预测,发现均包含一个DIOX_N功能域和一个2OG-FeII_Oxy功能域(图2c、d),说明它归属于α-酮戊二酸依赖性双加氧酶家族。RuFLS1的DIOX_N功能域位于第38~152个氨基酸,2OG-FeII_Oxy功能域位于第190~287个氨基酸。而RuFLS2的DIOX_N功能域位于第40~148个氨基酸,2OG-FeII_Oxy功能域位于第192~292个氨基酸,不同成员间功能结构域的位置不完全相同。RuFLS1、RuFLS2无跨膜区域,均为膜外蛋白。
表2 RuFLSs的理化性质
通过DNAMAN软件对多个植物的FLS蛋白序列进行氨基酸比对,RuFLS1、RuFLS2与扁桃(Prunus dulcis,XM_034342566.1)、桃(Prunus persica,XM_007222519.2)、芍药(Paeonia lactifiora,KM259902.3)、高山草莓(Fragaria vesca subsp.,XM_011460471.1)、茶树(Camellia sinensis,DQ198089.1)的FLSs的氨基酸序列高度一致,均具有结合底物二氢槲皮素和亚铁离子重要的氨基酸残基位点。RuFLS1在多个底物结合位点发生突变,可能在黑莓进化过程中丢失部分蛋白功能。且基于黑莓转录组数据的差异基因表达分析发现RuFLS1基因无显著差异表达,这可能与RuFLS1基因蛋白关键位点氨基酸残基发生变化有关,暗示其可能并不是调控黄酮等重要次生代谢物的关键基因。而RuFLS2基因是显著差异表达的酶基因,推测其可能对黑莓果实成熟过程中黄酮物质的积累起重要作用,因此,以下实施例进一步考察RuFLS2基因的功能,以揭示RuFLS2对黑莓黄酮相关次生代谢物合成和代谢的作用。
对2个RuFLSs蛋白的保守基序进行预测,结果表明RuFLS1、RuFLS2均具有8个同样的保守基序,在序列中排列的位置基本一致(图3)。Motif3是DIOX_N超家族,是具有2-氧戊二酸/Fe(II)依赖性双加氧酶活性的蛋白质的高度保守N端区域。Motif 2为Fe(II)依赖性加氧酶超家族,Motif 1为2OG超家族,后两者结合为2OG-Fe(II)_Oxygenase超家族与结构域预测结果一致,也与植物中FLS蛋白家族的保守结构域是一致的。有2处保守基序“IGTKMN”、“YRNEGLR”在RuFLS1、RuFLS2中排列的位置有差异,这可能导致RuFLS1和RuFLS2功能的差异。
使用NJ法利用MEGA构建系统进化树,结果显示,RuFLS1、RuFLS2与高山草莓(Fragaria vesca subsp.)、甜樱桃(Prunus avium)、扁桃(Prunus dulcis)、桃(Prunuspersica)、梅花(Prunus mume)等Rosaceae物种的F3H亲缘关系较近,Brassicaceae的FLS聚为一支,Liliaceae和Vitaceae的FLS聚为一支,符合物种进化的关系(图4),同为Rosaceae植物,苹果(Malus domestica)等的FLS蛋白与RuFLS1、RuFLS2蛋白亲缘关系较远,推测RuFLS1、RuFLS2并非单功能FLS酶,可能同时具有FLS、F3H活性的双功能酶。
实施例2:RuFLS2在黑莓及转转基因苗烟草中的表达水平检测
一、植物过量表达载体构建
利用GATEWAY技术构建过表达载体,即通过LR反应将入门载体(linker-7)中的目的片段转移至35S强启动子的过量表达载体pBI121-des-3HA中。首先进行入门载体线性化。反应体系为实施例1中的黑莓RuFLS基因克隆的50μL PCR反应体系,DNA模板为FLS2的阳性菌液,Topo-F/R引物是常用的线性化引物。然后进行PCR产物纯化回收。然后进行LR反应构建基因目的载体。LR反应体系为入门载体50ng,表达载体75ng,LR Clonase II Plusenzyme mix 0.5μL,总反应体积加超纯水至2.5μL。反应程序为25℃,1.5h。将反应产物转化大肠杆菌菌株Top10、挑菌、摇菌、送公司测序验证,获取阳性克隆后用加含有卡那的LB抗生素扩培,用于提取质粒。
