CN114774439B - 茶树CsFAAH6基因及其应用 - Google Patents

茶树CsFAAH6基因及其应用 Download PDF

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CN114774439B
CN114774439B CN202210474208.1A CN202210474208A CN114774439B CN 114774439 B CN114774439 B CN 114774439B CN 202210474208 A CN202210474208 A CN 202210474208A CN 114774439 B CN114774439 B CN 114774439B
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杨天元
范晓娟
张照亮
罗小草
谢云霞
廉晓红
王俊杰
赵鑫鹏
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Anhui Agricultural University AHAU
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Abstract

本发明公开了茶树CsFAAH6基因及其应用，茶树CsFAAH6基因的核苷酸序列如序列表中SEQ ID NO.1所示。茶树CsFAAH6基因编码的蛋白的氨基酸序列如序列表中SEQ IDNO.2所示。CsFAAH6在茶树成熟叶片高表达；不同品种茶树在不同月份新稍(一芽二叶)中茶氨酸含量与CsFAAH6表达量之间显著负相关；瞬时沉默CsFAAH6表达能够显著提高茶氨酸的含量，表明CsFAAH6具有降解茶氨酸的生理功能及其分子机制。本发明将丰富人们对茶树茶氨酸的代谢机制的认识，为通过分子辅助育种手段培育出高茶氨酸茶树新品种提供了理论依据和靶标基因。

Description

茶树CsFAAH6基因及其应用
技术领域
本发明涉及基因工程技术领域，具体涉及茶树CsFAAH6基因及其应用。
背景技术
我国是世界上第一大产茶和消费国，2020年根据中国茶叶流通协会数据显示目前中国茶叶种植面积和产量分别达到316.5万公顷和298.6万吨。茶氨酸是赋予绿茶鲜爽味和健康功效的主要呈味物质，茶树新梢中茶氨酸的积累受环境和遗传共同调控，茶氨酸含量主要受到根部合成和储存、根部向茶树新梢长距离运输以及在新梢中的积累和降解等因素动态调控。研究茶树中CsFAAH6介导的茶氨酸胞内代谢调控茶氨酸积累的功能与机制，有助于了解茶树新梢胞内茶氨酸代谢与积累的动态调控分子机制，也可以通过调控CsFAAH6的表达来提升茶树新梢茶氨酸含量，为提升茶叶品质与茶农经济收益提供基因资源和理论支撑。
发明内容
本发明的目的在于提供茶树CsFAAH6基因及其应用，瞬时沉默CsFAAH6能够显著提高茶树新梢茶氨酸的含量，为通过分子辅助育种手段培育高茶氨酸茶树新品种提供理论支撑和功能基因资源。
本发明是基于发明人的下列发现而完成的：脂肪酸酰胺水解酶FAAH(Fatty acidamidehydrolase)编码基因CsFAAH6表达量与茶树新梢和根部茶氨酸含量显著负相关。发明人分析了茶树CsFAAHs家族6条基因(CsFAAH1/2/3/4/5/6)的表达量与茶氨酸含量之间的相关性，最终确定了CsFAAH6是具有茶氨酸水解酶编码的重要候选基因。
在本发明的第一方面，提出了一种茶树CsFAAH6基因，所述茶树CsFAAH6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CsFAAH6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pCAMBIA1305.1-CsFAAH6，所述表达载体通过将SEQ ID NO：1所示的片段酶切至载体pCAMBIA1305.1获得。
在本发明另一方面，提出了茶树CsFAAH6基因用于改变茶树新梢和根部茶氨酸含量。
在本发明另一方面，提出了茶树CsFAAH6基因用于改变茶树新梢和根部茶氨酸含量的方法，包括以下步骤：
克隆茶树CsFAAH6基因；
构建茶树表达载体；
将茶树表达载体转入茶树新梢和根部。
进一步的，所述茶树表达载体为pCAMBIA1305.1-CsFAAH6。
进一步的，将茶树表达载体转入茶树新梢和根部后，利用反义寡核苷酸瞬时沉默新梢和根部的CsFAAH6表达，茶树新梢和根部的的茶氨酸含量提高。
与现有技术相比，本发明的有益效果是：
