CN114606245B - 茶树CsVAAT3基因及其应用 - Google Patents
茶树CsVAAT3基因及其应用 Download PDFInfo
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- CN114606245B CN114606245B CN202210455505.1A CN202210455505A CN114606245B CN 114606245 B CN114606245 B CN 114606245B CN 202210455505 A CN202210455505 A CN 202210455505A CN 114606245 B CN114606245 B CN 114606245B
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Abstract
本发明公开了茶树CsVAAT3基因及其应用,茶树CsVAAT3基因的核苷酸序列如序列表中SEQ ID NO.1所示。茶树CsVAAT3基因编码的蛋白的氨基酸序列如序列表中SEQ IDNO.2所示。茶树CsVAAT3的表达模式与茶树根系氮素水平呈现显著正相关,CsVAAT3在茶树根中高表达,将该基因构建的pDR196‑CsVAAT3质粒转化到液泡氨基酸吸收缺陷型酵母菌株ypq2,能够恢复酵母突变体在高浓度茶氨酸培养基上的生长能力。该基因的克隆有助于解析茶树根系茶氨酸储存的分子机制,为培育高茶氨酸含量的茶树新品种提供了重要的靶标基因资源以及理论基础。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及茶树CsVAAT3基因及其应用。
背景技术
茶树(Camellia sinensis(L.)O.Kuntze)是重要的叶用经济作物,茶叶中的茶氨酸含量与茶叶品质密切相关。然而,关于茶树体内液泡茶氨酸转运蛋白基因还尚未鉴定且茶氨酸在根部液泡储存的分子机制仍不清楚。茶树中的茶氨酸主要在根部合成,并且根中的茶氨酸含量随着季节的变化而改变,当茶树根部合成大量的茶氨酸时,茶树根部细胞内存在茶氨酸储存的生理过程,而液泡作为许多代谢物的储存场所,研究液泡膜茶氨酸转运蛋白基因的生理功能有助于了解茶氨酸储存的分子机制,为培育高茶氨酸茶树新品质提供理论基础和靶标基因。
发明内容
本发明的目的在于提供茶树CsVAAT3基因及其应用,其能够响应不同浓度氮素的处理且在酵母中能够将茶氨酸转运到酵母液泡中,为茶树合成更多的茶氨酸提供了新思路,为实现培育出高茶氨酸含量的茶树新品种理论和靶标基因资源。
为实现上述目的,本发明提供如下技术方案:
在本发明的第一方面,提出了一种茶树CsVAAT3基因,所述茶树CsVAAT3基因为液泡膜定位转运蛋白基因,所述茶树CsVAAT3基因的核苷酸序列,如序列表SEQ ID NO.1所示。
进一步的,本发明还提出了茶树CsVAAT3基因编码的蛋白序列,所述蛋白序列如序列表SEQ ID NO.2所示。
在本发明另一方面,提出了一种茶树表达载体pDR196-EGFP-CsVAAT3,所述表达载体通过将SEQ ID NO:1所示的片段酶切至载体pDR196-EGFP获得。
在本发明另一方面,提出了茶树CsVAAT3基因用于在酵母中将茶氨酸转运到酵母液泡中。
在本发明另一方面,提出了在酵母中将茶氨酸转运到酵母液泡中的方法,包括以下步骤:
克隆茶树CsVAAT3基因;
构建茶树表达载体;
茶树CsVAAT3转化酵母突变体。
进一步的,所述茶树表达载体为pDR196-EGFP-CsVAAT3。
进一步的,所述酵母突变体为高浓度茶氨酸敏感性菌株。
与现有技术相比,本发明的有益效果是:
1)本发明中,克隆茶树定位在液泡膜上的氨基酸转运蛋白,并在酵母中验证了其转运茶氨酸的功能,该转运蛋白在茶树中表达量随着氮水平的增加而增加。本发明还提供了含有CsVAAT3基因的重组质粒、转基因工程菌。本发明丰富了茶树中液泡膜茶氨酸转运蛋白的研究,有助于解析茶树茶氨酸储存的分子机制且能为培育茶氨酸含量更高的茶树提供理论基础。
2)茶树CsVAAT3的表达模式与茶树根系氮素水平呈现显著正相关,CsVAAT3在茶树根中高表达,将该基因构建的pDR196-CsVAAT3质粒转化到液泡氨基酸吸收缺陷型酵母菌株ypq2,能够恢复酵母突变体在高浓度茶氨酸培养基上的生长能力。
附图说明
图1为CsVAAT3在茶树不同组织的表达模式图;
