CN111575254A - 一种脂氧合酶、编码基因CsLOX3及其应用 - Google Patents
一种脂氧合酶、编码基因CsLOX3及其应用 Download PDFInfo
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- CN111575254A CN111575254A CN202010407416.0A CN202010407416A CN111575254A CN 111575254 A CN111575254 A CN 111575254A CN 202010407416 A CN202010407416 A CN 202010407416A CN 111575254 A CN111575254 A CN 111575254A
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- tea
- cslox3
- lipoxygenase
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Abstract
本发明属于茶叶加工技术领域,尤其涉及一种脂氧合酶、编码基因CsLOX3及其应用。其中脂氧合酶基因CsLOX3的核苷酸序列如序列表中SEQ ID NO.1所示;脂氧合酶基因CsLOX3编码的蛋白的氨基酸序列如序列表中SEQ ID NO.2所示。本发明提供了一种来源于茶叶且与茶叶香气相关的脂氧合酶基因CsLOX3,且脂氧合酶基因CsLOX3在不同适制性红茶绿茶品种中的表达差异显著。本发明通过体外寡核苷酸反义抑制实验发现并证明了,CsLOX3的表达能够显著影响茶叶中挥发性物质以及茉莉酸类物质的含量,为茶叶的加工处理以及培育优良茶树品种提供新的技术手段。
Description
技术领域
本发明属于茶叶加工技术领域,尤其涉及一种脂氧合酶、编码基因CsLOX3及其应用。
背景技术
红茶是一种全发酵茶,用适宜的茶树新芽叶为原料,经萎凋、揉捻(切)、发酵、干燥等一系列工艺过程精制而成,是国际茶叶市场上的主要销售茶类,颇受消费者的喜爱。茶叶的多酚类物质在红茶发酵过程中发生氧化还原等一系列反应,使茶叶颜色由绿变红,形成“红叶红汤”的基本特征和香型,从而使红茶具有独特的品味。
茶叶香气是指气味物质不同浓度和比例对嗅觉神经产生影响,使人觉察到的茶叶特有的气味。茶叶香气是对茶叶品质起重要作用的因素之一,影响茶叶香气的因素有很多,如茶树品种、栽培条件、自然环境、加工工艺、储藏方法等,已检测到茶叶中的挥发性成分高达几百种,以醛类、酮类、醇类、酯类等香气化合物为主。这些成分以不同比例的组合,构成了不同香型和类型的茶叶。茶叶中的香气成分物质虽然种类很多,但其含量却微乎其微,鲜叶中仅占0.001%-0.05%(占干物),红茶中仅占0.01%-0.03%(占干物)。
目前的研究表明,红茶中的香气物质主要来源于3个方面,包括萜烯类化合物如香叶醇、类胡萝卜素降解产生的化合物,如紫罗兰酮等,以及茶叶细胞膜脂质过氧化产生的氧化脂质(Oxylipins)类化合物。多数植物的绿叶中单半乳糖二酰甘油(MGDG),双半乳糖二酰甘油(DGDG)占绿叶膜脂的80%以上。这两种叶绿体膜脂在茶叶加工过程中,被激活的磷酸酯酶A1(PLA1)作用下分解产生α-亚麻酸(α-Linolenic acid,18:3),后者经脂氧合酶(LOX)催化生成13S-氢过氧亚麻酸(13-HPOT)。13-HPOT可以在不同的生理状态或环境胁迫下产生不同的分解产物。一方面,13-HPOT由脂氢过氧化物裂解酶(HPL)催化分解为C6-C9挥发性物质,包括3-己烯醛和己醛等具有青草香气的挥发性物质;另一方面,13-HPOT先后在丙二烯氧化酶(AOS)和丙二烯氧化环化酶(AOC)催化下转化为氧化植物二烯酸(OPDA),叶绿体中生成的OPDA被转运蛋白运输至过氧化物酶体中,经由氧化植物二烯酸还原酶(OPDAReductase)以及3次β-氧化最终生成茉莉酸(JA)。JA再被JAMT催化的甲基化反应产生甲基茉莉酸酯。图1:茉莉酸类物质的合成途径,在图1中可以看到糖脂MGDG等降解下的脂肪酸可以在LOX的基因的作用下经过多步的催化反应可以生成C6的挥发物和茉莉酸甲酯。
发明内容
