CN114573672B - 西瓜苦味物质葫芦素e的转运蛋白及其应用 - Google Patents
西瓜苦味物质葫芦素e的转运蛋白及其应用 Download PDFInfo
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Abstract
本申请涉及西瓜苦味物质葫芦素E的转运蛋白及其应用。本申请在西瓜基因组中鉴定出一种能够运输苦味物质葫芦素E的转运蛋白，为西瓜分子辅助育种提供了理论依据，同时也为利用合成生物学技术合成葫芦素E提供了支持。
Description
技术领域
本申请涉及西瓜苦味物质葫芦素E的转运蛋白及其应用，属于生物技术领域。
背景技术
苦味是由一种高度氧化的三萜化合物-葫芦素导致的，广泛存在于黄瓜、甜瓜、西瓜、南瓜和西葫芦等葫芦科植物中。西瓜的苦味主要是由一类称为葫芦素E的三萜化合物导致的，且葫芦素E主要存在于西瓜的根和果实中。由于西瓜果实中积累葫芦素E会严重影响西瓜的口感及品质，因此，科研人员希望采用更多育种手段来改善西瓜的品质。
人们知道，作物的品质性状与代谢产物合成、调控及运输过程密不可分。研究表明，一个由10个合成基因组成的基因簇参与了西瓜苦味的合成，其中包括1个环化酶，8个P450基因，和1个乙酰基转移酶。此外，两个主效转录因子ClBr和ClBt分别控制西瓜根和果实中葫芦素E的合成。然而，葫芦素E的完整的合成和转运机制还有待进一步揭示。
发明内容
为了解决上述技术问题，本申请提供一种葫芦素E的转运蛋白，其为如下之一：
a)氨基酸序列如SEQ ID NO.1所示；
b)将a)经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
c)与a)或b)具有80％或80％以上的同源性的蛋白质；
d)在a)、b)或c)的N端和/或C端连接标签得到的融合蛋白质。
本申请还提供与上述蛋白相关的生物材料，为下述A1)至A8)中的任一种：
A1)编码上述蛋白的核酸分子；
A2)含有A1)所述核酸分子的表达盒；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述表达盒的重组载体；
A5)含有A1)所述核酸分子的重组微生物；
A6)含有A2)所述表达盒的重组微生物；
A7)含有A3)所述重组载体的重组微生物；
A8)含有A4)所述重组载体的重组微生物。
在一些实施方式种，A1)所述核酸分子的序列如SEQ ID NO.2所示。
本申请还提供上述的蛋白或其相关生物材料在调控葫芦素E合成或转运中的应用。
在一种实施方式中，转运蛋白基因与葫芦素E的合成基因共调控。
在一种实施方式中，葫芦素E合成基因为Bi(即ClBi)。
本申请还提供上述的蛋白或其相关生物材料在植物育种中的应用。
在一种实施方式中，所述育种为培育苦味增加、或降低、或无苦味的植物。
本申请还提供上述的蛋白或其相关生物材料在调控葫芦素E合成基因Bi的表达水平中的应用。
一种调控植物苦味的方法，包括调控植物中上述的蛋白的表达量和/或活性的步骤。
在一些实施方式中，调控为降低或提高。
本申请还提供一种抑制植物病原菌的方法，包括采用本申请转运的蛋白或上述相关生物材料将植物细胞内合成的葫芦素E运输到细胞外的步骤。
在一种实施方式中，植物病原菌为细菌病原菌或真菌病原菌等，可存在于根际(根际病原菌)或叶际(叶际病原菌)或果实等组织，且可被葫芦素E抑制。
在一种实施方式中，本申请的植物为葫芦科植物，例如西瓜属的植物，具体的，例如西瓜。
附图说明
图1、候选转运蛋白与葫芦素E合成基因成簇分布。
图2、葫芦素E合成基因和候选转运蛋白基因在不同西瓜材料及组织中的表达情况。
图3、候选转运蛋白基因ClMATE与葫芦素E调控基因的互作关系。其中，A：酵母单杂交实验；B：烟草双荧光素酶激活实验；C：凝胶电泳迁移实验。
图4、候选转运蛋白基因ClMATE在烟草叶片中的亚细胞定位分析。
图5、候选转运蛋白基因ClMATE在黄瓜原生质体中的亚细胞定位分析。
图6、原位杂交试验分析候选转运蛋白基因ClMATE在根中的表达分布。
图7、西瓜幼苗的培养液中富集葫芦素E。
图8、利用酵母微粒体囊泡体系验证ClMATE的功能。
图9、利用卵母细胞表达体系验证ClMATE的功能。
图10、利用Crispr-Cas9技术获得ClMATE突变体。
图11、CmMATE突变体表型分析。其中，A：野生型与突变体材料中根以及培养液中葫芦素E含量比较，每组柱形图从左到右依次为：野生型、WCR-1、WCR-2；B：野生型与突变体材料中ClBi基因表达量比较。
具体实施方式
下述实施例中以具体试验为例，所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、西瓜中葫芦素E的转运蛋白挖掘与获得
1、候选基因的筛选
