CN113151315A - 烟草多酚代谢途径蛋白基因NtPPOE及其应用 - Google Patents
烟草多酚代谢途径蛋白基因NtPPOE及其应用 Download PDFInfo
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- CN113151315A CN113151315A CN202110268040.4A CN202110268040A CN113151315A CN 113151315 A CN113151315 A CN 113151315A CN 202110268040 A CN202110268040 A CN 202110268040A CN 113151315 A CN113151315 A CN 113151315A
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Abstract
本发明涉及烟草多酚代谢途径蛋白基因NtPPOE及其应用,该基因碱基序列如SEQ ID NO.1所示。烟草多酚代谢途径NtPPOE由602个氨基酸残基组成,包括三个保守功能域:178‑388位第一个保守蛋白功能域,395‑445位其第二个保守蛋白功能域,466‑597为其第三个保守蛋白功能域。该蛋白基因NtPPOE与植物叶片中多酚中绿原酸含量高度负相关相关,降低该蛋白基因NtPPOE表达后,叶片中绿原酸含量显著升高。本发明通过对特定烟草多酚代谢途径蛋白基因NtPPOE的初步研究,发现其与绿原酸含量高度相关,在将该基因沉默后,烟草中绿原酸含量极显著升高。基于这一特性,可为烟叶中多酚类物质含量调控烟草新品种培育一定的应用基础和参考借鉴。
Description
技术领域
本发明属于生物技术领域,特别是属于烟草基因工程技术领域,具体涉及烟草多酚代谢途径蛋白基因NtPPOE及其应用。
背景技术
烟叶原料是卷烟工业的基础。近年来,我国烟叶原料生产整体水平有了明显的提高,但是烟叶原料质量距卷烟工艺配方的需要还有一定的差距,尤其香气品质不能满足工业企业的需求。香气优雅、风格突出的高档次烟叶还有很大需求空间。烤烟中多酚类化合物含量与品质存在一致性关系,多酚类化合物积累直接影响烤烟的外观质量和内在质量,有研究者提出将多酚物质含量与蛋白质氮含量比值作为“芳香值”,以此作为判断烟叶中香气吃味的数据标准,因此提高烟叶中多酚类物质含量对提升烟叶香气品质具有重要意义。
烟叶多酚类物质含量很高,最多能达到干重的6%以上,其中绿原酸、莨菪亭和芸香苷是烟叶中含量最多的多酚类化合物,占烟草中多酚含量的80%以上,同时,仅仅绿原酸一种物质,就占到多酚的75-90%,因此,绿原酸是提高烟叶香气的一个重要潜在物质。
然而多酚类物质属于次生代谢物,传统栽培技术措施无法对其含量进行有效调控。多酚在植物中受到多酚氧化酶(polyphenol oxidase,PPO)的基因调控,植物中的PPO基因多以基因家族形式存在(Beecher and Skinne,2001;Shetty et al.,2011;Arisas etal.,2012)。在茄科经济作物中,茄子中找到个6个PPO基因(Shetty,2011);番茄中发现7个PPO基因;马铃薯中有2个PPO基因;红三叶草中找到至少6个PPO基因(Winters,2009)。Cai等(2013)发现高粱中存在8个不同的PPO基因;但葡萄藤中只发现一个PPO基因(Mayer,2006)。
PPO活性具有植物组织差异性。PPO广泛存在于植物的多种器官中,如叶片、根、块茎及花中,幼嫩部位含量较高而在成熟部位含量较低(谢春艳和宾金华,1999)。马铃薯中PPO在块茎、根及花中含量较高,而在叶片中含量较低;PPO的活性随着块茎的发育不断升高(Thygesen et al.,1995)。PPO活性在烟草苗期活性较高;进入旺长期后,叶片中PPO活性继续升高;进入成熟期后,PPO活性逐渐下降;并且PPO活性呈现上部叶>中部叶>下部叶的规律(武德传et al.,2006)。PPO活性受到外界环境的调控(Thygesen et al.,1995;武德传etal.,2006;谢春艳和宾金华,1999)。嘎拉苹果多酚氧化酶反应的最适pH值为6.0,最适反应温度是45℃。Palma-Orozco(2012)从马曼果中分离出两种同工酶(PPO1和PPO2),最适pH为7.0,最适温度是35℃。Rahman(2012)从花椰菜中分离出一种PPO,最适pH为8.0,最适温度为55℃。Han-Ju(2012)从油菜花中分离出的PPO最适pH为5.5,在60℃到70℃之间活性较为稳定。
通过调控PPO类基因表达量而调节烟叶中多酚类物质含量是一种理论上可行,现实中急需的一种提高烟叶香气品质的有效技术手段。
发明内容