二、农杆菌感受态的转化
取150ng待转化质粒,加入100μL农杆菌GV3101感受态细胞中,轻轻混匀,冰浴20min,液氮速冻60s;然后在37℃热激4min,迅速冰浴2min;加入800μL LB液体培养基(已加利福平),28℃100rpm摇菌3h;最后4000rpm离心3min,留少许上清,吹打混匀,涂布于平板,28℃培养48h;PCR检测挑选阳性克隆,4℃保存备用。
三、烟草侵染及阳性株筛选
上述验证成功的菌液继续扩大培养至50mL,测量菌液的OD值,0.5-0.8为宜。将菌液转入50mL离心管,5000rpm离心10min,弃上清,用40mL MS0(4.43g/L MS,pH 5.8)重悬菌液,加入乙酰丁香酮(AS)至浓度为20mM。挑选生长旺盛的野生型烟草组培苗,选择中间部位的叶片,剪去叶缘和叶脉,其余部分剪成1-1.5cm左右的小块,将叶片浸入制备好的菌液6-12min,期间轻摇数次,取出,吸干菌液,平铺于MS1(4.43g/L MS+25g/L蔗糖+5.6g/L琼脂+2.0mg/L 6-BA+0.1mg/L NAA+100μmol/L AS,pH 5.8)培养基,暗处25℃培养2-3d,之后进行光照16h黑暗8h,25℃继续培养,待形成愈伤组织后转移至MS2培养基(4.43g/L MS+25g/L蔗糖+5.6g/L琼脂+2.0mg/L 6-BA+0.1mg/L NAA+100mg/L特美汀+100mg/L卡那,pH 5.8)。待愈伤组织处形成幼芽,将幼芽切下,转入MS生根培养基(4.43g/L MS+25g/L蔗糖+5.6g/L琼脂+100mg/L特美汀,pH 5.8)。待长成后独立的株系取叶片提取RNA和检测,以验证筛选阳性克隆植株。
四、qRT-PCR分析方法
采用TB Green Premix Taq II(Tli RNA SEH Plus)(Takara,Dalian,China)进行qRT-PCR反应。15μL反应体系:TB Green Premix Taq II荧光染料7.5μL、cDNA模板1μL、上、下游引物各0.6μL和ddH2O 5.3μL。qRT-PCR程序:95℃2min,95℃10s,60℃10s,72℃15s,Melt 6s,40个循环。黑莓以18S为内参基因(表1),转基因苗烟草以NtActin为内参基因(表1)进行实时荧光定量PCR,最后采用2-ΔΔCT法进行定量数据分析。
1、RuFLS2在黑莓的根、茎、叶、花、结果枝和果实中的表达水平
qRT-PCR分析RuFLS2在黑莓的根、茎、叶、花、结果枝和果实中的表达水平,结果表明:RuFLS2在果实中的表达量较高,在茎、花、叶中的表达水平普遍较低,推测RuFLS2可能在黑莓果实中起主要作用(图4a)。基于此,又对RuFLS2基因在不同果实发育阶段进行了表达量检测,发现RuFLS2基因在果实发育过程中表现出显著的差异性,在果实发育前期表达量较低,在红紫果时期急剧增长而后迅速下降,但依旧高于果实发育前期(图4b)。
2、RuFLS2在转基因烟草中表达水平和对上下游基因的影响
随机挑选3个生长相对旺盛的转基因烟草株系进行表达水平检测,发现RuFLS2在转基因植株中均高表达(图5a)。为探索RuFLS2异源表达对烟草黄酮生物合成通路上关键酶基因的影响,我们检测了烟草NtFLS和上游基因NtF3H以及另一分支上的NtDFR、NtANS(Anthocyanidin synthase,ANS)和NtLAR(Leucoanthocyanidin reductase,LAR)的表达水平(图5)。结果显示转基因烟草NtFLS基因的表达水平显著提高(图5b),三个株系的转基因烟草F3H基因的表达水平变化趋势与FLS的变化趋势一致(图5c)。DFR与FLS引导黄酮合成通路上的不同分支,属于竞争关系,DFR的表达水平在不同的转基因株系中与FLS的变化趋势相反。