本发明在前期研究的基础上提出假设，并克隆处理CsFAAH6基因，通过反义寡核苷酸沉默实验，发现CsFAAH6表达量与茶树新稍中的茶氨酸呈显著负相关，抑制CsFAAH6表达能够减少茶氨酸在茶树体内的降解，提高茶树新梢中茶氨酸的积累量。本发明提供了含有CsFAAH6的重组质粒、转基因工程菌(pEASY-Blunt::CsFAAH6质粒转化大肠杆菌感受态细胞DH5α得到的新的工程菌)。同时本发明丰富了茶树茶氨酸胞内代谢调控的理论内容，也为提高茶树新梢茶氨酸含量和提升茶叶品质提供了靶标基因。
附图说明
图1中，A为茶树不同组织器官表型图；B为茶树CsFAAH6在茶树不同组织种的表达模式图；
图2为茶树CsFAAH6的亚细胞定位图；
图3中A和B为CsFAAH6基因茶树新梢瞬时沉默试验过程图；C为沉默试验后CsFAAH6基因表达量检测图；D为CsFAAH6基因沉默茶树梢中茶氨酸含量图；
图4中A为‘舒茶早’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；B为‘中茶108’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；C为‘皖茶91’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；D为‘福鼎大白茶’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；E为‘陕茶1号’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；F为‘柿大茶’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；G为‘铁观音’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；H为‘仙寓早’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图；I为‘迎霜’茶树CsFAAH6表达量与茶树新梢茶氨酸含量相关性图。
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、CsFAAH6基因的克隆与序列结构分析
茶树CsFAAH6基因，为茶树脂肪酸水解酶编码基因，其克隆与序列结构分析，具体如下：
‘舒茶早’品种茶树种植于安徽省安徽农业大学农萃园，取幼嫩根用于RNA的提取。总RNA的抽提采用RNAprep 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 ddH2O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CsFAAH6基因。其中上游引物：(5’-ATGGGCATTTTCAAGGCCAA-3’)，下游引物：(5’-TCAATCCTTTTTGAGCAGATCA-3’)。20μl PCR反应体系为：10×Ex taq buffer 2.5μl，dNTP 2.0μl，上、下游引物各1μl，Ex taq 0.2μl，模板1μl，ddH2O 15.8μl。
反应程序如下为：98℃10sec，98℃10sec，57℃30sec，72℃2min，72℃10min，35个循环。PCR产物CsFAAH6基因经纯化回收后，连接到pEASY-Blunt载体(Promega，Shanghai，China)上得到pEASY-Blunt::CsFAAH6质粒，转化大肠杆菌感受态细胞DH5α，送通用公司测序，得到的CsFAAH6基因的核苷酸序列如序列表SEQ ID NO.1所示，具体如下：
ATGGGCATTTTCAAGGCCAAAGGCGTAGTCTACAAGCCTGTCGACGATGTCGATCTCGGTCCTCACAGCGATGAGTTTTATCTCCGTGCTAACGTCAAAGCTCCTCGCATGGCTGGATTGCTGGTTAAAATTTTTGTTTGGTTCCTCGAGTCGCGGATTTTCGGGGGTATTTTGTTGTACATGTTGAAGAGAAACAACCTAATTCACAAGCTTGTTTCATATGCAGAGTTGGAAGAGTCACCTGTATTTGTTCCTTCACACCCTTATGAAGGCCTTAAAGAACAAGAAGTCAAATTAGTAGAGGATGATCTCTCTCCATCTGACAAAATTCAGAAGGCCATGGAATGCATACAATGCTCAGAAAGTATACAAGAAAATTCGGAGCTTAGTTTCCATCGCTGGACAGTATTGGATTATTCAAGAGCTTACATTTCAGGAGAGATTACTCCTCTCATGGTGGCGGAGCGATTTATAGCTGCTGTCCATGAATCGTCTGAACCTGCATTGCACATGTCATTCTTTATTGATTATAATGTTGGAGACATATTAAGGCAAGCTACTGAGTCAACTCAGCGGTACAAACAAGGAGAACCATTATCACCTCTAGATGGAGTCCCAATCGCAATCAAAGACGAAATAGATTGTATGCCCTATCCAACTACAGGGGGTACAAAGTGGTTGCAAAAGGTAAGACATTGTGCAGATGATGCATGCTGTGTTAAGCGCCTGAGATTATGTGGTGCCATACTTGTTGGGAAGACAAATATGCATGAGCTCGGGGCTGGAACCAGTGGTATCAATCCTCATTATGGGGTACCTAGAAATCCATATGATCCCAACAAGGTCTCTGGGGGTTCTTCTAGTGGATCTGCAGCTGTGGTTTCTGCAGGGTTGTGCCCTGTTGCCCTAGGTGTTGATGGGGGAGGATCTGTGAGAATGCCTGCTGCTCTTTGTGGTGTTGTTGGTCTGAAGCCAACTTTTGGACGTGTGCCCCATTCTGGTGTTATTCCTCTGAACTGGACAGTTGGGATGGTCGGTATCCTAGCAGGCACAGTTGAAGATGCATTTATTACTTATGCAGCTATCAGTGGTCAATTTCCATCATGCCAACCCACAGATGCAGTGAAAAAAATTAATTTCCCACTCCTGAAGACACCAAACTGTATATCTAACATCAAGATGGCTAAATATGGGGAGTGGTTTAATGATTGCACCGACGACATCAGAGTCTGTTGTTCCCATGCTCTGGACCAGCTTCACAAGCATTATGGATGGGAGACCATGGACGTGACCATACCAGAGATAGAGGTGATGCGCCTGGCGCATTATTCAACAATTGGATCGGAGTGTAGCAATTCAATTGCTTGTCATCTTGAAAACATGAATGTGGCAGAAATAGGGTTGGATGCAAGAGTAGCACTCTCTGTTTATGGTTCTTTCAGCAGCAGGGAGTATTTGAATGCCCAGAAAATTAGGAACCGACAGATGCAGTTTCATAAGAAAATATTTGCCATGGCAGATGTTATTGTTACACCAACGACAGGTGTGACTGCCTACCCAATATTCGATGATGCTTTGAAAACTGGGGAACTTGACTACATAAATGGAGCTGCACTTGTTCGGTATCAGATATCAGGAAATTTCTTGGGATTGCCAGCAGTAACCATACCTATTGGATACGACAAAGTTGGCTTGCCTATAGGCCTTCAATTTATTGGGAAGCCATGGTCCGAAGCTACGCTGATCCACATAGCGTTCGCAATGCAGGCCATCTCGGACTCAAAAAAACCACAGATTTTCTATGATCTGCTCAAAAAGGATTGA
CsFAAH6基因编码的蛋白序列，如序列表SEQ ID NO.2所示具体如下：
MGIFKAKGVVYKPVDDVDLGPHSDEFYLRANVKAPRMAGLLVKIFVWFLESRIFGGILLYMLKRNNLIHKLVSYAELEESPVFVPSHPYEGLKEQEVKLVEDDLSPSDKIQKAMECIQCSESIQENSELSFHRWTVLDYSRAYISGEITPLMVAERFIAAVHESSEPALHMSFFIDYNVGDILRQATESTQRYKQGEPLSPLDGVPIAIKDEIDCMPYPTTGGTKWLQKVRHCADDACCVKRLRLCGAILVGKTNMHELGAGTSGINPHYGVPRNPYDPNKVSGGSSSGSAAVVSAGLCPVALGVDGGGSVRMPAALCGVVGLKPTFGRVPHSGVIPLNWTVGMVGILAGTVEDAFITYAAISGQFPSCQPTDAVKKINFPLLKTPNCISNIKMAKYGEWFNDCTDDIRVCCSHALDQLHKHYGWETMDVTIPEIEVMRLAHYSTIGSECSNSIACHLENMNVAEIGLDARVALSVYGSFSSREYLNAQKIRNRQMQFHKKIFAMADVIVTPTTGVTAYPIFDDALKTGELDYINGAALVRYQISGNFLGLPAVTIPIGYDKVGLPIGLQFIGKPWSEATLIHIAFAMQAISDSKKPQIFYDLLKKD
2、CsFAAH6基因的表达差异分析
(1)茶树不同组织CsFAAH6基因表达