图2为CsVAAT3在不同氮水平处理下不同时间点的表达量图;
图3为茶树CsVAAT3在酵母中的亚细胞定位图;
图4为CsVAAT3在酵母中向液泡内转运茶氨酸的图;
图5为不同酵母菌株细胞内茶氨酸含量图;
图6为不同酵母菌株液泡内茶氨酸含量图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
1、CsVAAT3基因的克隆与序列结构分析
茶树CsVAAT3基因,为液泡氨基酸转运蛋白基因,其克隆与序列结构分析,具体如下:
茶树国家级良种舒茶早种植于安徽省庐阳区合肥安徽农业大学农业产业园,取幼嫩根用于RNA的提取。总RNA的抽提采用RNA prep Pure Plant Kit(Tiangen,Beijing,China)试剂盒按照说明操作,用分光度计检测其RNA含量和质量。
反转录生成第一链:取1μg RNA作模板,根据PrimeScript II 1st Strand cDNASynthesis Kit(Takara Biotech,China)试剂盒说明配置,加入Oligo dT Primer(50μM)0.6μl,Random 6mers(50μM)0.4μl,dNTP Mixture(10mM each)1μl,RNase Free dH2O补足至10μl,65℃变性5min,立即在冰上放置。然后在上述反应液中加入5×PrimerScriptbuffer 4μl,RNase Inhibitor(40U)0.5μl,PrimerScript RTase(200U)1μl dH2O补足20μl,42℃温育45min,95℃5min灭活反转录酶。经过优化后,取适量的反转录产物用于随后的PCR。以cDNA第一链作为RT-PCR模板,常规方法做PCR,扩增CsVAAT3基因。其中上游引物:(5’-ATGAAGCCATTGAAATTTTCAG-3’),下游引物:(5’-TGATGATACAATAGATTTGAC-3’)。20μlPCR反应体系为:10×Ex taq buffer 2.5μl,dNTP 2.0μl,Mg2+1.5μl,上、下游引物各1μl,Extaq 0.2μl,模板1μl,ddH2015.8μl。
反应程序如下为:98℃10sec,98℃10sec,57℃30sec,72℃2min,72℃10min,35个循环。PCR产物CsVAAT3基因经纯化回收后,连接到pEASY-Blunt载体(Promega,Shanghai,China)上得到pEASY-Blunt::CsVAAT3质粒,转化大肠杆菌感受态细胞DH5α,送通用公司测序,得到的CsVAAT3基因的核苷酸序列如序列表SEQ ID NO.1所示,具体如下:
ATGAAGCCATTGAAATTTTCAGACTGTGAAAAAGAGAAGGGTTGTGTTAAATGGGTTGAGAAGTACTTCAAGGACTGTCTCTGCAACCTCAACGACCAACTCTCATTCGGTATTGGTTTAGCAAGTCTGGTTTGTTGGGGTGTTGCTGAAATCCCTCAAATCATCACCAACTTCCACAACAAGTCCGGCCATGGCCTGTCTCTCTCATTTCTCTGCACTTGGATTGTTGGTGACATCTTCAACCTAGTGGGTTGCTTTCTCGAGCCTGCCACGTTGCCGACCCAGTTCTACACTGCATTGCTATACACAACAATCACAGTAATATTGGTGTTGCAATGCATATATTATGATCACTTTCTCCAATGGTGGAAGCATCGGAACATTCAAGTCAATCTGGTAAAAGATGAGTCAAAACCTTTGAAATCCGATTATGTCGACTCAAGTAGAGCTTCAACAAATACCCCTAGTGTTGAAGTACCTAAACGGAGAGAATTCTATTATACGTCAGCAAGATCATTGGCTAGGAGCAATACGCCACCATTCCAATCTTCTTATATTAGGGCTAAAAGTGGTCCTTCTGCTTTGGAGGTTTACAGTGATTCATCATCTGAAGATGACACAACTCCAGCTCCCTCCAACAAGTCCCAGCCTCAGCGAATTCCACGTTCCTTGGGTTATGGAACCTTCCTGGCTGCCTCAGCCAAATTGCCCATCCAAAGCCGGGCTTTAACAGAAGAAGCATACATGAAACGATCTGGGATGACATTATTGCAGGAGAATGGATTAATGCAAGACTTGGCATGGGGACAATGGTTGGGATGGTTGATGGCAGCCATATACATGGGCGGTCGAGTCCCACAAATTTGGTTGAATATCAAAAGAGGGAGTGTGGAGGGCTTGAACCCTTTCATGTTCATCCTTGCCCTCATTGCCAATGTCACTTACACTGGAAGTATTCTAGTGAGAAGCACTGAATGGGAGAAGATAAAACCTAACTTGCCTTGGTTGCTGGATGCAGTAGTCTGTGTGCTGCTTGATCTCTTTATCATCCTTCAGTACGTTTACTACAGGTATTTGAAGCAAAAGAGGAAGATCGACTCCATTGAAGCATATTATGTAGACCCTGTGGATGTCAAATCTATTGTATCATCATAA
CsVAAT3基因编码的蛋白序列,如序列表SEQ ID NO.2所示具体如下:
MKPLKFSDCEKEKGCVKWVEKYFKDCLCNLNDQLSFGIGLASLVCWGVAEIPQIITNFHNKSGHGLSLSFLCTWIVGDIFNLVGCFLEPATLPTQFYTALLYTTITVILVLQCIYYDHFLQWWKHRNIQVNLVKDESKPLKSDYVDSSRASTNTPSVEVPKRREFYYTSARSLARSNTPPFQSSYIRAKSGPSALEVYSDSSSEDDTTPAPSNKSQPQRIPRSLGYGTFLAASAKLPIQSRALTEEAYMKRSGMTLLQENGLMQDLAWGQWLGWLMAAIYMGGRVPQIWLNIKRGSVEGLNPFMFILALIANVTYTGSILVRSTEWEKIKPNLPWLLDAVVCVLLDLFIILQYVYYRYLKQKRKIDSIEAYYVDPVDVKSIVSS
2、CsVAAT3基因的表达差异分析
(1)茶树不同组织CsVAAT3基因表达
茶树国家级良种‘舒茶早’种植于安徽省庐阳区合肥安徽农业大学农业产业园,14个组织器官用来分析基因表达。这14个组织器官包括芽(Bud)、1叶(1st Leaf)、1叶脉(1stMain Vein)、2叶(2nd Leaf)、2叶脉(2nd Main Vein)、3叶(3rd Leaf)、3叶脉(3rd MainVein)、4叶(4th Leaf)、4叶脉(4th Main Vein)、5叶(5th Leaf)、5叶脉(5thMain Vein)、维管束(Vascular Bundle)、2叶和3叶之间的嫩茎(Stem)和根(Root)。同时这些样品用于总RNA的提取以及cDNA第一链合成。反转录产物(cDNA第一条链)稀释5倍作为模板,使用2×AceQUniversal qPCRMaster Mix(Vazyme,Nanjing,China),配制10μl反应体系:1.0μl稀释5倍的逆转录产物,上下游引物各0.4μl(10pmol/μl),5μl 2×AceQ Universal qPCRMaster Mix,3.2μl ddH20,每个反应配3个重复。然后在Bio-rad CFX-384仪器上以程序:①95℃;②95℃10sec,60℃30sec,72℃30sec运行39个循环;③从65℃到95℃,以0.1℃/sec绘制熔解曲线。上游引物:(5’-TCTCTGCAACCTCAACGACC-3’),下游引物:(5’-GCTCGAGAAAGCAACCCACT-3’),以茶树CsGADPH基因为内参,上游引物:(5’-TTGGCATCGTTGAGGGTCT-3’),下游引物:(5’-CAGTGGGAACACGGAAAGC-3’)通过仪器自带分析软件,计算出CsVAAT3基因在不同组织中的相对表达水平。
(2)茶树在不同氮素水平下的CsVAAT3基因表达模式
一年生茶树扦插苗(龙井43)取自安徽省巢湖市年晟生态农业公司苗圃基地。采用大小均匀的茶树幼苗进行水培。在安徽农业大学茶树生物学与利用国家重点实验室的生长温室中,温室设置为温度25℃,光照时间14h,黑暗时间10h,相对湿度设置为70-75%,水培过程进行通气处理。将茶树幼苗在全基础营养液中生长1个月,以生长发育良好的根。以正常氮水平为对照,将茶苗在0N、1/5N、1N、5N、10N的营养液中生长,在不同时间点(10d、20d、30d)收集根组织样品,并立即在液氮中速冻,存储于-80℃的超低温冰箱用于分析CsVAAT3基因的表达量。RNA的提取和定量PCR方法同上。