针对现有技术存在的问题,本发明提供了一种脂氧合酶、编码基因CsLOX3及其应用,目的在于解决现有技术中的一部分问题或至少缓解现有技术中的一部分问题。
本发明是这样实现的,一种脂氧合酶基因CsLOX3,核苷酸序列见SEQ ID NO.1所示,或与SEQ ID NO.1限定的DNA序列杂交且编码相同功能蛋白质的DNA序列;或与SEQ IDNO.1限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA分子。
本发明还披露了一种脂氧合酶,氨基酸序列见SEQ ID NO.2所示,或由SEQ IDNO.2所示序列经过若干个氨基酸残基的取代和/或缺失和/或添加,且具有相同蛋白功能的氨基酸序列;或由SEQ ID NO.2所示的氨基酸序列衍生的,具有98%以上同源性的,且具有相同蛋白功能的氨基酸序列。
本发明还披露了如上述的脂氧合酶基因CsLOX3在制备和/或作为脂氧合酶中的应用。
本发明还披露了如上述的脂氧合酶基因CsLOX3在调整茶叶中挥发性物质含量中的应用。
进一步地,所述茶叶中挥发性物质为己醛、2-己烯醛、1-己醇,2-乙基中的至少一种。
本发明还披露了如上述的脂氧合酶基因CsLOX3在调整茶叶中茉莉酸类物质含量和/或水杨酸含量中的应用。
进一步地,所述茉莉酸类物质为茉莉酸、茉莉酸氨基酸和甲基茉莉酸酯中的至少一种。
本发明还披露了如上述的脂氧合酶基因CsLOX3在调整茶叶中茉莉酸前提物质OPDA含量中的应用。
本发明还披露了如上述的脂氧合酶基因CsLOX3在作为标志物筛选适合制作绿茶的茶树品种和/或适合制作红茶或黑茶的茶树品种中的应用。
本发明还披露了如上述的脂氧合酶基因CsLOX3在茶树育种中的应用。
本发明还披露了一种促进茶叶中CsLOX3基因表达量的方法,对茶叶进行揉捻处理。具体为将茶叶拢成球状先轻揉后重揉再轻揉。
综上所述,本发明的优点及积极效果为:
本发明提供了一种来源于茶叶的与茶叶香气相关的脂氧合酶基因CsLOX3,且脂氧合酶基因CsLOX3编码的蛋白具有脂氧合酶的功能。
本发明通过检测红茶加工过程中脂氧合酶基因CsLOX3的表达差异发现,红茶的揉捻操作促进该基因的表达。
本发明通过体外寡核苷酸反义抑制实验发现并证明了,CsLOX3的表达能够显著影响茶叶中挥发性物质以及茉莉酸类物质的含量,为茶叶的加工处理以及培育优良茶树品种提供新的技术手段。
附图说明
图1是本发明实施例的茉莉酸类物质的合成途径;
图2是本发明实施例的舒茶早不同组织中脂氧合酶基因CsLOX3的表达差异;其中,L1:一叶;L2:二叶;L3:三叶;FL:花;FR:果;S:茎;R:根;B:芽;
图3是本发明实施例的红茶加工过程中脂氧合酶基因CsLOX3的表达差异;
图4是本发明实施例的CsLOX3在不同适制性茶树品种中的表达;
图5是本发明实施例的体外寡核苷酸反义抑制实验中脂氧合酶基因CsLOX3的表达量;
图6是本发明实施例的体外寡核苷酸反义抑制实验3d后茉莉酸类物质测定结果;
图7是本发明实施例的体外寡核苷酸反义抑制实验3d后挥发物测定结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明,各实施例及试验例中所用的设备和试剂如无特殊说明,均可从商业途径得到。此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
本发明披露了一种脂氧合酶、编码基因CsLOX3及其应用,具体如下各实施例所示。本发明涉及的脂氧合酶基因CsLOX3,其核苷酸序列如序列表中SEQ ID NO.1所示,氨基酸序列如序列表中SEQ ID NO.2所示。
本发明中涉及的材料:
1、茶树样品:舒茶早以及其它各个茶树品种,植株种植于安徽省庐阳区合肥安徽农业大学农业产业园,叶片采摘条件为25℃~28℃。
红茶的制作过程主要为采摘、萎凋、揉捻、发酵、干燥。具体为:采摘舒茶早植株的一芽一叶(此时为鲜叶样本);在室温摊放16h左右(完成后为萎凋样本);将茶叶拢成球状先轻揉后重揉再轻揉(完成后为揉捻样本);用纱布包住茶叶30℃发酵12h(完成后为发酵样本),发酵期间要每隔2h散开一次然后继续发酵;110℃干燥15min左右(完成后为干燥样本)。
2、大肠杆菌:DH5α。
3、载体:pGEM-T Easy。
