CN114875044A - 野葡萄VyVTE1基因及其编码的蛋白和应用 - Google Patents
野葡萄VyVTE1基因及其编码的蛋白和应用 Download PDFInfo
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Abstract
本发明涉及一种野葡萄VyVTE1基因及其编码的蛋白和应用,属于基因工程技术领域。本发明的野葡萄VyVTE1基因编码的蛋白的氨基酸序列如SEQ ID NO.1所示。本发明的野葡萄VyVTE1基因能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达,促进转基因植株抗旱性增强。
Description
技术领域
本发明涉及一种野葡萄VyVTE1基因及其编码的蛋白和应用,属于基因工程技术领域。
背景技术
植物生育酚由一个色原烷醇环和一个聚丙烯侧链组成的饱和链式结构,可依据色原烷醇环系统中甲基的位置与数量,分为α、β、γ、δ四种不同形式的生育酚,存在于大多数光合植物,其中α-T含量和活性均最高。已有研究证明266种植物光合器官中均存在生育酚,且其含量维管植物要高于非维管植物。高等植物叶和种子是富含生育酚的主要器官。生育酚在光合作用中主要作为抗氧化剂,也是植物防御系统的组成部分。在低光照条件下,拟南芥生育酚环化酶基因突变体(vte1)(α-和γ-T合成受阻)与野生型叶绿素含量与光合产量相差无几,然而,强光照5天后,vte1突变体的叶绿素含量与光合产量均明显下降,表明低光照条件下生育酚缺失不会影响植物的生长与光合能力,但强光条件下能够限制植物的生长与光合作用。拟南芥突变体与转基因植物暗处理发现生育酚对于植物具有光保护作用,当胁迫伤害严重时,植物维持较好的生存状态与生育酚紧密相联;然而,氧化伤害不严重时,叶黄素循环、抗氧化物质等光合保护机制会补偿生育酚的损失,对植物光合起保护作用。蓝藻α-T缺失突变体中修复PSII活性的D1蛋白会受到抑制,这是由于α-T 缺失,单线态氧大量产生,D1蛋白“从头合成”受阻,造成PSII光抑制受损。γ-TMT过表达的芥菜相较野生型能够维持较高含量的α-T,重镉胁迫下能够维持较好的生存状态和光合性能。
葡萄是世界第二大水果,拥有悠久的栽培历史,种类繁多,具有重要的食用价值和经济价值。近年来,随着全球气候的恶变,世界各地干旱事件频繁发生,在非干旱季节或非干旱地区干旱危害也频繁出现。干旱对葡萄生长发育过程和产量品质有严重的影响,已经成为制约葡萄生长和提高果品质量的主要因素之一,尤其是近年来全球气候的变化,使葡萄产业受到很大的威胁。在面临缺水问题的大背景之下,发掘抗旱葡萄资源、研究葡萄抗旱基因对提高葡萄抗旱性、培育抗旱新品种及节水栽培等都具有重要的科学价值和意义。
发明内容
本发明的目的是提供一种VyVTE1基因,为葡萄抗旱能力改良提供新的候选基因。
本发明还提供了含有VyVTE1基因编码的蛋白以及VyVTE1基因在提高植物抗旱性中的应用。
为了实现以上目的,本发明所采用的技术方案是:
野葡萄VyVTE1基因,所述野葡萄VyVTE1基因的开放阅读框为1509bp,核苷酸序列如SEQ ID NO.1所示。本发明的野葡萄VyVTE1基因能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达,促进转基因植株抗旱性增强。
所述野葡萄VyVTE1基因的cDNA的核苷酸序列如SEQ ID NO.2所示,编码序列全长1670bp。
所述野葡萄VyVTE1基因编码的蛋白,氨基酸序列如SEQ ID NO.3所示。野葡萄VyVTE1基因可编码一个含502个氨基酸的蛋白。
上述的野葡萄VyVTE1基因在提高植物抗旱性中的应用。
进一步地,上述的应用,包括以下步骤:利用基因工程手段在植物中过量表达野葡萄 VyVTE1基因。相对于转化空载体的植物,过量表达VyVTE1基因导致转基因植物中抗逆相关物质的积累和抗旱相关基因的表达,转基因植物抗旱性增强。
进一步地,所述植物为玉米或葡萄。
附图说明
图1为本发明的实验例1中VyVTE1基因的表达特性分析结果示意图,其中图1A为VyVTE1基因在葡萄不同组织中的表达分析结果图,图1B为VyVTE1基因在干旱处理后的表达;
图2为本发明的实验例2中VyVTE1转基因拟南芥植株的抗旱性鉴定结果图;