图1-2是茶树CsVAAT3在不同组织和不同氮水平处理下的表达模式。由图1可知,通过qRT-PCR检测结果显示CsVAAT3在各个组织中都有表达,但根中的表达相对较高;如图2所示,在不同氮水平茶苗水培处理下,CsVAAT3的表达量随着氮水平的增加而显著增加,这暗示着CsVAAT3能够将茶树细胞质中的茶氨酸转运至液泡中储存起来。
3、茶树CsVAAT3的亚细胞定位
(1)pDR196-EGFP-CsVAAT3载体构建
以pEASY-Blunt::CsVAAT3质粒为模板,上游引物:(5’-CCCCAGCCTCGACTAGTATGAAGCCATTGAAATTTTCAG-3’),下游引物:(5’-AGCTTGATATCGAATTCTGATGATACAATAGATTTGAC-3’),进行PCR扩增。PCR产物用1.2%的琼脂糖胶电泳条带进行回收。首先将基因PCR回收产物与载体质粒进行双酶切,酶切产物用1.2%的琼脂糖胶电泳条带再进行回收,利用T4酶连技术,加入2μl载体和6μl基因酶切后回收产物,1μl T4 DNA Ligase Mix及1μl T4 DNALigase Buffer,4℃过夜后转化DH5α,送生工公司测序。
(2)FM4-64转染酵母
酵母突变体YPQ2是高浓度茶氨酸敏感性菌株,在高浓茶氨酸环境下生长受阻,用来验证所选基因在酵母中的亚细胞定位。将YPQ2菌株在YPDA固体培养基上划线,28℃恒温培养箱倒置培养2-3天。挑取长势良好的单菌落于2mL离心管中,加入1mL YPDA液体培养基,在28℃摇床,180r/min培养1天。吸取200μl生长良好的菌液转移至30mL的YPDA液体培养基,在28℃摇床,180r/min培养12-24h。待菌液摇至OD为0.8-1.2,吸取1mL至1.5mL离心管,12000rpm离心1min收集菌体,去上清,用灭菌水清洗一遍。在管底加入5μl转化的质粒DNA,用枪头吸打混匀。加入500μl PEG mixture,5μl DTT(1M,现配现用),涡旋混匀。室温静置20min,45℃水浴20min,冰浴20min。吸取底部沉淀菌体50μl,涂于SD-U的固体培养基上,28℃恒温培养箱倒置培养2-3天。酵母菌落PCR验证阳性菌落。菌落PCR反应程序:98℃变性10min,94℃预变性5min,{98℃变性10s,60℃退火10s,72℃延伸2min 30s},30个循环,72℃延伸10min,16℃保温。取1ml验证正确的酵母菌液,6000r/min,丢弃上清,使用HBSS溶液重悬后,加入1μl FM4-64染色,使用激光共聚焦显微镜观察荧光记录并拍照。
图3是CsVAAT3在酵母中的亚细胞定位图。如图3所示,其中,GFP:绿色荧光蛋白:pDR196-EGFP-CsVAAT3;Bright Field:pDR196-EGFP-CsVAAT3的明场图片;Merged:FM4-64融合图片。由图3可知:通过FM4-64的染色,在酵母液泡膜上能够检测到CsVAAT3的绿色荧光,表明CsVAAT3蛋白定位在液泡膜上。
4、CsVAAT3基因在酵母体内功能验证
(1)CsCsVAAT3-pDR196载体构建
以pEASY-Blunt::CsVAAT3质粒为模板,上游引物:(5’-CCCCAGCCTCGACTAGTATGAAGCCATTGAAATTTTCAG-3’),下游引物:(5’-AGCTTGATATCGAATTCTGATGATACAATAGATTTGAC-3’)。PCR产物用1.2%的琼脂糖胶电泳条带进行回收。首先将基因PCR回收产物与载体质粒进行双酶切,酶切产物用1.2%的琼脂糖胶电泳条带再进行回收,利用T4酶连技术,加入2μl载体和6μl基因酶切后回收产物,1μl T4 DNA Ligase Mix及1μl T4 DNA Ligase Buffer,4℃过夜后转化DH5α,送生工公司测序。
(2)CsVAAT3转化酵母突变体