本发明的目的在于提供一种烟草多酚代谢途径蛋白基因NtPPOE及其应用,从而调节烟叶中多酚类物质含量,从而为高香气物质烟叶培育提供基础。
本发明是通过以下技术方案实现的:
烟草多酚代谢途径蛋白基因NtPPOE,其碱基序列如SEQ ID NO.1所示。
进一步的,其氨基酸序列如SEQ ID NO.2所示,由602个氨基酸残基组成,其中第178-388位、395-445位、466-597位氨基酸为保守的PPO结构域。
进一步的,引物序列为:
NtPPOE-F:5’-GTGTTGAAACAAATAATT-3’,
NtPPOE-R:5’-GTACAAGTACCATCTATG-3’。
进一步的,以烟草K326的cDNA为模板,以NtPPOE-F、NtPPOE-R为引物,进行PCR扩增。
上述任一项烟草多酚代谢途径蛋白基因NtPPOE的应用,所述蛋白基因NtPPOE与植物叶片中多酚类物质含量相关。
进一步的,利用基因沉默技术或者基因超表达方法,通过调节烟草中的蛋白基因NtPPOE表达量,来调节控制烟叶中多酚类物质含量。
进一步的,通过转基因技术、瞬时表达技术或基因组编辑技术,构建含有蛋白基因NtPPOE的病毒诱导沉默载体、RNAi干涉载体、超表达载体或基因组编辑载体,转化烟草,筛选获得多酚类物质含量变化的烟草新品种。
进一步的,利用病毒诱导的基因沉默技术,干扰蛋白基因NtPPOE的表达使其沉默,蛋白基因NtPPOE沉默植株中多酚类物质含量显著上升,进而获得叶片中多酚类物质含量升高的植物新品种。
本发明的有益效果是:
本发明通过对特定烟草多酚代谢途径蛋白基因NtPPOE的初步研究,发现其与绿原酸含量高度相关,在将该基因沉默后,烟草中多酚类物质含量发生了明显提高。基于这一特性,可为烟草高香气物质烟叶新品种培育提供一定的应用基础和参考借鉴。
附图说明
图1为本发明烟草TRV2-PDS、TRV2-GFP及TRV2-NtPPOE载体转化组的表型对比图;
图2为与对照植株相比,蛋白基因NtPPOE沉默植株中该基因的相对表达量;
图3为病毒诱导的基因沉默技术的烟叶及对照烟叶中的主要多酚类物质含量比较。
具体实施方式
以下通过实施例来对本发明的技术方案进行详细的说明,以下的实施例仅是示例性的,仅能用来解释和说明本发明的技术方案,而不能解释为是对本发明技术方案的限制。
生物材料:
本氏烟草,一种常用烟草材料,下述实施例中烟草种植于郑州烟草院种植基地,育苗钵中育苗,待发芽后两周进行分苗,种于塑料钵(10cm×10cm)中,22℃、16h光/8h暗条件下进行日常肥水管理等。
下述实施例中所采用的VIGS载体是一种来自烟草脆裂病毒的病毒载体(tobaccorattle virus,TRV),所具体利用的TRV2由郑州烟草研究院基因中心保存,其带有卡那筛选标记和35S启动子,同时TRV2带有EcoR I和BamH I等多克隆位点,可以用来携带和转化外源基因。
实验试剂:
LB液体培养基,1L含量中含有:10g细菌蛋白胨(bacteriological peptone),10g氯化钠(NaCl),5g酵母抽提物(yeast extract),高温高压灭菌。
YEB液体培养基,1L含量中含有:5g牛肉浸膏(beef extract),5g细菌蛋白胨(bacteriological peptone),5g蔗糖(sucrose),1g酵母抽提物(yeast extract),2mL 1M硫酸镁(MgSO4),高温高压灭菌。
1M 2-(N-吗啉)乙磺酸(MES)储备液:ddH2O溶解,过滤灭菌,-20℃储存备用。
200mM乙酰丁香酮(Acetosyringone,As)储备液:二甲基亚砜(DSMO)溶解,-20℃储存备用;
MMA(100mL):1mL(1M)MgCl2;1mL(1M,pH5.6)MES;75μL(200mM)As。
实施例1
本实施例就烟草NtPPOE基因克隆及沉默载体的构建过程简要介绍如下。
(1)烟草蛋白基因NtPPOE的克隆
根据前期对于烟草基因组及相关蛋白基因NtPPOE的分析,选择特异编码序列为目标蛋白基因片段,设计PCR扩增用引物序列如下:
NtPPOE-F:5’-GTGTTGAAACAAATAATT-3’,
NtPPOE-R:5’-GTACAAGTACCATCTATG-3’;
以烟草K326叶片的cDNA为模板,进行PCR扩增获得NtPPOE基因。
PCR扩增程序为:95℃预变性3min,95℃变性15s,55℃退火15s,72℃延伸30s,34个循环后,72℃彻底延伸5min。
对PCR扩增产物进行琼脂糖凝胶电泳检测,并回收电泳产物备用。
(2)构建重组TRV2-NtPPOE载体
将步骤(1)中的PCR扩增产物进行EcoRI、BamHI双酶切,同时对空载体TRV2进行EcoRI、BamHI双酶切,分别回收酶切产物,利用T4 DNA连接酶进行连接。