图3为本发明的实验例3中转VyVTE1基因拟南芥植株的生理特性分析结果图,其中图3A为转基因拟南芥与对照在干旱胁迫后的存活率示意图,图3B为转基因拟南芥与对照在干旱胁迫后的相对电导率示意图,图3C为转基因拟南芥与对照在干旱胁迫后的叶片相对失水率示意图,图3D为转基因拟南芥与对照在干旱胁迫后的叶绿素含量示意图;
图4为本发明的实验例4中转基因拟南芥植株中抗旱相关基因的表达分析结果图。
具体实施方式
以下结合具体实施方式对本发明的技术方案作进一步的说明。
实施例1野葡萄VyVTE1基因的克隆
用plus植物总RNA提取试剂盒(天根)提取燕山葡萄叶片组织总RNA。普通反转录用PrimeScriptII1st Strand cDNA Synthesis Kit(TaKaRa)合成cDNA第一链。具体操作步骤如下:在PCR管中加入:Random 6mers(50μM)1μl,dNTP Mixture(10mM each)1 μl,TotalRNA 2μg,双纯水补齐至10μl,充分混匀,瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5min,冰上急冷。
根据VyVTE1基因序列设计引物:
FL-VyVTE1-F:CACTCTCATTTGGTGTGCACTGAAG,
FL-VyVTE1-R:GATATCAAATTGGTCCCGATCCCTC。
进行目的基因VyVTE1的PCR扩增,PCR反应体系如下:1μl的反转录模板;正反向引物各1μl;dNTP Mix 2.5μl,DNA Polymerase 1.0μl,双纯水补齐至50μl。反应程序为:95℃,30s;进入循环,共计40个循环(95℃,5s;57℃,30s;72℃,30s);72℃, 5min。
扩增产物经1%琼脂糖凝胶电泳后,切下目的片段,凝胶回收纯化,并克隆到pMD18-T 载体上,经菌落PCR检测,送至金唯智公司测序验证,得到pMD18-T-VyVTE1质粒。测序结果表明,VyVTE1基因编码序列全长为1670个核苷酸,核苷酸序列如SEQ ID NO.2 所示,开放阅读框为1509个核苷酸,核苷酸序列SEQ ID NO.1所示,可编码一个含502 个氨基酸的蛋白,氨基酸序列如SEQ ID NO.3所示。
实施例2野葡萄VyVTE1基因过量表达载体的构建
为研究葡萄VyVTE1基因的功能,将VyVTE1基因共1509bp的ORF片段正确插入植物过量表达载体pCAMBIA2300-GFP上。
根据前期克隆到的VyVTE1基因ORF序列,设计可以扩增VyVTE1基因ORF的上下游引物VyVTE1-ORF-F和VyVTE1-ORF-R;根据pCAMBIA2300-GFP载体上的酶切位点,在引物VyVTE1-ORF-F的5’端加上酶切位点XbaI,具体序列为GGGTCTAGAATGGAAACAAACACTTACTCTATTTGGCG,在引物VyVTE1-ORF-R的5’端加上酶切位点KpnI,具体序列为GGGGGTACCCTACAGGCCAGGGGGCTTAAAAAATG。
以pMD18-T-VyVTE1质粒为模板,用VyVTE1-ORF-F与VyVTE1-ORF-R进行扩增,回收目的条带后连接到pMD19-T克隆载体,转化TOP10感受态细胞,在附加Amp的LB 培养基上进行蓝白斑筛选,分别经过菌液PCR与质粒酶切检测,pMD19-T-VyVTE1阳性克隆送公司测序。用XbaI、KpnI双酶切重组克隆载体pMD19-T-VyVTE1与植物表达载体 pCAMBIA2300-GFP,回收线性化载体与目标片段,连接并转化TOP10,经Kan抗生素筛选,挑取单克隆摇菌,菌液检测后提质粒酶切检测,形成植物表达载体 pCAMBIA2300-VyVTE1。
实施例3葡萄VyVTE1基因在拟南芥中的过量表达
采用电击转化法将pCAMBIA2300-VyVTE1质粒转化是农杆菌GV3101感受态细胞中,涂布在含有抗生素(含60mg/L的Gent,100mg/L的Kan)的LB平板上,置于28℃培养。挑取单菌落进行PCR检测,阳性克隆即为含有重组植物表达载体pCAMBIA2300-VyVTE1 的农杆菌。
将含有重组植物表达载体的农杆菌划线培养在LB平板(含60mg/L的Gent,100mg/L的Kan)上划线,置于28℃条件下培养24h;挑取单克隆在10ml LB液体培养基(附加相应的抗生素)中,在28℃条件下培养24h;取5ml菌液转移至50ml新鲜的LB液体培养基中,在28℃条件下继续培养,至菌液OD600达到0.6左右;转移至离心瓶或离心管中,室温条件下,转速为4000rpm离心10min,去除上清液收集菌体;重悬于渗透缓冲液 (0.5×MS,5%蔗糖,0.03%Silwet L-77(GE Health)),调OD600至0.8;将拟南芥花序上已有的果荚去掉,花序完全浸入渗透液中10-30s(或用移液器直接将渗透液滴在花序上),立即去掉拟南芥叶或茎秆上的渗透液,将植株平放在托盘中,用塑料薄膜覆盖托盘,24h 后取下薄膜,于温室中继续培养;为提高转化效率,7天之后用同样方法再次侵染;经过转化的拟南芥植株进行正常管理,待果荚现白色时进行收种子。