茶树国家级良种舒茶早品种种植于安徽省庐阳区合肥安徽农业大学农业产业园，16个组织器官用来分析基因表达。这16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(3rdMain Vein)、4叶(4th Leaf)、4叶脉(4th Main Vein)、5叶(5thLeaf)、5叶脉(5th Main Vein)、6叶(6thLeaf)、6叶脉(6th Main 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×AceQ Universal qPCR
Figure BDA0003624579510000061
Master 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 qPCR/>
Figure BDA0003624579510000062
Master 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℃5min②95℃10sec，60℃30sec，72℃30sec运行40个循环③从65℃到95℃，以0.1℃/sec绘制熔解曲线。上游引物：(5’-GTTCTTTCAGCAGCAGGGAG-3’)，下游引物：(5’-CGAACAAGTGCAGCTCCATT-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CsFAAH6在不同组织中的相对表达水平。
图1是茶树不同组织中CsFAAH6基因的表达。由图1可知，通过qRT-PCR检测结果显示CsFAAH6在各个组织中都有表达，成熟的叶部表达量显著高于其它部位。
3、CsFAAH6在茶树原生质体中的亚细胞定位
(1)pCAMBIA1305.1-CsFAAH6载体构建
以pEASY-Blunt::CsFAAH6质粒为模板，上游引物：(5’-GGACTAGTATGGGCATTTTCAAGGCC-3’)，下游引物：(5’-CGGGATCCATCCTTTTTGAGCAGATCATAGAA-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)茶树原生质体的制备
①收集长势健康的‘舒茶早’茶树花瓣。
②利用锋利的剃须刀片将花瓣切成1mm左右放入含有20mL酶解液的培养皿中(90mm×1.5mm)。
③将培养皿置于20～25℃的摇床中(40r/min)30～90min让原生质体细胞从花瓣中充分酶解下来。
④将含有原生质体的酶解液转移到50ml离心管中，100×g离心3min收集原生质体。用10ml预冷的W5溶液清洗2次，操作要柔和，避免破坏细胞。用适宜体积的W5溶液重悬体并在冰上放置最少30min。在此期间内用血球计数器计数并测算原生质体的浓度(若浓度过大，可以稀释10～20倍再测算)。
(3)原生质体的转化
①将原生质体在100×g条件下离心1min，弃掉上清，用新配制的MMg溶液重悬并将浓度调整3～5×105细胞/mL。
②将20μL pCAMBIA1305.1-CsFAAH6质粒加入到5mL的离心管底部(如果用多个质粒共同转化，需将转化的质粒预混合)，然后加入200μL MMg溶液(含有6～10×104个原生质体细胞)，轻弹离心管底部使混合液充分混匀。
③加入220μL新配制的PEG4000溶液，缓慢反转离心管混匀，室温下放置5～30min。
④100×g离心1min收集原生质体，并用2mL W5溶液漂洗2次。
⑤用0.6ml W5溶液重悬原生质体并转移到24孔板上培养(板孔用0.5～0.8ml 1％的BSA浸泡至少30min后倒掉BSA溶液使用)。于20～23℃下培养14～18h后，用激光共聚焦显微镜检测荧光信号。
如图2所示，CsFAAH6融合GFP信号在茶树原生质体细胞中的线粒体特异表达，而空载体GFP信号则充满了整个茶树原生质体细胞。
4、抑制CsFAAH6基因表达能够显著提高茶树叶片茶氨酸含量
为验证CsFAAH6在茶树体内是否具有降解茶氨酸的功能，利用反义寡核苷酸瞬时沉默叶片中的CsFAAH6。首先将CsFAAH6正(sODN)、反义(AsODN)寡核苷酸引物各4管分别加入250μL ddH2O混匀合一管，引物的终浓度为40μM，取330μL加入1.5mL离心管内做五个重复。将提前黑暗处理的茶树新稍保留一、二、三叶斜切适当高度后插入装有引物的离心管内，用封口膜封管。将处理好的样品插在板子上放入装有少量水的泡沫盒中用保鲜膜密封放在人工气候室培养24h后取样，提取RNA做荧光定量PCR分析基因的表达量，其余样品冷冻干燥后提取茶氨酸用高效液相HPLC检测茶氨酸含量。