将连接产物转化大肠杆菌感受态DH5α,转化操作结束后将转化产物涂布在含50mg/L Kan的LB固体培养基上,在37℃培养过夜。
挑选阳性单菌落扩增后进一步进行PCR鉴定,并结合测序验证,确保获得构建正确的重组载体TRV2-NtPPOE。
烟草蛋白基因NtPPOE,包括1809个碱基,碱基序列如SEQ ID NO.1所示,具体如下:
ATGGCGTCAAGTGTTATTCCACCAGTGTGCAATAGCACAACAGTCAAAACTCCCTTTACTTCAACCACCAAGTCTTCTTCTTTAGCTTCCACTCCAAAACCCTCTCAACTTTTCCTCCGTGGAAAACGTAACCACAGCTTCAAAGTCTCATGCAAGGTCTCCAATGGTGATGAAAACCAAAGTGTTGAAACAAATAATTCTGTTGATAGGAGAAATGTGCTTCTAGGTTTAGGAGGTCTATATGGTGCTGCTAATGTTGTACCATTGGCTTCAGCCACTCCCATTCCAGCCCCTACTACTTCATGTAGCAAGACTGGTGCCACAATTAAACCCGGTTTACCAGTACCTTATTCTTGTTGTCCCCCTCCGCTAAAAATTGATCCTAAGGATATTCCTCATTACACGTTTCCAACAGGATCGAAGCTCCGTATTCGACCAGCTTCTCATGCCGTGGATGAAGAGTACATGGCTAAGTACAACTTAGCCATTACTAAAATGAAGGAGCTCGACGTCACTGATCCAGATGATCCACGTGGGTTCGCGGCGCAAGCCAAAATCCACTGTGCTTATTGCAACGGTGCATACACCGTTGCTGGCAAAGAGCTACAAATTCACTTCTCGTGGCTTTTTTTCCCATTCCATAGATGGTACTTGTACTTCTATGAGAGAATCTTGGGTTCTTTAATCAATGATCCTACTTTTGGTTTGCCATATTGGAACTGGGATCATCCAAAGGGCATGCGTTTGCCACACATGTTTGATCAACCAAATGTGTACCCTGATCTTTACGATCCAAGACGTAACCAAGAGCACCGTGGTTCGGTAATCATGGACCTTGGTCATTTTGGTCAAGACGTGAAAGGAACTGACTTACAAATGATGAGAAATAACCTTACTCTAATGTATCGTCAAATGATTACCAATTCACCGTGTCCACAACTGTTTTTTGGTAAGCCATATTGTACGGAAGTTGGACCCAAACCAGGGCAGGGAGCTATTGAAAACATCCCTCATACTCCTGTCCACATTTGGGTTGGTAGTAAGCCTAATGAGAATAACTGTAAAAACGGTGAAGATATGGGAAATTTCTATTCAGCTGGTAAGGATCCTGCTTTCTATAGTCACCATGCAAATGTAGATCGCATGTGGACAATATGGAAGACATTAGGAGGAAAACGCAAGGACATCAACAAGCCAGATTATTTGAACAGTGAGTTCTTCTTCTACGACGAAAAGAAAAACCCTTTTCTCGTGAAAGTCCGTGACTGTTTGGACAATAAGAAAATGGGATATGATTTCCAAGCAATGCCAACCCCATGGCGCAATTTTAAGCCATTGAAGAAGAGCAAGAGCAAGGTCAATGCACGTTCAGTTCCTCCAGTTACCCAAACATTCCCTATTGCAAAGATTGACAAAGCCATAACATTTTCCATCAAAAGGGAAACTTCAGGTACTTTCAAGTCATGTTATTTAAAAGTTTAAACTGTTAGAAATAACACACTTTTAATTACTAAACTTAATTAGATCATATAGGTGGATAAAAATAAAGTTTTTGCGGTTAGATTTAAACCCATGACCTCTTTTGAATCTCTCGTGCCATTGTTAAGTTGCTAGAGAGCACATATTTTTAATTTGTTAGAGTACACCTTCAATTATTTAATTATATTATGTCTTTAACAGGCCGGACTCAGCAGGAGAAAGACGCAGAAGAGGAGATGTTAACTTTCTTGGAACTGAACATCGATCAGCGAAAGCACATAAGGTTTGATGTGTTCATTAACGCAGATGCAAACTCCAACTGGTATGAGCTAGACAGGGCAGAGTTTGCAGGAAGTTACACTGCCTTGCCTCATGTTCATTCAGATCCCACTAAACCACATGTCGCCCCTATTGCAAAATTCCAGCTGGCCATTACCGAGTTGCTCGAGGAAATTGGCCTTGAAGATGAAGATGATATAGTGGTGACTCTGGTCCCGAAAACTGGGGGCGAATTTGTCGCCATTAAATCTGCTGTGATTACACTTGAAGCTTGTTGA