4、LB培养基:称取10g的NaCl,5g的酵母提取物,10g的胰蛋白胨,加入950mL去超纯水搅拌溶解,加水定容至1000mL,高压蒸汽灭菌15min,即获得LB液体培养基,LB固体培养基为在LB液体培养基中加入15g的琼脂粉即可。
5、氨苄青霉素母液(Amp+,50mg/ml):称取0.5g氨苄青霉素Amp,溶于10mL灭菌水,过滤除菌,分装小管,-20℃保存。
6、配置体外寡核苷酸反义抑制缓冲液:将核苷酸粉末离心(8000rpm,5min),每管加入2ml 50Mmol/L的蔗糖溶液,在涡旋器上震荡数分钟,观察粉末是否完全溶解,若未见颗粒物,即可停止震荡,获得体外寡核苷酸反义抑制缓冲液(以下简称缓冲液),再用移液枪移取250ul缓冲液分装于96孔板中,备用。
实施例1舒茶早不同组织、不同茶树品种以及红茶加工过程中脂氧合酶基因CsLOX3的表达差异
1、舒茶早不同组织中脂氧合酶基因CsLOX3的表达差异
分别采摘舒茶早植株的根、茎、花、果、芽、一叶、二叶、三叶,采用高通量测序的方法检测各个样本中脂氧合酶基因CsLOX3的表达量。
2、红茶加工过程中脂氧合酶基因CsLOX3的表达差异
分别取红茶加工过程中采摘、萎凋、揉捻、发酵、干燥时期的样品,按实施例3中所述的方法获得cDNA,进行荧光定量PCR检测。
正向引物:TTGTTCTGGGTCGGGCTATG
反向引物:TGAACCCTCTGATCTGTC
3、不同适制性茶树品种中CsLOX3基因的表达差异
分别取不同适制性茶树品种的三叶,采用高通量测序的方法检测不同品种三叶的表达量。
测试结果与分析
在图2中,可以看到舒茶早不同组织中脂氧合酶基因CsLOX3的表达差异,从结果来看,CsLOX3在芽和一叶中表达量较高。
在图3中,可以看到红茶加工过程中脂氧合酶基因CsLOX3的表达差异,综合转录组和定量结果来看,揉捻促进CsLOX3的表达。
在图4中,可以看到CsLOX3在不同适制性茶树品种中的表达,发现CsLOX3在适合做绿茶的品种中表达量比在适合做红茶和黑茶的品种中的表达量高。因此,可以将CsLOX3基因表达量作为判断茶树品种适制绿茶和红茶的一个参数。
实施例2体外寡核苷酸反义抑制实验
1、根据LOX3序列设计合成寡核苷酸反义的引物,交付合成;
2、按以上步骤配置体外寡核苷酸反义抑制缓冲液。
3、用剪刀剪下大小基本一致,颜色鲜艳,色泽健康,无虫无病的一芽二叶,插入装有缓冲液的96孔板中,要确保一芽二叶尾部没入缓冲液中。
4、将96孔板放入光照培养箱中按光照16h/黑暗8h进行光照培养,培养箱温度为28℃。
5、依次取处理前样品0h,处理后样品1d,2d,3d的茶样品。
测试结果与分析
在图5中,可以看到体外寡核苷酸反义抑制实验中脂氧合酶基因CsLOX3的表达量,抑制后CsLOX3的表达量降低了90%以上。
在图6中,可以看到体外寡核苷酸反义抑制实验3d后茉莉酸测定结果,大部分茉莉酸类化合物的含量都得到了降低,其中茉莉酸含量降低了46%。
在图7中,可以看到体外寡核苷酸反义抑制实验3d后C6-C9挥发物测定结果,如己醛、2-己烯醛、1-己醇,2-乙基的含量都降低了90%以上。
实施例3脂氧合酶基因CsLOX3的克隆
本实施例中通过基因克隆获得目标基因的精确序列。
1、设计特异性引物,其引物序列如SEQ ID NO.3和SEQ ID NO.4所示:
F:ATGGTGGTTCTAGGTTTGAT,SEQ ID NO.3;
R:TCAAATTGAGATGCTATTTG,SEQ ID NO.4;
2、按照植物总RNA提取试剂盒和第一链cDNA合成试剂盒说明书,提取茶树样品的总RNA,并反转录为cDNA。
3、以反转录的cDNA为模板,用上述特异性引物进行扩增,PCR体系:正向引物2微升,反向引物2微升,模板2微升,MIX25微升,ddH2O19微升;PCR程序:第一步:94℃保持5min。扩增程序为98℃预变性10s,98℃变性10s,57℃退火温度30s,68℃延伸1min,35个循环,68℃继续延伸5min,获得的PCR产物置于4℃保存。
4、将PCR产物利用PCR纯化试剂盒纯化,并连接到pGEM-T Easy后转化DH5α,进行菌落PCR验证,具体方法如下。获得阳性菌落,提取菌落质粒,获得含有脂氧合酶基因CsLOX3的T载体,同时将菌液送至公司测序验证,即可获得包含脂氧合酶基因CsLOX3序列的DH5α大肠杆菌。