如图3所示，4个生物学重复的叶片均有不同程度的CsFAAH6基因沉默，而叶片茶氨酸含量检测结果显示，与对照(sODN)相比较，处理组有4个生物学重复叶片茶氨酸含量均显著增加，结合CsFAAH6在成熟叶中的高表达，以及CsFAAH6的线粒体亚细胞定位特征，这些结果表明CsFAAH6参与了茶树新稍中茶氨酸降解的细胞学过程。
5、茶树CsFAAH6表达量与茶树新梢茶氨酸含量呈现显著负相关
检测9个茶树品种的3月24号、4月8号和4月22号春季三个不同时期新梢(一芽二叶)的茶氨酸的含量和CsFAAH6的表达量，如图4所示，将3个时期(2020年3月24日、4月8日、4月22日)9不同品种的茶树样品一芽二叶磨样，9个不同品种茶树样品提取RNA做荧光定量PCR分析CsFAAH6基因的表达情况和利用高效液相色谱(HPLC)检测9个不同茶树品种新梢茶氨酸含量，并分析不同时期不同品种基因表达量与茶氨酸含量间的相关性规律。
如图4所示，茶氨酸的含量随着CsFAAH6的表达量的上升而显著下降，呈现显著的负相关趋势。除仙寓早品种以外，8个品种的相关性系数均超过了-0.9。不同茶树品种新梢茶氨酸含量与CsFAAH6的相关性也进一步验证了CsFAAH6具有降解茶氨酸的功能。
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CsFAAH6基因及其应用
<130> NO
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1824
<212> DNA
<213> 茶树(Camellia sinensis L. O. Kuntze)
<400> 1
atgggcattt tcaaggccaa aggcgtagtc tacaagcctg tcgacgatgt cgatctcggt 60
cctcacagcg atgagtttta tctccgtgct aacgtcaaag ctcctcgcat ggctggattg 120
ctggttaaaa tttttgtttg gttcctcgag tcgcggattt tcgggggtat tttgttgtac 180
atgttgaaga gaaacaacct aattcacaag cttgtttcat atgcagagtt ggaagagtca 240
cctgtatttg ttccttcaca cccttatgaa ggccttaaag aacaagaagt caaattagta 300
gaggatgatc tctctccatc tgacaaaatt cagaaggcca tggaatgcat acaatgctca 360
gaaagtatac aagaaaattc ggagcttagt ttccatcgct ggacagtatt ggattattca 420
agagcttaca tttcaggaga gattactcct ctcatggtgg cggagcgatt tatagctgct 480
gtccatgaat cgtctgaacc tgcattgcac atgtcattct ttattgatta taatgttgga 540
gacatattaa ggcaagctac tgagtcaact cagcggtaca aacaaggaga accattatca 600
cctctagatg gagtcccaat cgcaatcaaa gacgaaatag attgtatgcc ctatccaact 660
acagggggta caaagtggtt gcaaaaggta agacattgtg cagatgatgc atgctgtgtt 720
aagcgcctga gattatgtgg tgccatactt gttgggaaga caaatatgca tgagctcggg 780
gctggaacca gtggtatcaa tcctcattat ggggtaccta gaaatccata tgatcccaac 840
aaggtctctg ggggttcttc tagtggatct gcagctgtgg tttctgcagg gttgtgccct 900
gttgccctag gtgttgatgg gggaggatct gtgagaatgc ctgctgctct ttgtggtgtt 960
gttggtctga agccaacttt tggacgtgtg ccccattctg gtgttattcc tctgaactgg 1020
acagttggga tggtcggtat cctagcaggc acagttgaag atgcatttat tacttatgca 1080
gctatcagtg gtcaatttcc atcatgccaa cccacagatg cagtgaaaaa aattaatttc 1140