烟草多酚代谢途径蛋白NtPPOE,包括602个氨基酸,氨基酸序列如SEQ ID NO.2所示,具体如下:
ASSVIPPVCNSTTVKTPFTSTTKSSSLASTPKPSQLFLRGKRNHSFKVSCKVSNGDENQSVETNNSVDRRNVLLGLGGLYGAANVVPLASATPIPAPTTSCSKTGATIKPGLPVPYSCCPPPLKIDPKDIPHYTFPTGSKLRIRPASHAVDEEYMAKYNLAITKMKELDVTDPDDPRGFAAQAKIHCAYCNGAYTVAGKELQIHFSWLFFPFHRWYLYFYERILGSLINDPTFGLPYWNWDHPKGMRLPHMFDQPNVYPDLYDPRRNQEHRGSVIMDLGHFGQDVKGTDLQMMRNNLTLMYRQMITNSPCPQLFFGKPYCTEVGPKPGQGAIENIPHTPVHIWVGSKPNENNCKNGEDMGNFYSAGKDPAFYSHHANVDRMWTIWKTLGGKRKDINKPDYLNSEFFFYDEKKNPFLVKVRDCLDNKKMGYDFQAMPTPWRNFKPLKKSKSKVNARSVPPVTQTFPIAKIDKAITFSIKRETSGRTQQEKDAEEEMLTFLELNIDQRKHIRFDVFINADANSNWYELDRAEFAGSYTALPHVHSDPTKPHVAPIAKFQLAITELLEEIGLEDEDDIVVTLVPKTGGEFVAIKSAVITLEAC
实施例2
在实施例1基础上,利用农杆菌介导的VIGS技术,进一步将所构建的重组TRV2-NtPPOE载体转化烟草植株,并就相关植物表型变化情况做进一步验证分析,具体实验过程简介如下。
(1)转化农杆菌
需要说明的是,参考实施例1操作及现有技术,同时制备TRV2-GFP、TRV2-PDS重组载体作为转基因正负对照,具体转化过程为:
将TRV2-GFP(载体对照)、TRV2-PDS(VIGS效率对照)及TRV2-NtPPOE的阳性克隆质粒,分别通过电击转化方式转化进入农杆菌GV3101感受态细胞中,利用含50mg/L Kan和50mg/L Rif的YEB平板进行培养筛选,在28℃倒置培养2d后,利用菌落PCR筛选带有目的基因的农杆菌。
(2)制备转染用菌液
将步骤(1)中筛选所得阳性农杆菌克隆在5mL的YEB液体培养基(含50mg/L Kan和50mg/L Rif)中,28℃、250rpm条件下培养过夜。
取50uL过夜培养物接种至50mL的YEB液体培养基(含50mg/L Kan)中,培养至OD600=1.0-1.5左右,然后4000转/分离心5min,收集菌体,再用MMA(1mL(1M)MgCl2;1mL(1M,pH5.6)MES;75μL(200mM)As)重悬,调节OD600=1.0左右。
最后室温放置3h左右后,作为转染用菌液。
(3)瞬时转化
以3-4周苗龄的本氏烟草叶片为实验材料,利用1mL规格注射器,将步骤(2)中所制备转染用菌液注射至烟草叶片中,注射后的烟草继续在人工培养箱内培养,观察表型变化。
注射3周后的烟草表型变化情况如图1所示。可以看出,含TRV2-PDS的农杆菌浸染植株新生叶有漂白现象,说明侵染成功;而TRV2-GFP组则无明显变化,相对应的TRV2-NtPPOE组烟草植株无显著变化,表明蛋白基因NtPPOE对烟草其他基本生理状态无明显影响。
进一步通过qRT-PCR对蛋白基因NtPPOE的表达情况进行了检测,结果如图2所示,可以看出,TRV2-NtPPOE的侵染植株中,NtPPOE的表达量显著降低,基因表达量下降约80%。
进一步地,对实验组(TRV2-NtPPOE浸染植株)和对照组(TRV2-GFP浸染植株)中的植物多酚类物质含量情况进行了检测,结果如图3,结果可以看出,沉默植株的生长表型正常,绿原酸含量升高12倍左右,这一结果表明蛋白基因NtPPOE是一个很好调控多酚类物质靶标基因,可以在不影响烟株表型的情况下有效调控烟叶中多酚类物质,是提高烟叶香气品质的重要靶标基因。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变形,本发明的范围由所附权利要求及其等同限定。
序列表
<110> 河南中烟工业有限责任公司
<120> 烟草多酚代谢途径蛋白基因NtPPOE及其应用
<130> WPM201836
<140> 2021102680404
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2036
<212> DNA