连接条件:4℃过夜连接。
转化体系:
得到的连接产物加入50μL DH5α中,轻轻混合均匀置于冰中,冰浴30min;42℃水浴锅中热激45sec,立即置于冰中,冰浴3min;加入1mL新鲜的LB培养基,37℃,200rpm振荡培养1h;将培养后的菌液离心,保留300μL上清,重悬混匀,涂板,37℃培养过夜。
本发明中涉及的检测方法
红茶加工过程中香气挥发物的测定:
1、取四所述样品并揉捻均匀,用干净的白色纱布包起来,洒上少许水,放入37℃的恒温培养箱中进行发酵过夜,约12h。取出装入GC-MS专用顶空萃取瓶中,同时加入1ul葵酸乙酯(99%,上海TCI化成工业有限公司)作为内参,密封进样瓶,-20℃,保存备测。
2、提取茶样挥发物:将进样瓶没入60℃水浴锅中,用铁架台固定,将萃取头垂直插入瓶盖,注意萃取头不要碰触到瓶壁和茶样品,萃取1h后取出萃取头插入GC-MS进样口吸附5min,同时启动仪器开始收集数据。
3、GC-MS检测条件:
色谱条件:进样口温度为240℃,载气为高纯氮气,纯度大于99.99%,流速0.8ml/min,不分流进样;色谱柱型号为HP-5MS石英毛细管柱(60m×0.32mm×0.25um);柱温起始为40℃,保持3min,以2℃/min升至90℃,保持5min,再以3℃/min升至160℃,最后以10℃/min升至250℃,保持5min;
质谱条件:离子源为EI源,离子源温度230℃,电子能量70eV,四极杆温度150℃,接口温度280℃,电子倍增器电压1680V,扫描范围m/z为35-350amu。
茉莉酸及茉莉酸甲酯的测定:
将茶叶样品在液氮中研磨成粉末,并在避光条件下提取激素,方法如下。
配制激素提取缓冲液配方,甲醇:ddH2O:乙酸=80:19:1(V:V:V)。茶样品液氮研磨,尽量磨碎,称取约0.3g茶树根样品和0.1g茶树芽叶样品于2ml离心管中,加入750μl抽提液,颠倒混匀,置于冰上,此过程需在避光条件下进行。然后4℃,避光条件下抽提16h以上。4℃,13000rpm离心10min,吸上清于一新离心管中,再次加入适量抽提液(一般为750μl),4℃,避光抽提4h以上,离心,合并两次上清液。0.22um滤膜过滤(有机系滤膜),用氮气慢慢吹干,加200μl甲醇,颠倒几次后4℃溶解3~6h(此过程在避光条件下进行)。溶解液在4℃,13000rpm离心15min后,轻轻吸取上清150~180μl于内插管中,放置于质谱专用样品瓶中等待上机检测。用液质联用技术分析激素样品,液相色谱仪型号为岛津LC-20AD,定量分析采用外标法。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 安徽农业大学
<120> 一种脂氧合酶、编码基因CsLOX3及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1755
<212> DNA
<213> 核苷酸序列(CsLOX3)
<400> 1
atggtggttc taggtttgat agagatgggt agcgacagcg gtggtggtct ggcggtggcg 60
acgatagtag cgaagctcac agttaaagaa ggtgattacg gggatgggga tggtgatggc 120
atggatcggt gcggttttaa cgaattgaga ggtattttgc atagccttaa tcccaatcat 180
gctcaagcaa atggaattaa agggcaattg aaaggagaat ttgtgccaag atatgtggac 240
tgggctaaat caccccttgt tctgggtcgg gctatggatt tggactatac agttgaatgg 300
ccattgaaga gtaaacttgc cccaaagata tacggcccac cagaatcggc aatcaccaaa 360
gagctaattg agcgacagat cagagggttc atgacccttg aagagagtgc aaagggacaa 420