将收获的转基因种子用0.2%的TritonX-100浸泡10min;再用10%的次氯酸钠表面消毒,12min;灭菌水洗涤五次,每2min一次;用水将种子铺在含50mg/L卡那霉素的 MS(0.5×MS,1%蔗糖,1%琼脂,pH 5.7)平板上,4℃黑暗培养两天;然后转移至22℃, 16h光照的条件下培养。经除草剂筛选后仍然长势良好,真叶叶片及生长点呈深绿色且根部伸扎入培养基的幼苗即可初步确定为阳性苗。
对上述初步筛选得到的VyVTE1转基因阳性植株及对照植株(转入 pCAMBIA2300-GFP空载体),进一步在DNA水平进行鉴定,采用SDS提取法提取拟南芥叶片中的总DNA。分别以上述所提取的VyVTE1转基因植株及对照植株的DNA为模板,在VyVTE1基因上设计上游引物(5'AGTCCCATTTATGTGCCTACG3'),下游引物 (5'TTTCACGATGCCACTCTATCC3')组成引物对,进行PCR检测;反应体系(25μL) 为:10×buffer 2.5μL;d NTPs 0.5μL;Taq酶0.3μL;ddH2O16.2μL;Primer F 1.5μL;PrimerR 1.5μL;DNA 2.5μL。反应程序为:94℃预变性5min;35个循环,94℃变性30S,58℃退火30S,72℃延伸1min;72℃延伸10min,4℃保存,PCR产物在1%琼脂糖凝胶上进行电泳检测。结果表明:在初步筛选得到的VyVTE1转基因植株能扩增出目的基因大小的特异性片段,而对照植株没有扩增出任何片段,进一步地确定筛选到的初步筛选到的VyVTE1 转基因植株为转基因阳性植物。可见通过利用强启动子(花椰菜花叶病毒35S启动子)驱动原理的转基因技术,将VyVTE1基因的超量表达载体转入拟南芥中,可以获得转基因拟南芥植株。
实验例1野葡萄VyVTE1基因的表达特性分析
燕山葡萄组培苗继代培养16d后,选择生长健壮表现一致的幼苗用于干旱胁迫处理。干旱胁迫处理时,将葡萄组培苗从培养基中拔出,置于滤纸上暴露在室温为(32±1)℃、相对湿度为55%、光周期为光照14h/黑暗10h的条件下处理,在0、2、6、12、24h取叶片组织的作为样品进行分析的。正常培养的组培苗作为干旱胁迫处理的对照。
在大田生长8~10a的燕山葡萄,在转色期取葡萄果实,于盛花期取根系(第一新生侧根)、茎(新展开叶下第4~5片叶的茎段)、叶(新展开叶下第4~5片)、花序和卷须 (新生枝条的第1个枝)等组织作为样品进行分析。
用plus植物总RNA提取试剂盒(天根)提取燕山葡萄组织总RNA。普通反转录用PrimeScriptII1st Strand cDNA Synthesis Kit(TaKaRa)合成cDNA第一链。具体操作步骤如下:在PCR管中加入:Random 6mers(50μM)1μl,dNTP Mixture(10mM each)1μl, TotalRNA 2μg,RNase free dH2O补齐至10μl,充分混匀,瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5min,冰上急冷。
根据VyVTE1基因序列设计实时荧光定量PCR引物,正向引物序列为 qRT-VyVTE1-F(5'ATGGAGTTGTTAGCTGGGAAAT3'),反向引物序列为 qRT-VyVTE1-R(5'GTAGGGGCACGCAATGTAGT3')。以VyGAPDH基因为内参,其正向引物序列为qRT-VyGAPDH-F(5'CCCTTGTCCTCCCAACTCT3'),反向引物序列为 qRT-VyGAPDH-R(5'CCTTCTCAGCACTGTCCCT3')。