<213> 人工序列(NtPPOE)
<400> 1
atggcgtcaa gtgttattcc accagtgtgc aatagcacaa cagtcaaaac tccctttact 60
tcaaccacca agtcttcttc tttagcttcc actccaaaac cctctcaact tttcctccgt 120
ggaaaacgta accacagctt caaagtctca tgcaaggtct ccaatggtga tgaaaaccaa 180
agtgttgaaa caaataattc tgttgatagg agaaatgtgc ttctaggttt aggaggtcta 240
tatggtgctg ctaatgttgt accattggct tcagccactc ccattccagc ccctactact 300
tcatgtagca agactggtgc cacaattaaa cccggtttac cagtacctta ttcttgttgt 360
ccccctccgc taaaaattga tcctaaggat attcctcatt acacgtttcc aacaggatcg 420
aagctccgta ttcgaccagc ttctcatgcc gtggatgaag agtacatggc taagtacaac 480
ttagccatta ctaaaatgaa ggagctcgac gtcactgatc cagatgatcc acgtgggttc 540
gcggcgcaag ccaaaatcca ctgtgcttat tgcaacggtg catacaccgt tgctggcaaa 600
gagctacaaa ttcacttctc gtggcttttt ttcccattcc atagatggta cttgtacttc 660
tatgagagaa tcttgggttc tttaatcaat gatcctactt ttggtttgcc atattggaac 720
tgggatcatc caaagggcat gcgtttgcca cacatgtttg atcaaccaaa tgtgtaccct 780
gatctttacg atccaagacg taaccaagag caccgtggtt cggtaatcat ggaccttggt 840
cattttggtc aagacgtgaa aggaactgac ttacaaatga tgagaaataa ccttactcta 900
atgtatcgtc aaatgattac caattcaccg tgtccacaac tgttttttgg taagccatat 960
tgtacggaag ttggacccaa accagggcag ggagctattg aaaacatccc tcatactcct 1020
gtccacattt gggttggtag taagcctaat gagaataact gtaaaaacgg tgaagatatg 1080
ggaaatttct attcagctgg taaggatcct gctttctata gtcaccatgc aaatgtagat 1140
cgcatgtgga caatatggaa gacattagga ggaaaacgca aggacatcaa caagccagat 1200
tatttgaaca gtgagttctt cttctacgac gaaaagaaaa acccttttct cgtgaaagtc 1260
cgtgactgtt tggacaataa gaaaatggga tatgatttcc aagcaatgcc aaccccatgg 1320
cgcaatttta agccattgaa gaagagcaag agcaaggtca atgcacgttc agttcctcca 1380
gttacccaaa cattccctat tgcaaagatt gacaaagcca taacattttc catcaaaagg 1440
gaaacttcag gtactttcaa gtcatgttat ttaaaagttt aaactgttag aaataacaca 1500
cttttaatta ctaaacttaa ttagatcata taggtggata aaaataaagt ttttgcggtt 1560
agatttaaac ccatgacctc ttttgaatct ctcgtgccat tgttaagttg ctagagagca 1620
catattttta atttgttaga gtacaccttc aattatttaa ttatattatg tctttaacag 1680
gccggactca gcaggagaaa gacgcagaag aggagatgtt aactttcttg gaactgaaca 1740