cactgtatgg gtcgaggact cttcttctta acacctgatg gcaccttgag gcctttagcc 480
atagagctga ctcggccacc ggtagacggc aagcctcagt ggaagcaagt gttcacacct 540
accggggatg ctactggttg ctggctttgg aggcttgcca aggttcatgc ccttgctcat 600
gactctggtt atcaccagtt agtcagtcac tggctaagaa ctcactgcat gacagagcct 660
tacataattg cgagcaacag gcaacttagt gcaatgcacc cgatttacaa gctattgcac 720
cctcattttc ggtacacaat ggagatcaat gctttggctc gacaagccct cattaatgct 780
ggtggaatta ttgagacttg cttctcaccc aaaaagtact ctatcgagct tagttctgtt 840
gcctatgatc aacagtggcg tttcgatctc caagcactac cgactgactt aattagcagt 900
cagttcacaa ctgatacggt cggtttggct gttgtacaac cctttttcgc gattgatgat 960
tttataactt tgatgattgc cttatccaat tctcagggaa tggctgtgga ggatccaaca 1020
gctctgcatg gcctaaggct aaccattgag gactaccctt tcgcgaatga tggtctgcta 1080
gtttgggatg ccataaagca atgggttaca gactatgtca aacactatta cctagatgca 1140
agctttgtac agtctgacaa agagcttcaa gcatggtgga cagaaatccg aacagtaggt 1200
catggtgaca agaaagacga aacatggtgg ccagtgttga aaaccccaca agatctaatc 1260
ggaattctca caactatgat ctgggtaaca tccggtcacc attcagctgt caacttcggg 1320
caatatatct atgcaggtta tttccccaat aggcctacca ttgcgcgaac caaaatgcct 1380
actgaagatc caactgatga agagtggaag tgtttcatca acaaccctga agttgccctc 1440
ttgatgtgct ttccttctca aattcaagct acaaaagtca tggctgtttt ggatgtgtta 1500
tctaatcatt caacggatga agagtatctc ggtaaggaca tggaggcatc atggatagaa 1560
aatccaatta taaaggcggc atttgaacgg tttaatggga aattgaagga gctagaagga 1620
gtcattgatc gtaggaatgt tgataagaac ttgaagaata gatgtggagc tggtgtggtt 1680
ccttacgagc ttttgaagcc attttctgaa cctggagtga cagggagggg agttccaaat 1740
agcatctcaa tttga 1755
<210> 2
<211> 584
<212> PRT
<213> 氨基酸序列(CsLOX3)
<400> 2
Met Val Val Leu Gly Leu Ile Glu Met Gly Ser Asp Ser Gly Gly Gly
1 5 10 15
Leu Ala Val Ala Thr Ile Val Ala Lys Leu Thr Val Lys Glu Gly Asp
20 25 30
Tyr Gly Asp Gly Asp Gly Asp Gly Met Asp Arg Cys Gly Phe Asn Glu