利用比较基因组学分析，在西瓜第1条染色体上发现一个被注释成MATE家族的转运蛋白基因Cla008357，该基因与葫芦素E合成基因成簇存在(图1)。接下来，利用西瓜野生材料和栽培材料不同组织的转录组数据，分析发现，Cla008357基因与葫芦素E合成基因DHO、CYP87D20表达模式高度一致(图2)。我们将候选转运蛋白基因Cla008357命名为ClMATE。
2、调控因子ClBr和ClBt与候选基因ClMATE互作
利用酵母单杂交体系，烟草双荧光素酶激活试验，以及凝胶电泳迁移试验，验证苦味主效调控因子ClBr和ClBt与候选转运蛋白ClMATE的互作情况。
酵母单杂交试验：利用引物对候选基因ClMATE的启动子区域进行PCR扩增，使用In-Fusion HD Cloning Kit(Clontech)同源重组试剂盒，将扩增产物分别构建到pHIS2的诱饵载体上。使用同样方法将转录因子ClBr和ClBt分别克隆到pGADT7载体上。随后将构建好的重组载体pHIS2和pGADT7共转化到酵母菌株AH109中，对照组共转化空载的pGADT7与重组载体pHIS2。转化后的酵母分别在亮氨酸，色氨酸双缺陷型(SD-Leu-Trp)和亮氨酸，色氨酸，组氨酸三缺陷型(SD-Leu-Trp-His)的固体培养基上生长，三重缺陷的培养基中可加入一定量3-AT(sigma)来抑制对照组中组氨酸的本底表达。30℃培养4～5天后，观察结果。结果表明，转录因子ClBr和ClBt可以直接与ClMATE基因的启动子区域结合(图3)。
烟草双荧光素酶激活试验：利用同源重组方法(In-Fusion HD Cloning)将转录因子ClBr和ClBt分别构建到双元载体pCAMBIA1300的35S启动子下游区域，即为效应蛋白载体。利用相同的方法将候选转运蛋白基因ClMATE构建到pGreen II 0800-LUC载体上，即为报告基因载体。随后将构建好的重组载体分别转化到农杆菌感受态GV3101中。不同的效应蛋白和报告基因组合对烟草进行瞬时共注射实验。对照组为空载pCAMBIA1300与已经构建好的报告基因载体。注射三天后取样，使用双荧光素酶酶报告体系的试剂盒(Promega)，对萤火虫荧光素酶活性(Firefly luciferase)和海肾萤光素酶活性(Renillia luciferase)进行测定。结果表明，转录因子ClBr和ClBt可以直接激活ClMATE基因的表达(图3)。
凝胶电泳迁移试验：将转录因子ClBr和ClBt分别构建到原核表达载体pET32a上。构建好的重组质粒转化到大肠杆菌感受态BL21(DE3)中，并诱导表达，纯化获得目标蛋白ClBr和ClBt。随后目标蛋白ClBr和ClBt分别与生物素标记的候选转运蛋白基因ClMATE的DNA探针进行孵育，电泳。结果表明，转录因子ClBr和ClBt可以直接与ClMATE基因的启动子区域结合(图3)。
以上结果说明，转录因子ClBr和ClBt可调控ClMATE基因表达，与葫芦素E合成基因共调控。
实施例2、候选转运蛋白的亚细胞定位分析
对于转运蛋白而言，其不同亚细胞器膜的定位差异，与本身具有的生理功能密切相关。我们采用烟草亚细胞定位体系、黄瓜原生质体亚细胞定位体系以及原位杂交试验对候选转运蛋白ClMATE进行亚细胞定位分析。
烟草亚细胞定位：将候选转运蛋白ClMATE构建到pSuper1300载体上，并与GFP组成融合蛋白ClMATE-GFP。同时将已知的拟南芥细胞质膜定位蛋白PIP2A构建到pSuper1300载体上，并与RFP组成融合蛋白PIP2A-RFP。随后将重组载体分别转化到农杆菌感受态细胞GV3101中。挑取单克隆，并培养农杆菌浓度至OD600为0.6～0.8，将分别含有ClMATE-GFP和PIP2A-RFP的农杆菌混合，共侵染烟草叶片下表皮。侵染2～4天后，用共聚焦荧光显微镜(Leica)观察荧光信号，发现GFP和RFP荧光信号重合(图4)，说明ClMATE定位在细胞质膜上。
黄瓜原生质体亚细胞定位：使用PEG介导的瞬时转化方法将重组质粒ClMATE-GFP和PIP2A-RFP共转化到原生质体中。具体流程：取15μg的重组质粒与200μL的原生质体悬浮液混合于2mL的离心管中，颠倒轻柔混匀；向离心管中加入200μL，40％PEG4000溶液(40％PEG4000,0.15M Mannitol,100mM CaCl2)，轻柔颠倒混匀，静置8分钟左右；加入1.0mL的W5溶液，终止反应，轻柔混匀；在低温离心机中80～100x g离心6分钟，弃掉上清，重复一遍；转化过的原生质体使用W5溶液进行重悬，放置于弱光下培养12～24小时。随后观察荧光信号，发现GFP和RFP荧光信号重合(图5)，说明ClMATE定位在细胞质膜上。