RuFLS-1、RuFLS-2株系的NtFLS基因上调显著高于RuFLS-3,NtDFR基因在RuFLS-1、RuFLS-2株系中表达量上调幅度显著低于RuFLS-3(图5b,d),ANS是DFR的下游基因,在转基因烟草中的表达水平也显著上调(图5e)。LAR也是DFR的下游基因(图5f),但NtLAR在转基因株系中的表达水平差异较大。由此可见RuFLS2的异源表达上调了烟草NtFLS基因的表达,NtDFR与NtFLS相互竞争,LAR基因的表达则不受RuFLS2影响。
实施例3:黑莓发育过程中总酚和类黄酮含量测定
总酚含量测定参照Cheok等人的方法(Cheok,C.Y.;Chin,N.L.;Yusof,Y.A.;Law,C.L.Extraction of total phenolic content from Garcinia mangostanaLinn.hull.I.Effects of solvents and UV-Vis spectrophotometer absorbancemethod.Food Bioprocess Technol.2012,5,2928-2933.DOI:10.1007/s11947-011-0627-2),使用Plant total phenol test kit商业化试剂盒(A143-1-1,南京建成生物工程研究所),取鲜样用液氮磨成粉,称取0.1g,加入2mL提取液(60%乙醇水溶液),涡旋混匀抽提3min,60℃提取30min,4000rpm·min-1离心10min,取上清液置于离心管,按要求加入试剂,混匀,室温静置10min,波长760nm测定各管吸光值,生物学重复三次,计算总酚含量。
类黄酮含量(TFC)检测采用氯化铝热量法(Mohamed,A.F.;Abeer,A.A.;Ahlam,A.H.;Sharifa,A.B.Color,flavonoids,phenolics and antioxidants of Omanihoney.Heliyon.2018,4,e00874.DOI:10.1016/j.heliyon.2018.e00874.),使用Plantflavonoids test kit商业化试剂盒(A142-1-1,南京建成生物工程研究所),称取0.05g样品,生理盐水洗净擦干后用液氮研磨成粉状,加入2mL 60%乙醇提取液,60℃震荡提取2h,10000rpm·min-1室温离心10min,取上清液待测。按照标准曲线绘制项流程操作,各样品重复检测三次,取其均值,代入标准曲线计算类黄酮含量。
花色苷含量(TAC)使用pH示差法测定(Cheng,F.R.;Cui,H.X.;Fang,J.L.;Yuan,K.;Jin,S.H.;Zhu,X.T.;Xu,Y.Content determination of functional composition andantioxidant activity from six purple plants.Pharmacogn.Mag.2021,17,342-347;DOI:10.4103/PM.PM_203_20.),稍作修改:取黑莓果实50g匀浆,取3g匀浆液加入30mL 50%乙醇提取液,摇匀,35℃下60Hz超声处理20min,5000rpm离心5min,取上清液0.5mL置于10ml离心管,加入pH1.0缓冲液4.5mL,室温反应20min,双蒸水调零,510nm处测吸光值,计算花色苷含量。
结果显示,总酚、类黄酮含量变化趋势相似,整体呈现前期急剧下降后期平缓的趋势,在紫果期略有上升,青果时期含量显著高于其他时期,总酚达到40.68mg·g-1FW,类黄酮达到11.50mg·g-1FW。花色苷在果实发育前期积累较少,趋近于无,红紫果至紫果时期大幅度上升,紫果时期达到最大,为2.60mg·g-1FW,说明花色苷主要在果实发育的后期合成积累(表3)。
表3黑莓果实不同发育阶段的总酚、黄酮和花色苷含量的变化