实时荧光定量PCR按照TaKaRaPremix Ex TaqTMII(Perfect Real Time)说明在Bio-Rad IQ5 Real-Time PCR Detection System(Bio-RadLaboratories,Hercμles,CA)上进行。25μl的反应体系:1μl的反转录模板;正反向引物各1μl;12.5μl的Premix Ex TaqTM;9μl的双纯水;反应程序为:95℃,30s;进入循环,共计40个循环(95℃,5s;57℃,30s;72℃,30s)。结果采用2-ΔΔC(t)法进行分析,大田生长的燕山葡萄的各组织中VyVTE1基因的相对表达量见图1A,干旱胁迫处理组和对照组的组培苗的叶片组织中VyVTE1基因的相对表达量见图1B。
由图1可知,VyVTE1主要在叶片中大量表达,其次是在根、卷须、果中表达量较高,在茎和花中的表达量最低。经过干旱处理后,VyVTE1表达量逐渐增加,在干旱处理后的6 h达到峰值,随后在12h和24h表达量又有所降低。
以下实验例2~4中的VyVTE1转基因T3代植株均为实施例3中筛选的VyVTE1转基因植株的转基因T3代阳性纯合植株。
实验例2转基因拟南芥植株的抗旱性鉴定
将VyVTE1转基因T3代植株和对照植株(转入pCAMBIA2300-GFP空载体)在MS培养基上生长7天后,转移至营养钵中,正常浇水20天使其生长成健壮的幼苗。然后停止给拟南芥幼苗浇水即进行干旱处理,直到第14天部分拟南芥植株叶片出现明显的失水萎焉症状。之后对所有植株进行复水,72小时后观察植株生长状况。干旱处理前后及复水后拟南芥植株的表现型通过拍照记录,结果见图2。由图2可知,对照植株(图2中标记为 EV)表现出对干旱敏感,呈现失水萎蔫的状态;3个VyVTE1转基因株系(图2中标记为 OE#1、OE#2和OE#3)相对于对照植株更加抗旱,这说明过量表达VyVTE1导致拟南芥植株更加抗旱。
实验例3转基因拟南芥植株生理生化特性分析
对VyVTE1转基因T3代植株和对照植株(转入pCAMBIA2300-GFP空载体)分别进行失水率测定、相对电导率测定以及叶绿素含量的测定,并统计存活率。
失水率的测定:VyVTE1转基因T3代植株和对照植株正常生长3周后,分别取约0.2g的莲座叶进行失水率的测定。将采取的莲座叶放置于干燥的滤纸上,每隔10min测量一次叶片的鲜重(FW),直到测至50min时失水率测定结束。将每一次测定的失水量与第一次测定的鲜重的比值作为失水率,结果如图3。
相对电导率的测定:将叶片装入离心管中,用超去离子水定容至10ml,室温下振荡1小时后测定溶液的电导值,记为煮前C1。随后将溶液连同叶片置于沸水中煮沸10min后,等温度降至室温后测定电导值,记为C2。将C1与C2的比值(C1/C2)作为相对电导率,结果如图3。
叶绿素含量的测定:将新鲜的各株系拟南芥叶片剪成0.2cm左右的细丝或小块混合均匀后,称取0.1-0.2g,放入50ml的离心管中,在容量瓶或试管中加入0.5ml纯丙酮和10-15 ml 80%的丙酮,并仔细将粘附在瓶壁边缘的叶子碎末洗到丙酮溶液中,盖上瓶塞,室温下置摇床浸提过夜,次日取出容量瓶,观察叶组织已全部变白时,表示叶绿素已浸提干净,然后用80%丙酮定容至25ml,离心后,波长645nm,663nm,652nm比色测定,计算叶绿素总含量,计算公式如下:叶绿素总含量(mg/g鲜重)=(20.21×A645+8.02×A663) ×V提×D÷m÷1000=0.01×(20.21×A645+8.02×A663)×D÷m,其中V提:提取液体积,10mL;D:稀释倍数;m:样本质量,g。计算结果如图3。
存活率统计:干旱处理20天后存活的拟南芥植株除以处理前所有植株的总数,上述结果乘以100%即为存活率,结果见图3。
由图3可知,相对于转化空载体的拟南芥植株,超量表达VyVTE1基因导致转基因拟南芥中抗逆相关物质的积累,转基因植株抗旱性增强。
实验例4转基因拟南芥抗旱相关基因表达分析
用plus植物总RNA提取试剂盒提取VyVTE1转基因T3代植株和对照植株(转入pCAMBIA2300-GFP空载体)叶片总RNA。普通反转录用PrimeScriptII1st Strand cDNASynthesis Kit(TaKaRa)合成cDNA第一链,具体操作步骤如下:
在PCR管中加入:Random 6mers(50μM)1μl,dNTP Mixture(10mM each)1μl, TotalRNA 2μg,RNase free dH2O补齐至10μl,充分混匀,瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5min,冰上急冷。
拟南芥AtActin为内参基因
正向引物序列qRT-AtActin-F:5’-CGGTGGTTCTATCTTGGCATC-3’,