ccactcctga agacaccaaa ctgtatatct aacatcaaga tggctaaata tggggagtgg 1200
tttaatgatt gcaccgacga catcagagtc tgttgttccc atgctctgga ccagcttcac 1260
aagcattatg gatgggagac catggacgtg accataccag agatagaggt gatgcgcctg 1320
gcgcattatt caacaattgg atcggagtgt agcaattcaa ttgcttgtca tcttgaaaac 1380
atgaatgtgg cagaaatagg gttggatgca agagtagcac tctctgttta tggttctttc 1440
agcagcaggg agtatttgaa tgcccagaaa attaggaacc gacagatgca gtttcataag 1500
aaaatatttg ccatggcaga tgttattgtt acaccaacga caggtgtgac tgcctaccca 1560
atattcgatg atgctttgaa aactggggaa cttgactaca taaatggagc tgcacttgtt 1620
cggtatcaga tatcaggaaa tttcttggga ttgccagcag taaccatacc tattggatac 1680
gacaaagttg gcttgcctat aggccttcaa tttattggga agccatggtc cgaagctacg 1740
ctgatccaca tagcgttcgc aatgcaggcc atctcggact caaaaaaacc acagattttc 1800
tatgatctgc tcaaaaagga ttga 1824
<210> 2
<211> 607
<212> PRT
<213> 茶树(Camellia sinensis L. O. Kuntze)
<400> 2
Met Gly Ile Phe Lys Ala Lys Gly Val Val Tyr Lys Pro Val Asp Asp
1 5 10 15
Val Asp Leu Gly Pro His Ser Asp Glu Phe Tyr Leu Arg Ala Asn Val
20 25 30
Lys Ala Pro Arg Met Ala Gly Leu Leu Val Lys Ile Phe Val Trp Phe
35 40 45
Leu Glu Ser Arg Ile Phe Gly Gly Ile Leu Leu Tyr Met Leu Lys Arg
50 55 60
Asn Asn Leu Ile His Lys Leu Val Ser Tyr Ala Glu Leu Glu Glu Ser
65 70 75 80
Pro Val Phe Val Pro Ser His Pro Tyr Glu Gly Leu Lys Glu Gln Glu
85 90 95
Val Lys Leu Val Glu Asp Asp Leu Ser Pro Ser Asp Lys Ile Gln Lys
100 105 110
Ala Met Glu Cys Ile Gln Cys Ser Glu Ser Ile Gln Glu Asn Ser Glu
115 120 125
Leu Ser Phe His Arg Trp Thr Val Leu Asp Tyr Ser Arg Ala Tyr Ile
130 135 140
Ser Gly Glu Ile Thr Pro Leu Met Val Ala Glu Arg Phe Ile Ala Ala
145 150 155 160
Val His Glu Ser Ser Glu Pro Ala Leu His Met Ser Phe Phe Ile Asp
165 170 175
Tyr Asn Val Gly Asp Ile Leu Arg Gln Ala Thr Glu Ser Thr Gln Arg
180 185 190
Tyr Lys Gln Gly Glu Pro Leu Ser Pro Leu Asp Gly Val Pro Ile Ala
195 200 205
Ile Lys Asp Glu Ile Asp Cys Met Pro Tyr Pro Thr Thr Gly Gly Thr
210 215 220
Lys Trp Leu Gln Lys Val Arg His Cys Ala Asp Asp Ala Cys Cys Val