酵母突变体YPQ2、AVT2、AVT6是高浓度茶氨酸敏感性菌株,在高浓茶氨酸环境下生长受阻,用来验证所选基因的抗胁迫能力。将YPQ2菌株在YPDA固体培养基上划线,28℃恒温培养箱倒置培养2-3天。挑取长势良好的单菌落于2mL离心管中,加入1mL YPDA液体培养基,在28℃摇床,180r/min培养1天。吸取200μl生长良好的菌液转移至30mL的YPDA液体培养基,在28℃摇床,180r/min培养12-24h。待菌液摇至OD为0.8-1.2,吸取1mL至1.5mL离心管,12000rpm离心1min收集菌体,去上清,用灭菌水清洗一遍。在管底加入5μl转化的质粒DNA,用枪头吸打混匀。加入500μl PEG mixture,5μl DTT(1M,现配现用),涡旋混匀。室温静置20min,45℃水浴20min,冰浴20min。吸取底部沉淀菌体50μl,涂于SD-U(含有100mM K+)的固体培养基上,28℃恒温培养箱倒置培养2-3天。酵母菌落PCR验证阳性菌落。菌落PCR反应程序:98℃变性10min,94℃预变性5min,{98℃变性10s,60℃退火10s,72℃延伸2min 30s},30个循环,72℃延伸10min,16℃保温。
(3)茶树CsVAAT3酵母打点功能验证
挑取单菌落于20mL的SD-U固体培养基中,28℃、180r/min的情况下,摇菌至OD600值为1.0左右(约48h);吸取4mL菌液,5000rpm离心2min,去上清(无菌操作)后用无菌水重新悬浮菌体,检测OD600值;用无菌水稀释菌液至OD600值为0.6(各个样品之间OD600值相差不要超过0.02);(无菌操作)将菌液依次稀释为100、10-1、10-2、10-3。例如10-1为从原菌液(100)中吸取100μl到新的1.5mL离心管中,并加入900μl无菌水,混匀;10-2为从上一稀释液(10-1)中吸取100μl到新的1.5mL离心管中,并加入900μl无菌水,混匀;10-3为从上一稀释液(10-2)中吸取100μl到新的1.5mL离心管中,并加入900μl无菌水,混匀(全程无菌操作)。
将配好的固体培养基放在已经画好的网格线上,利用10μl移液器吸取2μl的菌液,并将菌液滴在网格的交叉线上,同一横排点同一菌液的不同浓度,同一纵列点同一浓度的不同菌液。等菌液完全风干后再进行封板,在30℃恒温培养箱中倒置培养。注意打点时,移液枪应垂直于平皿。一般情况下,培养三天即可观察酵母生长情况。
图4是CsVAAT3在酵母异源体系高浓度茶氨酸处理下酵母生长表型。如图4所示,将酵母空载体pDR196和pDR196-CsVAAT3的质粒转化高浓度茶氨酸敏感型酵母液泡氨基酸突变体YPQ2、AVT2、AVT6,在较高茶氨酸(50mM)处理下,野生型BY4743以及突变体生长受到了明显的抑制,CsVAAT3转化的酵母突变体却是正常的生长状态。。表明CsVAAT3是一种能够将茶氨酸隔离在酵母液泡中的功能基因。
(4)酵母细胞及液泡内茶氨酸含量测定
利用野生型酵母BY4743,突变体YPQ2以及转基因酵母菌株YPQ2-pDR196和YPQ2-CsVAAT3来测定酵母全细胞及液泡内茶氨酸含量。挑取以上菌株的单克隆与50ml锥形瓶,每个菌株三个平行重复,摇至OD600一致后,6000r/min,3min,去除上清,用不含有氮源的培养基清洗三次,后悬浮在无氮源培养基中,28℃、200r/min培养3h,后向每个摇菌瓶中加入对应浓度的茶氨酸,28℃、200r/min培养3h。此后在冰面上上操作,用无氮源的培养基将各个摇菌瓶中的OD600调一致。吸取4ml菌液在5ml离心管中,6000r/min离心3min,后用ddH2O清洗两次,向处理组中加入3ml处理液(2.5mM磷酸钾、0.6M山梨醇、10mM葡萄糖、200μM无水氯化铜),向对照组中加入3ml ddH2O,30℃,放置15min,用ddH2O清洗3次,然后用500μl ddH2O重悬,98℃中煮沸15min,12000r/min,30min离心取上清。利用HPLC检测溶液中的茶氨酸含量。
图5和图6是酵母细胞和酵母液泡中的茶氨酸含量图。如图5所示,在野生型BY4743、突变体YPQ2以及YPQ2-pDR196液泡中的茶氨酸含量显著低于YPQ2-CsVAAT3液泡中的茶氨酸含量(图6),在全细胞中YPQ2-CsVAAT3的茶氨酸含量也显著高于突变体YPQ2以及YPQ2-pDR196(图5),这些结果说明,CsVAAT3所表达的蛋白在具有将茶氨酸储存在酵母液泡中的功能。
以上内容仅仅是对本发明结构所作的举例和说明,所属本技术领域的技术人员对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代,只要不偏离本发明的结构或者超越本权利要求书所定义的范围,均应属于本发明的保护范围。
序列表
<110> 安徽农业大学
<120> 茶树CsVAAT3基因及其应用
<130> NO
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1155