35 40 45
Leu Arg Gly Ile Leu His Ser Leu Asn Pro Asn His Ala Gln Ala Asn
50 55 60
Gly Ile Lys Gly Gln Leu Lys Gly Glu Phe Val Pro Arg Tyr Val Asp
65 70 75 80
Trp Ala Lys Ser Pro Leu Val Leu Gly Arg Ala Met Asp Leu Asp Tyr
85 90 95
Thr Val Glu Trp Pro Leu Lys Ser Lys Leu Ala Pro Lys Ile Tyr Gly
100 105 110
Pro Pro Glu Ser Ala Ile Thr Lys Glu Leu Ile Glu Arg Gln Ile Arg
115 120 125
Gly Phe Met Thr Leu Glu Glu Ser Ala Lys Gly Gln His Cys Met Gly
130 135 140
Arg Gly Leu Phe Phe Leu Thr Pro Asp Gly Thr Leu Arg Pro Leu Ala
145 150 155 160
Ile Glu Leu Thr Arg Pro Pro Val Asp Gly Lys Pro Gln Trp Lys Gln
165 170 175
Val Phe Thr Pro Thr Gly Asp Ala Thr Gly Cys Trp Leu Trp Arg Leu
180 185 190
Ala Lys Val His Ala Leu Ala His Asp Ser Gly Tyr His Gln Leu Val
195 200 205
Ser His Trp Leu Arg Thr His Cys Met Thr Glu Pro Tyr Ile Ile Ala
210 215 220
Ser Asn Arg Gln Leu Ser Ala Met His Pro Ile Tyr Lys Leu Leu His
225 230 235 240
Pro His Phe Arg Tyr Thr Met Glu Ile Asn Ala Leu Ala Arg Gln Ala
245 250 255
Leu Ile Asn Ala Gly Gly Ile Ile Glu Thr Cys Phe Ser Pro Lys Lys
260 265 270
Tyr Ser Ile Glu Leu Ser Ser Val Ala Tyr Asp Gln Gln Trp Arg Phe
275 280 285
Asp Leu Gln Ala Leu Pro Thr Asp Leu Ile Ser Ser Gln Phe Thr Thr
290 295 300
Asp Thr Val Gly Leu Ala Val Val Gln Pro Phe Phe Ala Ile Asp Asp
305 310 315 320
Phe Ile Thr Leu Met Ile Ala Leu Ser Asn Ser Gln Gly Met Ala Val
325 330 335
Glu Asp Pro Thr Ala Leu His Gly Leu Arg Leu Thr Ile Glu Asp Tyr
340 345 350
Pro Phe Ala Asn Asp Gly Leu Leu Val Trp Asp Ala Ile Lys Gln Trp
355 360 365
Val Thr Asp Tyr Val Lys His Tyr Tyr Leu Asp Ala Ser Phe Val Gln
370 375 380
Ser Asp Lys Glu Leu Gln Ala Trp Trp Thr Glu Ile Arg Thr Val Gly
385 390 395 400
His Gly Asp Lys Lys Asp Glu Thr Trp Trp Pro Val Leu Lys Thr Pro
405 410 415
Gln Asp Leu Ile Gly Ile Leu Thr Thr Met Ile Trp Val Thr Ser Gly