反向引物序列qRT-AtActin-R:5’-GTCTTTCGCTTCAATAACCCTA-3’。AtCOR15A基因
正向引物序列qRT-AtCOR15A-F:5’-CAGCGGAGCCAAGCAGAGCAG-3’,
反向引物序列qRT-AtCOR15A-R:5’-CATCGAGGATGTTGCCGTCACC-3’。
AtERD15基因
正向引物序列qRT-AtERD15-F:5’-CCAGCGAAATGGGGAAACCA-3’,
反向引物序列qRT-AtERD15-R:5’-ACAAAGGTACAGTGGTGGC-3’。
AtRD29A基因
正向引物序列qRT-AtRD29A-F:5’-GTTACTGATCCCACCAAAGAAGA-3’,
反向引物序列qRT-AtRD29A-R:5’-GGAGACTCATCAGTCACTTCCA-3’。
AtP5CS1基因
正向引物序列qRT-AtP5CS1-F:5’-CGACGGAGACAATGGAATTGT-3’,
反向引物序列qRT-AtP5CS1-R:5’-GATCAGAAATGTGTAGGTAGC-3’。
实时荧光定量PCR按照TaKaRa Premix Ex TaqTMII(Perfect RealTime)说明在Bio-Rad IQ5 Real-Time PCR Detection System(Bio-Rad Laboratories,Hercμles,CA)上进行。25μl的反应体系:1μl的反转录模板;正反向引物各1μl;12.5μl的 Premix Ex TaqTM;9μl的双纯水。反应程序为:95℃,30s;进入循环,共计40个循环(95℃,5s;57℃,30s;72℃,30s)。结果采用2-ΔΔC(t)法进行分析,如图4所示。根据已有的知识,拟南芥中的AtCOR15A、AtERD15、AtRD29A、AtP5CS1为抗旱标志基因。由图4可知,由图4可知,在VyVTE1转基因T3代植株的3个株系中(图2中标记为OE#1、OE#2 和OE#3),AtCOR15A、AtERD15、AtRD29A、AtP5CS1抗旱标志基因的表达量显著高于对照植株(图2中标记为EV),这表明过量表达VyVTE1导致拟南芥中AtCOR15A、 AtERD15、AtRD29A、AtP5CS1抗旱标志基因表达量增加,从而增加了转基因植株的抗旱性。
<110> 河南科技大学
<120>野葡萄VyVTE1基因及其编码的蛋白和应用
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1509
<212> DNA
<400> 1
atggaaacaa acacttactc tatttggcgg gctcctgttt ttcaacacgt tgattcgtat 60
tcgcatttca gattttcgtg gaatcctaga tcgatcgggt ctccatgccg tcctctgaag 120
ctgaggcttc gaagaagctc acagattttc gcattgaact cgacttcaac aagtgaaggt 180
aaccgttctt ctgcggcgga gagtggagag accgagagtt tgggttctgt gagtcccatt 240
tatgtgccta cgccttccaa tcgagaactt cgcactccac acagcgggta ccatcttgat 300
ggaagtcccc gccagttttt tgaggggtgg tacttcaagg tctcaatacc agaacacaag 360
cagagcttct ggtttatgta ttctgtggag aatcctgcat ttcagaagaa gttggggaca 420
ttcgaagaat tacaatatgg tcctcgattt acaggagttg gggctcagat tcttggtgcc 480
gatgacaagt atatttgtca attctcagaa gaatctacta acttttgggg gtgtaggcat 540
gagctaatgc tggggcatac atttgttggc agaaaagact tgcggcctcc aaataaggag 600
gtccctcctg aggaattcaa tagaagagtg atagaaggtt tccaagtcga cccactttgg 660
catcaaggtt tcatccgtga tgatggcaga tcaaattatg tggatactgt aaagactgca 720