225 230 235 240
Lys Arg Leu Arg Leu Cys Gly Ala Ile Leu Val Gly Lys Thr Asn Met
245 250 255
His Glu Leu Gly Ala Gly Thr Ser Gly Ile Asn Pro His Tyr Gly Val
260 265 270
Pro Arg Asn Pro Tyr Asp Pro Asn Lys Val Ser Gly Gly Ser Ser Ser
275 280 285
Gly Ser Ala Ala Val Val Ser Ala Gly Leu Cys Pro Val Ala Leu Gly
290 295 300
Val Asp Gly Gly Gly Ser Val Arg Met Pro Ala Ala Leu Cys Gly Val
305 310 315 320
Val Gly Leu Lys Pro Thr Phe Gly Arg Val Pro His Ser Gly Val Ile
325 330 335
Pro Leu Asn Trp Thr Val Gly Met Val Gly Ile Leu Ala Gly Thr Val
340 345 350
Glu Asp Ala Phe Ile Thr Tyr Ala Ala Ile Ser Gly Gln Phe Pro Ser
355 360 365
Cys Gln Pro Thr Asp Ala Val Lys Lys Ile Asn Phe Pro Leu Leu Lys
370 375 380
Thr Pro Asn Cys Ile Ser Asn Ile Lys Met Ala Lys Tyr Gly Glu Trp
385 390 395 400
Phe Asn Asp Cys Thr Asp Asp Ile Arg Val Cys Cys Ser His Ala Leu
405 410 415
Asp Gln Leu His Lys His Tyr Gly Trp Glu Thr Met Asp Val Thr Ile
420 425 430
Pro Glu Ile Glu Val Met Arg Leu Ala His Tyr Ser Thr Ile Gly Ser
435 440 445
Glu Cys Ser Asn Ser Ile Ala Cys His Leu Glu Asn Met Asn Val Ala
450 455 460
Glu Ile Gly Leu Asp Ala Arg Val Ala Leu Ser Val Tyr Gly Ser Phe
465 470 475 480
Ser Ser Arg Glu Tyr Leu Asn Ala Gln Lys Ile Arg Asn Arg Gln Met
485 490 495
Gln Phe His Lys Lys Ile Phe Ala Met Ala Asp Val Ile Val Thr Pro
500 505 510
Thr Thr Gly Val Thr Ala Tyr Pro Ile Phe Asp Asp Ala Leu Lys Thr
515 520 525
Gly Glu Leu Asp Tyr Ile Asn Gly Ala Ala Leu Val Arg Tyr Gln Ile
530 535 540
Ser Gly Asn Phe Leu Gly Leu Pro Ala Val Thr Ile Pro Ile Gly Tyr
545 550 555 560
Asp Lys Val Gly Leu Pro Ile Gly Leu Gln Phe Ile Gly Lys Pro Trp
565 570 575
Ser Glu Ala Thr Leu Ile His Ile Ala Phe Ala Met Gln Ala Ile Ser
580 585 590
Asp Ser Lys Lys Pro Gln Ile Phe Tyr Asp Leu Leu Lys Lys Asp
595 600 605

Claims (3)

1.一种茶树CsFAAH6基因，其特征在于：所述茶树CsFAAH6基因的核苷酸序列，如序列表SEQ ID NO.1所示。
2.根据权利要求1所述的一种茶树CsFAAH6基因，其特征在于：所述茶树CsFAAH6基因编码的蛋白序列，如序列表SEQ ID NO.2所示。
3.权利要求1所述的茶树CsFAAH6基因的用途，其特征在于：抑制CsFAAH6基因表达能够提高茶树叶片茶氨酸含量。
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