420 425 430
His His Ser Ala Val Asn Phe Gly Gln Tyr Ile Tyr Ala Gly Tyr Phe
435 440 445
Pro Asn Arg Pro Thr Ile Ala Arg Thr Lys Met Pro Thr Glu Asp Pro
450 455 460
Thr Asp Glu Glu Trp Lys Cys Phe Ile Asn Asn Pro Glu Val Ala Leu
465 470 475 480
Leu Met Cys Phe Pro Ser Gln Ile Gln Ala Thr Lys Val Met Ala Val
485 490 495
Leu Asp Val Leu Ser Asn His Ser Thr Asp Glu Glu Tyr Leu Gly Lys
500 505 510
Asp Met Glu Ala Ser Trp Ile Glu Asn Pro Ile Ile Lys Ala Ala Phe
515 520 525
Glu Arg Phe Asn Gly Lys Leu Lys Glu Leu Glu Gly Val Ile Asp Arg
530 535 540
Arg Asn Val Asp Lys Asn Leu Lys Asn Arg Cys Gly Ala Gly Val Val
545 550 555 560
Pro Tyr Glu Leu Leu Lys Pro Phe Ser Glu Pro Gly Val Thr Gly Arg
565 570 575
Gly Val Pro Asn Ser Ile Ser Ile
580
<210> 3
<211> 20
<212> DNA
<213> 人工序列(CsLOX3-F)
<400> 3
atggtggttc taggtttgat 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(CsLOX3-R)
<400> 4
tcaaattgag atgctatttg 20
Claims (10)
1.一种脂氧合酶基因CsLOX3,核苷酸序列见SEQ ID NO.1所示,或与SEQ ID NO.1限定的DNA序列杂交且编码相同功能蛋白质的DNA序列;或与SEQ ID NO.1限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA分子。
2.一种脂氧合酶,氨基酸序列见SEQ ID NO.2所示,或由SEQ ID NO.2所示序列经过若干个氨基酸残基的取代和/或缺失和/或添加,且具有相同蛋白功能的氨基酸序列;或由SEQID NO.2所示的氨基酸序列衍生的,具有98%以上同源性的,且具有相同蛋白功能的氨基酸序列。
3.如权利要求1所述的脂氧合酶基因CsLOX3在制备和/或作为脂氧合酶中的应用。
4.如权利要求1所述的脂氧合酶基因CsLOX3在调整茶叶中挥发性物质含量中的应用。
5.根据权利要求4所述的应用,其特征在于:所述茶叶中挥发性物质为己醛、2-己烯醛、1-己醇,2-乙基中的至少一种。
6.如权利要求1所述的脂氧合酶基因CsLOX3在调整茶叶中茉莉酸类物质含量和/或水杨酸含量中的应用。
7.根据权利要求6所述的应用,其特征在于:所述茉莉酸类物质为茉莉酸、茉莉酸氨基酸和甲基茉莉酸酯中的至少一种。
8.如权利要求1所述的脂氧合酶基因CsLOX3在调整茶叶中茉莉酸前提物质OPDA含量中的应用。
9.如权利要求1所述的脂氧合酶基因CsLOX3在作为标志物筛选适合制作绿茶的茶树品种和/或适合制作红茶或黑茶的茶树品种中的应用。
10.一种促进茶叶中CsLOX3基因表达量的方法,其特征在于:对茶叶进行揉捻处理。
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CN116949064A (zh) * | 2023-09-12 | 2023-10-27 | 中国热带农业科学院三亚研究院 | 一种木薯低温响应茉莉酸合成基因及应用 |
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