tcgatcagcg aaagcacata aggtttgatg tgttcattaa cgcagatgca aactccaact 1800
ggtatgagct agacagggca gagtttgcag gaagttacac tgccttgcct catgttcatt 1860
cagatcccac taaaccacat gtcgccccta ttgcaaaatt ccagctggcc attaccgagt 1920
tgctcgagga aattggcctt gaagatgaag atgatatagt ggtgactctg gtcccgaaaa 1980
ctgggggcga atttgtcgcc attaaatctg ctgtgattac acttgaagct tgttga 2036
<210> 2
<211> 600
<212> PRT
<213> 人工序列(NtPPOE)
<400> 2
Ala Ser Ser Val Ile Pro Pro Val Cys Asn Ser Thr Thr Val Lys Thr
1 5 10 15
Pro Phe Thr Ser Thr Thr Lys Ser Ser Ser Leu Ala Ser Thr Pro Lys
20 25 30
Pro Ser Gln Leu Phe Leu Arg Gly Lys Arg Asn His Ser Phe Lys Val
35 40 45
Ser Cys Lys Val Ser Asn Gly Asp Glu Asn Gln Ser Val Glu Thr Asn
50 55 60
Asn Ser Val Asp Arg Arg Asn Val Leu Leu Gly Leu Gly Gly Leu Tyr
65 70 75 80
Gly Ala Ala Asn Val Val Pro Leu Ala Ser Ala Thr Pro Ile Pro Ala
85 90 95
Pro Thr Thr Ser Cys Ser Lys Thr Gly Ala Thr Ile Lys Pro Gly Leu
100 105 110
Pro Val Pro Tyr Ser Cys Cys Pro Pro Pro Leu Lys Ile Asp Pro Lys
115 120 125
Asp Ile Pro His Tyr Thr Phe Pro Thr Gly Ser Lys Leu Arg Ile Arg
130 135 140
Pro Ala Ser His Ala Val Asp Glu Glu Tyr Met Ala Lys Tyr Asn Leu
145 150 155 160
Ala Ile Thr Lys Met Lys Glu Leu Asp Val Thr Asp Pro Asp Asp Pro
165 170 175
Arg Gly Phe Ala Ala Gln Ala Lys Ile His Cys Ala Tyr Cys Asn Gly
180 185 190
Ala Tyr Thr Val Ala Gly Lys Glu Leu Gln Ile His Phe Ser Trp Leu
195 200 205
Phe Phe Pro Phe His Arg Trp Tyr Leu Tyr Phe Tyr Glu Arg Ile Leu
210 215 220
Gly Ser Leu Ile Asn Asp Pro Thr Phe Gly Leu Pro Tyr Trp Asn Trp
225 230 235 240
Asp His Pro Lys Gly Met Arg Leu Pro His Met Phe Asp Gln Pro Asn
245 250 255
Val Tyr Pro Asp Leu Tyr Asp Pro Arg Arg Asn Gln Glu His Arg Gly
260 265 270
Ser Val Ile Met Asp Leu Gly His Phe Gly Gln Asp Val Lys Gly Thr
275 280 285
Asp Leu Gln Met Met Arg Asn Asn Leu Thr Leu Met Tyr Arg Gln Met
290 295 300
Ile Thr Asn Ser Pro Cys Pro Gln Leu Phe Phe Gly Lys Pro Tyr Cys
305 310 315 320
Thr Glu Val Gly Pro Lys Pro Gly Gln Gly Ala Ile Glu Asn Ile Pro
325 330 335
His Thr Pro Val His Ile Trp Val Gly Ser Lys Pro Asn Glu Asn Asn