原位杂交试验：本试验分为探针制备，植物材料组织的制片以及杂交显色三个主要过程。利用引物将ClMATE基因的特异序列进行PCR扩增。扩增产物使用无RNA酶的酚氯仿异戊醇(25:24:1)进行抽提，并使用无水乙醇和醋酸钠纯化DNA片段。随后以获得的DNA为模版，利用RNA转录聚合酶试剂盒(Promega)制备地高辛标记的正义和反义探针。甜瓜种子萌发后两天的幼根作为组织样本被用于原位杂交实验。幼根被放置于4％的多聚甲醛(PFA)固定液中，真空抽气10分钟，直至组织材料沉降于离心管底部，随后更换新的固定液，4℃固定过夜。材料依次经过不同的乙醇浓度脱水，不同二甲苯浓度透明，梯度浸蜡和样品包埋等流程。随后对包埋样品进行切片处理，用于后续杂交显影实验。使用不同浓度的乙醇以及含有的蛋白酶K的PBS溶液对组织切片进行预处理。随后使用PFA进行重新固定和乙醇梯度脱水，并配置杂交液进行杂交。杂交液组成为：1.25×salt，50％甲酰胺，12.5％硫酸葡聚糖，1.25×Denhardt's以及1.5mg/mL的tRNA。制备好的杂交液与探针孵育，随后杂交。杂交后，使用5×SSC样品洗涤两次，随后使用20μg/mL的RNase A处理，以及0.2×SSC清洗。最后使用地高辛标记抗体(1:1000稀释)在室温杂交2小时，根据显影状态进行信号采集。结果表明，ClMATE主要在根尖表皮细胞中表达，且与葫芦素E合成基因ClBi表达部位相同(图6)。
以上结果证明，候选转运蛋白ClMATE是细胞质膜定位蛋白并且主要在根尖表皮细胞中表达。推测，该蛋白介导葫芦素E由细胞内到胞外的运输。为了进一步验证，我们利用水培的方法，对西瓜幼苗进行培养，发现液体培养基中富集了大量的葫芦素E(图7)。
实施例3、候选转运蛋白生化功能验证
我们利用酵母微粒体囊泡体系以及蟾蜍卵母细胞表达体系，对ClMATE的生化功能进行了验证。
酵母微粒体囊泡体系：将基因ClMATE构建到酵母表达载体pYES2上，随后将构建好的重组质粒转化到酵母菌株INVSc1中。挑取单克隆，30℃培养，诱导表达ClMATE蛋白。破碎酵母细胞后，密度梯度离心获取酵母微粒体，并与葫芦素B孵育。通过PVDF滤膜真空过滤后，对微粒体囊泡表面进行清洗。最后测定微粒体囊泡内葫芦素B的含量，与对照相比，含有ClMATE蛋白的微粒体囊泡内葫芦素E的含量显著增加(图8)，说明ClMATE能够介导葫芦素E运输。
蟾蜍卵母细胞表达体系：将基因ClMATE构建到卵母细胞表达载体pGEMHE上。随后利用mMessage mMachine Transcription Kit(Invitrogen)体外转录试剂盒完成cRNA制备。将cRNA注射到每个卵母细胞中，18℃环境下放置48小时，待蛋白表达后，向每个卵母细胞中注射葫芦素B。孵育1小时后，提取卵母细胞培养液中的葫芦素B，并测定其含量。与对照相比，含有ClMATE蛋白的卵母细胞培养液中的葫芦素E的含量显著增加(图9)，说明葫芦素E可以通过ClMATE蛋白将其运输到细胞外。
以上结果证明，ClMATE蛋白可以直接运输葫芦素B。
实施例4、候选转运蛋白遗传功能验证
为了进一步验证候选转运蛋白ClMATE功能，我们利用CRISPR-Cas9的基因编辑技术，对西瓜植株中ClMATE基因进行突变处理。西瓜转基因后代筛选过程中，我们鉴定到两种纯合突变类型的转化材料，突变类型分别为缺失1bp的纯合突变材料WCR-1，以及插入1bp的纯合突变材料WCR-2，两种纯合突变类型都导致ClMATE基因翻译提前终止(图10)。与野生型材料相比，ClMATE基因突变体材料中，葫芦素E含量在西瓜根及其培养液中显著降低，同时还发现葫芦素E合成基因ClBi的表达水平也显著降低(图11)。以上结果表明ClMATE基因参与了西瓜植株中葫芦素E由根内细胞到根外的运输，而且该基因还对葫芦素E的合成具有调节作用。
本申请已经尽力阐述了全部关键的技术细节，实施例仅仅为了示例本申请的构思，不应当反而成为限制本申请保护范围的理由。
SEQUENCE LISTING
<110> 中国农业科学院农业基因组研究所
<120> 西瓜苦味物质葫芦素E的转运蛋白及其应用
<130> 20220309
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 484
<212> PRT
<213> 人工合成
<400> 1
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Gln Tyr Gly Thr Asn Ser Val Thr Asn Ile Phe Val Gly Gln Leu Gly
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Glu Leu Glu Leu Ser Gly Ile Ser Ile Ala Ile Ser Val Ile Ala Thr
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Cys Gly Gln Ala Phe Gly Ala Gly Gln Ile His Met Leu Gly Val Tyr