果实颜色 | 总酚(mg·g-1FW) | 黄酮(mg·g-1FW) | 花色苷(mg·g-1FW) |
绿色果 | 40.68±1.00a | 11.50±0.37a | 0.02±0.002d |
青红色果 | 11.91±1.05b | 6.97±0.11b | 0.05±0.01d |
红色果 | 3.28±0.03c | 1.81±0.01c | 0.39±0.01c |
红紫色果 | 2.35±0.01c | 1.80±0.12c | 0.93±0.02b |
紫色果 | 3.10±0.01c | 1.95±0.10c | 2.60±0.03a |
注:不同字母表示黑莓不同发育阶段之间存在显著差异(p<0.05)。
关于黑莓相关数据为平均值±标准差(SD),使用SPSS 24.0统计软件程序(SPSS,Chicago,IL,USA)进行。使用单因素ANOVA比较平均值,Duncan事后多重比较计算黑莓不同组织部位(花,根,茎,叶,枝)、不同时期果实的黄酮、花色苷、总酚含量和RuFLS表达水平的差异显著性(P<0.05)。
实施例4:野生型及转基因烟草的代谢组分析
一、野生型及转基因烟草的LC-MS/MS分析
将6个烟草样本(野生型和转基因型各三株)4℃解冻后,分别加入预冷的甲醇/乙腈/水溶液(2∶2∶1,v/v),低温超声提取,取上清真空干燥,质谱分析时加入100μL乙腈水溶液(乙腈∶水=1∶1,v/v)复溶,取上清液进样分析。
样品采用Agilent 1290 Infinity LC超高效液相色谱系统(UHPLC)HILIC色谱柱进行分离,整个分析过程中样品置于4℃自动进样器中。随机顺序进行样本的连续分析以避免仪器检测信号波动而造成的影响。采用AB Triple TOF 6600质谱仪进行样本一级、二级谱图的采集。
二、统计分析
关于转基因烟草的代谢组分析,所有样本中应用了非监督的降维主成分分析(principal component analysis,PCA),使用r包模型(Warnes,G.R.;Bolker,B.;Lumley,T.;Johnson,R.C.Gmodels:Various R Programming Tools for ModelFitting.Available online:https://CRAN.R-project.org/package=gmodels.)以初步显示不同组别样本之间的差异。偏最小二乘判别分析(PLS-DA)使用r包ropls(http://www.r-project.org/)应用于对照组,通过筛选与分类成员相关的变量来更好地区分两组的代谢组学特征。潜在结构正交投影鉴别分析(OPLS-DA)在对照组中使用r包模型(http://www.r-project.org/),通过交叉验证和排列检验进一步验证。以预测能力(Q2)值验证代谢组学中的OPLS-DA模型。当Q2值大于0.4时,认为是可接受的预测模型。当Q2值大于0.9时,认为是良好的预测模型。随机测试200次,然后生成R2’值和Q2’值的分布。
PLS-DA可以使组间区分最大化,有利于寻找差异代谢物。本实验表明,WT对照组和FLS转基因组烟草代谢物在正离子模式下R2X=0.678,R2Y=0.999,Q2Y=0.763;在负离子模式下R2X=0.427,R2Y=0.998,Q2Y=0.688,符合我们对实验数据模型的预期,说明本研究所建立的PLS-DA模型参数合理且稳定,可以用于代谢组学分析。OPLS-DA是PLS-DA的衍生算法,两种有监督模式识别的多元统计分析均发现,FLS在置信区左侧,而WT在置信区右侧,说明两种模型均能有效区分转基因烟草与非转基因烟草的代谢产物。此外,通过置换验证OPLS-DA模型,发现OPLS-DA模型可靠性良好,后续的模型检验和差异代谢物筛选用OPLS-DA结果进行分析。