340 345 350
Cys Lys Asn Gly Glu Asp Met Gly Asn Phe Tyr Ser Ala Gly Lys Asp
355 360 365
Pro Ala Phe Tyr Ser His His Ala Asn Val Asp Arg Met Trp Thr Ile
370 375 380
Trp Lys Thr Leu Gly Gly Lys Arg Lys Asp Ile Asn Lys Pro Asp Tyr
385 390 395 400
Leu Asn Ser Glu Phe Phe Phe Tyr Asp Glu Lys Lys Asn Pro Phe Leu
405 410 415
Val Lys Val Arg Asp Cys Leu Asp Asn Lys Lys Met Gly Tyr Asp Phe
420 425 430
Gln Ala Met Pro Thr Pro Trp Arg Asn Phe Lys Pro Leu Lys Lys Ser
435 440 445
Lys Ser Lys Val Asn Ala Arg Ser Val Pro Pro Val Thr Gln Thr Phe
450 455 460
Pro Ile Ala Lys Ile Asp Lys Ala Ile Thr Phe Ser Ile Lys Arg Glu
465 470 475 480
Thr Ser Gly Arg Thr Gln Gln Glu Lys Asp Ala Glu Glu Glu Met Leu
485 490 495
Thr Phe Leu Glu Leu Asn Ile Asp Gln Arg Lys His Ile Arg Phe Asp
500 505 510
Val Phe Ile Asn Ala Asp Ala Asn Ser Asn Trp Tyr Glu Leu Asp Arg
515 520 525
Ala Glu Phe Ala Gly Ser Tyr Thr Ala Leu Pro His Val His Ser Asp
530 535 540
Pro Thr Lys Pro His Val Ala Pro Ile Ala Lys Phe Gln Leu Ala Ile
545 550 555 560
Thr Glu Leu Leu Glu Glu Ile Gly Leu Glu Asp Glu Asp Asp Ile Val
565 570 575
Val Thr Leu Val Pro Lys Thr Gly Gly Glu Phe Val Ala Ile Lys Ser
580 585 590
Ala Val Ile Thr Leu Glu Ala Cys
595 600
Claims (8)
1.烟草多酚代谢途径蛋白基因NtPPOE,其特征在于,碱基序列如SEQ ID NO.1所示。
2.根据权利要求1所述的烟草多酚代谢途径蛋白基因NtPPOE,其特征在于,氨基酸序列如SEQ ID NO.2所示,由602个氨基酸残基组成,其中第178-388位、395-445位、466-597位氨基酸为保守的PPO结构域。
3.根据权利要求1所述的烟草多酚代谢途径蛋白基因NtPPOE,其特征在于,引物序列为:
NtPPOE-F:5’-GTGTTGAAACAAATAATT-3’,
NtPPOE-R:5’-GTACAAGTACCATCTATG-3’。
4.根据权利要求3所述的烟草多酚代谢途径蛋白基因NtPPOE,其特征在于,以烟草K326的cDNA为模板,以NtPPOE-F、NtPPOE-R为引物,进行PCR扩增。
5.上述权利要求1至4中任一项烟草多酚代谢途径蛋白基因NtPPOE的应用,其特征在于,所述蛋白基因NtPPOE与植物叶片中多酚类物质含量相关。
6.根据权利要求5所述的烟草多酚代谢途径蛋白基因NtPPOE的应用,其特征在于,利用基因沉默技术或者基因超表达方法,通过调节烟草中的蛋白基因NtPPOE表达量,来调节控制烟叶中多酚类物质含量。
7.根据权利要求5所述的烟草多酚代谢途径蛋白基因NtPPOE的应用,其特征在于,通过转基因技术、瞬时表达技术或基因组编辑技术,构建含有蛋白基因NtPPOE的病毒诱导沉默载体、RNAi干涉载体、超表达载体或基因组编辑载体,转化烟草,筛选获得多酚类物质含量变化的烟草新品种。
8.根据权利要求7所述的烟草多酚代谢途径蛋白基因NtPPOE的应用,其特征在于,利用病毒诱导的基因沉默技术,干扰蛋白基因NtPPOE的表达使其沉默,蛋白基因NtPPOE沉默植株中多酚类物质含量显著上升,进而获得叶片中多酚类物质含量升高的植物新品种。
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