<212> DNA
<213> 茶树(Camellia sinensis L. O. Kuntze)
<400> 1
atgaagccat tgaaattttc agactgtgaa aaagagaagg gttgtgttaa atgggttgag 60
aagtacttca aggactgtct ctgcaacctc aacgaccaac tctcattcgg tattggttta 120
gcaagtctgg tttgttgggg tgttgctgaa atccctcaaa tcatcaccaa cttccacaac 180
aagtccggcc atggcctgtc tctctcattt ctctgcactt ggattgttgg tgacatcttc 240
aacctagtgg gttgctttct cgagcctgcc acgttgccga cccagttcta cactgcattg 300
ctatacacaa caatcacagt aatattggtg ttgcaatgca tatattatga tcactttctc 360
caatggtgga agcatcggaa cattcaagtc aatctggtaa aagatgagtc aaaacctttg 420
aaatccgatt atgtcgactc aagtagagct tcaacaaata cccctagtgt tgaagtacct 480
aaacggagag aattctatta tacgtcagca agatcattgg ctaggagcaa tacgccacca 540
ttccaatctt cttatattag ggctaaaagt ggtccttctg ctttggaggt ttacagtgat 600
tcatcatctg aagatgacac aactccagct ccctccaaca agtcccagcc tcagcgaatt 660
ccacgttcct tgggttatgg aaccttcctg gctgcctcag ccaaattgcc catccaaagc 720
cgggctttaa cagaagaagc atacatgaaa cgatctggga tgacattatt gcaggagaat 780
ggattaatgc aagacttggc atggggacaa tggttgggat ggttgatggc agccatatac 840
atgggcggtc gagtcccaca aatttggttg aatatcaaaa gagggagtgt ggagggcttg 900
aaccctttca tgttcatcct tgccctcatt gccaatgtca cttacactgg aagtattcta 960
gtgagaagca ctgaatggga gaagataaaa cctaacttgc cttggttgct ggatgcagta 1020
gtctgtgtgc tgcttgatct ctttatcatc cttcagtacg tttactacag gtatttgaag 1080
caaaagagga agatcgactc cattgaagca tattatgtag accctgtgga tgtcaaatct 1140
attgtatcat cataa 1155
<210> 2
<211> 384
<212> PRT
<213> 茶树(Camellia sinensis L. O. Kuntze)
<400> 2
Met Lys Pro Leu Lys Phe Ser Asp Cys Glu Lys Glu Lys Gly Cys Val
1 5 10 15
Lys Trp Val Glu Lys Tyr Phe Lys Asp Cys Leu Cys Asn Leu Asn Asp
20 25 30
Gln Leu Ser Phe Gly Ile Gly Leu Ala Ser Leu Val Cys Trp Gly Val
35 40 45
Ala Glu Ile Pro Gln Ile Ile Thr Asn Phe His Asn Lys Ser Gly His
50 55 60
Gly Leu Ser Leu Ser Phe Leu Cys Thr Trp Ile Val Gly Asp Ile Phe
65 70 75 80
Asn Leu Val Gly Cys Phe Leu Glu Pro Ala Thr Leu Pro Thr Gln Phe