cggtgggaat acagtactcg ccccagttat ggctggggta atgttgggtc taaacagaag 780
tccgcagctg gctggcttgc agcttttcct gtatttgaac cccatcggca aatatgcatg 840
gcgggaggac tctcaacagg ttggatagag tggcatcgtg aaaggtgtga atttgaaaat 900
gccccttctt attcagaaaa gaactggggt ggaggtttcc cacgaaaagg gttttgcgtc 960
caatggaatg cctttgaagg tgcagatgga gaagtttctt tgactgcagc tggtgggttg 1020
aggaaaatac ctggattgac tgaagtgttt gaaaatgctg cattggttgg agttcactct 1080
gatggaattt tctatgaatt tgtgccatgg aatggagttg ttagctggga aattaatcaa 1140
tgggcttact ggtacatatc tgcagagaat gaatcacata tggaagaatt agtggcaaca 1200
acaaaggatc caggtactac attgcgtgcc cctaccacgg cagctggcct tgctcctgcc 1260
tgcaaagata attgttctgg tggactaaaa ttgcaaatac ggaaacgaac atttaacgga 1320
agtaaaggaa agatgatttt ggatgttaca agaaacatgg ctgcagttga agttggggga 1380
ggaccgtggt tcaacacctg gacaggcaag actgctgcac cagagcttgt tcgccttgct 1440
cttcaggttc ctgttgatga agatgcgata tttggtttgg ctccattttt taagccccct 1500
ggcctgtag 1509
<210> 2
<211> 1670
<212> DNA
<400> 2
cactctcatt tggtgtgcac tgaagttagt ctccgatttc tctctcattt tctctctata 60
ctctccatgg aaacaaacac ttactctatt tggcgggctc ctgtttttca acacgttgat 120
tcgtattcgc atttcagatt ttcgtggaat cctagatcga tcgggtctcc atgccgtcct 180
ctgaagctga ggcttcgaag aagctcacag attttcgcat tgaactcgac ttcaacaagt 240
gaaggtaacc gttcttctgc ggcggagagt ggagagaccg agagtttggg ttctgtgagt 300
cccatttatg tgcctacgcc ttccaatcga gaacttcgca ctccacacag cgggtaccat 360
cttgatggaa gtccccgcca gttttttgag gggtggtact tcaaggtctc aataccagaa 420
cacaagcaga gcttctggtt tatgtattct gtggagaatc ctgcatttca gaagaagttg 480
gggacattcg aagaattaca atatggtcct cgatttacag gagttggggc tcagattctt 540
ggtgccgatg acaagtatat ttgtcaattc tcagaagaat ctactaactt ttgggggtgt 600
aggcatgagc taatgctggg gcatacattt gttggcagaa aagacttgcg gcctccaaat 660
aaggaggtcc ctcctgagga attcaataga agagtgatag aaggtttcca agtcgaccca 720
ctttggcatc aaggtttcat ccgtgatgat ggcagatcaa attatgtgga tactgtaaag 780