结合多元统计分析OPLS-DA的VIP值和单变量统计分析T检验P值来筛选不同比较组间的差异代谢物,结果显示FLS转基因组烟草代谢物在正离子模式下共有47个极显著的差异代谢物质,RuFLS2基因过表达烟草比野生型烟草有16个代谢产物上调,31个代谢产物下调;在负离子调控模式下共有37个差异代谢物质,其中18个代谢产物上调,19个代谢产物下调(图7a)。
通过京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)对WT组和转基因烟草FLS组的差异代谢物进行代谢通路差异富集,发现40个KEGG通路中均有富集。差异代谢物主要富集在4大类别(图7b):新陈代谢metabolism(包括10大亚类)、遗传信息处理genetic information processing(包括2个亚类)、环境信息处理environmental information processing(包括2个亚类)、人类疾病human diseases也包括2个亚类。其中有19种差异代谢物质分布在代谢通路(ko01110,Metabolic pathways),6种差异代谢物质分布在次级代谢产物的生物合成通路(ko01110,Biosynthesis ofsecondary metabolites),5种差异代谢物质分布在不同环境中的微生物代谢中(ko01120,Microbial metabolism in diverse environments)。
为考察过量表达RuFLS2基因对黄酮相关代谢物质的影响,重点对转基因FLS组和WT组烟草类黄酮合成相关的差异代谢物质进行分析。结果显示,转基因烟草类黄酮合成通路上检测到6种差异代谢物,除柚皮素-7-O-葡萄糖苷(naringenin-7-O-glucoside)外其他5种物质含量均有下调。花青素合成通路上检测到1种差异代谢物——矢车菊素(cyanidin),其含量上调。黄酮和黄酮醇合成通路上共检测到7种差异代谢物,其中芹菜素-7-葡萄糖苷(apigenin 7-glucoside)、山奈酚-3-O-芸香糖苷(kaempferol-3-O-rutinoside)、紫云英苷(astragalin)、槲皮素(quercetin)含量明显上调,木犀草素-7-葡萄糖苷(luteolin 7-glucoside)、槲皮素-3-葡萄糖苷(quercetin 3-glucoside)、女贞苷(ligustroflavone)含量下调。说明RuFLS2基因的过量表达消耗了上游的黄酮类物质并显著提高了植株中黄酮醇的含量(表4)。
表4转基因烟草中与类黄酮生物合成相关的主要差异代谢产物
Claims (8)
1.一种黑莓黄酮醇合成酶基因RuFLS2,其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的黑莓黄酮醇合成酶基因RuFLS2的表达蛋白,其氨基酸序列如SEQID NO.2所示。
3.含有权利要求1所述的黑莓黄酮醇合成酶基因RuFLS2的载体。
4.根据权利要求3所述的含有黑莓黄酮醇合成酶基因RuFLS2的载体,其特征在于,所述的载体是植物表达载体。
5.根据权利要求4所述的含有黑莓黄酮醇合成酶基因RuFLS2的载体,其特征在于,所述的植物表达载体是PBI121-RuFLS2。
6.权利要求1所述的黑莓黄酮醇合成酶基因RuFLS2在提高植物中黄酮醇类物质中的应用。
7.根据权利要求6所述的黑莓黄酮醇合成酶基因RuFLS2在提高植物中黄酮醇类物质中的应用,其特征在于,包括以下步骤:
1)构建黑莓黄酮醇合成酶基因RuFLS2的载体;
2)将所构建的黑莓黄酮醇合成酶基因RuFLS2的载体转化到植物或植物细胞中;
3)培育筛选得到黄酮醇类物质含量提高的转基因植物。
8.根据权利要求6或7所述的应用,其特征在于,所述的黄酮醇类物质为柚皮素-7-O-葡萄糖苷、矢车菊素、芹菜素-7-葡萄糖苷、山奈酚-3-O-芸香糖苷、紫云英苷、槲皮素中的一种或几种。
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