85 90 95
Tyr Thr Ala Leu Leu Tyr Thr Thr Ile Thr Val Ile Leu Val Leu Gln
100 105 110
Cys Ile Tyr Tyr Asp His Phe Leu Gln Trp Trp Lys His Arg Asn Ile
115 120 125
Gln Val Asn Leu Val Lys Asp Glu Ser Lys Pro Leu Lys Ser Asp Tyr
130 135 140
Val Asp Ser Ser Arg Ala Ser Thr Asn Thr Pro Ser Val Glu Val Pro
145 150 155 160
Lys Arg Arg Glu Phe Tyr Tyr Thr Ser Ala Arg Ser Leu Ala Arg Ser
165 170 175
Asn Thr Pro Pro Phe Gln Ser Ser Tyr Ile Arg Ala Lys Ser Gly Pro
180 185 190
Ser Ala Leu Glu Val Tyr Ser Asp Ser Ser Ser Glu Asp Asp Thr Thr
195 200 205
Pro Ala Pro Ser Asn Lys Ser Gln Pro Gln Arg Ile Pro Arg Ser Leu
210 215 220
Gly Tyr Gly Thr Phe Leu Ala Ala Ser Ala Lys Leu Pro Ile Gln Ser
225 230 235 240
Arg Ala Leu Thr Glu Glu Ala Tyr Met Lys Arg Ser Gly Met Thr Leu
245 250 255
Leu Gln Glu Asn Gly Leu Met Gln Asp Leu Ala Trp Gly Gln Trp Leu
260 265 270
Gly Trp Leu Met Ala Ala Ile Tyr Met Gly Gly Arg Val Pro Gln Ile
275 280 285
Trp Leu Asn Ile Lys Arg Gly Ser Val Glu Gly Leu Asn Pro Phe Met
290 295 300
Phe Ile Leu Ala Leu Ile Ala Asn Val Thr Tyr Thr Gly Ser Ile Leu
305 310 315 320
Val Arg Ser Thr Glu Trp Glu Lys Ile Lys Pro Asn Leu Pro Trp Leu
325 330 335
Leu Asp Ala Val Val Cys Val Leu Leu Asp Leu Phe Ile Ile Leu Gln
340 345 350
Tyr Val Tyr Tyr Arg Tyr Leu Lys Gln Lys Arg Lys Ile Asp Ser Ile
355 360 365
Glu Ala Tyr Tyr Val Asp Pro Val Asp Val Lys Ser Ile Val Ser Ser
370 375 380
Claims (5)
1.一种茶树CsVAAT3基因,其特征在于:所述茶树CsVAAT3基因为液泡膜定位转运蛋白基因,所述茶树CsVAAT3基因的核苷酸序列,如序列表SEQ ID NO.1所示。
2.一种茶树表达载体pDR196-EGFP-CsVAAT3,其特征在于:所述表达载体通过将SEQ IDNO:1所示的片段酶切至载体pDR196-EGFP获得。
3.一种根据权利要求1所述的茶树CsVAAT3基因的用途,其特征在于:所述茶树CsVAAT3基因用于在酵母中将茶氨酸转运到酵母液泡中。
4.在酵母中将茶氨酸转运到酵母液泡中的方法,其特征在于,包括以下步骤:
克隆权利要求1所述的茶树CsVAAT3基因;
构建茶树表达载体;
茶树CsVAAT3转化酵母突变体,所述酵母突变体为高浓度茶氨酸敏感性菌株,所述酵母突变体在高浓茶氨酸环境下生长受阻。
5.根据权利要求4所述的在酵母中将茶氨酸转运到酵母液泡中的方法,其特征在于:所述茶树表达载体为pDR196-EGFP-CsVAAT3。
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