actgcacggt gggaatacag tactcgcccc agttatggct ggggtaatgt tgggtctaaa 840
cagaagtccg cagctggctg gcttgcagct tttcctgtat ttgaacccca tcggcaaata 900
tgcatggcgg gaggactctc aacaggttgg atagagtggc atcgtgaaag gtgtgaattt 960
gaaaatgccc cttcttattc agaaaagaac tggggtggag gtttcccacg aaaagggttt 1020
tgcgtccaat ggaatgcctt tgaaggtgca gatggagaag tttctttgac tgcagctggt 1080
gggttgagga aaatacctgg attgactgaa gtgtttgaaa atgctgcatt ggttggagtt 1140
cactctgatg gaattttcta tgaatttgtg ccatggaatg gagttgttag ctgggaaatt 1200
aatcaatggg cttactggta catatctgca gagaatgaat cacatatgga agaattagtg 1260
gcaacaacaa aggatccagg tactacattg cgtgccccta ccacggcagc tggccttgct 1320
cctgcctgca aagataattg ttctggtgga ctaaaattgc aaatacggaa acgaacattt 1380
aacggaagta aaggaaagat gattttggat gttacaagaa acatggctgc agttgaagtt 1440
gggggaggac cgtggttcaa cacctggaca ggcaagactg ctgcaccaga gcttgttcgc 1500
cttgctcttc aggttcctgt tgatgaagat gcgatatttg gtttggctcc attttttaag 1560
ccccctggcc tgtagctgtc tggtctcgcc aagccttcta ttattctttg atgaattatc 1620
aagtgattta tggattatca cgagggaggg atcgggacca atttgatatc 1670
<210> 3
<211> 502
<212> PRT
<400> 3
Met Glu Thr Asn Thr Tyr Ser Ile Trp Arg Ala Pro Val Phe Gln His
1 5 10 15
Val Asp Ser Tyr Ser His Phe Arg Phe Ser Trp Asn Pro Arg Ser Ile
20 25 30
Gly Ser Pro Cys Arg Pro Leu Lys Leu Arg Leu Arg Arg Ser Ser Gln
35 40 45
Ile Phe Ala Leu Asn Ser Thr Ser Thr Ser Glu Gly Asn Arg Ser Ser
50 55 60
Ala Ala Glu Ser Gly Glu Thr Glu Ser Leu Gly Ser Val Ser Pro Ile
65 70 75 80
Tyr Val Pro Thr Pro Ser Asn Arg Glu Leu Arg Thr Pro His Ser Gly
85 90 95
Tyr His Leu Asp Gly Ser Pro Arg Gln Phe Phe Glu Gly Trp Tyr Phe
100 105 110
Lys Val Ser Ile Pro Glu His Lys Gln Ser Phe Trp Phe Met Tyr Ser
115 120 125
Val Glu Asn Pro Ala Phe Gln Lys Lys Leu Gly Thr Phe Glu Glu Leu
130 135 140
Gln Tyr Gly Pro Arg Phe Thr Gly Val Gly Ala Gln Ile Leu Gly Ala
145 150 155 160
Asp Asp Lys Tyr Ile Cys Gln Phe Ser Glu Glu Ser Thr Asn Phe Trp
165 170 175
Gly Cys Arg His Glu Leu Met Leu Gly His Thr Phe Val Gly Arg Lys
180 185 190
Asp Leu Arg Pro Pro Asn Lys Glu Val Pro Pro Glu Glu Phe Asn Arg
195 200 205
Arg Val Ile Glu Gly Phe Gln Val Asp Pro Leu Trp His Gln Gly Phe
210 215 220
Ile Arg Asp Asp Gly Arg Ser Asn Tyr Val Asp Thr Val Lys Thr Ala
225 230 235 240
Arg Trp Glu Tyr Ser Thr Arg Pro Ser Tyr Gly Trp Gly Asn Val Gly
245 250 255
Ser Lys Gln Lys Ser Ala Ala Gly Trp Leu Ala Ala Phe Pro Val Phe
260 265 270
Glu Pro His Arg Gln Ile Cys Met Ala Gly Gly Leu Ser Thr Gly Trp
275 280 285
Ile Glu Trp His Arg Glu Arg Cys Glu Phe Glu Asn Ala Pro Ser Tyr
290 295 300
Ser Glu Lys Asn Trp Gly Gly Gly Phe Pro Arg Lys Gly Phe Cys Val
305 310 315 320
Gln Trp Asn Ala Phe Glu Gly Ala Asp Gly Glu Val Ser Leu Thr Ala
325 330 335
Ala Gly Gly Leu Arg Lys Ile Pro Gly Leu Thr Glu Val Phe Glu Asn
340 345 350
Ala Ala Leu Val Gly Val His Ser Asp Gly Ile Phe Tyr Glu Phe Val
355 360 365
Pro Trp Asn Gly Val Val Ser Trp Glu Ile Asn Gln Trp Ala Tyr Trp
370 375 380
Tyr Ile Ser Ala Glu Asn Glu Ser His Met Glu Glu Leu Val Ala Thr
385 390 395 400
Thr Lys Asp Pro Gly Thr Thr Leu Arg Ala Pro Thr Thr Ala Ala Gly
405 410 415
Leu Ala Pro Ala Cys Lys Asp Asn Cys Ser Gly Gly Leu Lys Leu Gln
420 425 430
Ile Arg Lys Arg Thr Phe Asn Gly Ser Lys Gly Lys Met Ile Leu Asp
435 440 445
Val Thr Arg Asn Met Ala Ala Val Glu Val Gly Gly Gly Pro Trp Phe
450 455 460
Asn Thr Trp Thr Gly Lys Thr Ala Ala Pro Glu Leu Val Arg Leu Ala
465 470 475 480
Leu Gln Val Pro Val Asp Glu Asp Ala Ile Phe Gly Leu Ala Pro Phe
485 490 495
Phe Lys Pro Pro Gly Leu
500
Claims (6)
1.野葡萄VyVTE1基因,其特征在于:所述野葡萄VyVTE1基因的开放阅读框为1509bp,核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的野葡萄VyVTE1基因,其特征在于:所述野葡萄VyVTE1基因的cDNA的核苷酸序列如SEQ ID NO.2所示。
3.如权利要求1所述的野葡萄VyVTE1基因编码的蛋白,氨基酸序列如SEQ ID NO.3所示。
4.如权利要求1所述的野葡萄VyVTE1基因在提高植物抗旱性中的应用。
5.根据权利要求4所述的应用,其特征在于:包括以下步骤:利用基因工程手段在植物中过量表达野葡萄VyVTE1基因。
6.根据权利要求4或5所述的应用,其特征在于:所述植物为玉米或葡萄。
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