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Met Gln Arg Ser Trp Val Ile Met Leu Ile Cys Ala Leu Ile Ile Thr
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Asp Asp Val Ala Glu Leu Ala Gly Ser Phe Ser Met Leu Ile Leu Pro
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Gln Leu Phe Ser Phe Val Val Ala Phe Pro Thr Gln Lys Phe Leu Gln
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atggaggcag tgccacttct cggcgacgac aacggcggag actatgctcc ggcgaagaca 60
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ccggtgatat ttcagattgt ttgtcagtat ggaaccaact ctgttacaaa tatttttgtg 180
ggtcaacttg gggagcttga gctttctgga atttccattg ccatctctgt tattgccact 240
ttcgcttttg gtttcatgtt tggaatggga agtgcaacag aaacattgtg tggccaagca 300
tttggagctg gacaaatcca catgctgggt gtttatatgc agagatcatg ggtaataatg 360
ctcatatgtg ccttaataat cacaccaatc tatgtatttg ccactcccgt tttgaagctt 420
ttagggcaac aagatgacgt ggctgaactg gctgggagtt tttccatgct catactccca 480
cagctgttct cttttgtggt ggcttttcca acccaaaagt ttcttcaggc acagagcaaa 540
gtgtgggcat tggcctggat tggctttggg gcccttctgg cccatgtttt catgctgtgg 600
ctcttcattt ttcagtttgg ttggggcact actggggctg gtttggcctt gaacatctct 660
ggttggggga tttccattgc tcaagtcatt tatgtgatgg gttggtgtaa ggatgcttgg 720
catggattct cttggttggc tttcaaggat ttgtggggat ttgttaagct ctcattttcc 780
tctgctatta tgttttgttt ggagatttgg tacatgagta gtatcattat tcttgctggt 840
caccttccaa atgctgtcat ctctgttgat tcactttcca tttgcatgaa cttgaacgga 900
tgggaaaata tcattttcat tggaatcaat gtcgctatga gtgttagagt ctccaatgaa 960
cttggaaagg cacggcctcg agctgcaatg tactccgtct atgtgacgat cgtggaatct 1020
cttattctcg gcctcatatt tatggtcctg atattctttg tcaaggatta ttttgctgtc 1080
atcttcacaa gcagtgtagc tgtgcagaaa tatgtttcca cattagctta tcttcttggc 1140
ataaccatgg ttctcaacag tgttcaacca gtcatatcag gcgtggctat tggagctgga 1200
tggcaggcat tggtggctta tataaactta ggctgctatt acatttttgg tctccctctt 1260
ggtgttatct taggttatgt agcaaacttt ggagtgaagg ggctttgggg tggaatgata 1320
gccgggatcg cgatgcagac gattctgttg ctgcttgttc tatacaaaac caactggaat 1380
agagaagtgg aggaaacttc aggaaggatg cagaaatgga ctggacaaga catcaggaat 1440
agagaagaga gttga 1455
Claims (10)
1.一种蛋白，其为如下之一：
a）氨基酸序列如SEQ ID NO.1所示；
b）在a）的N端和/或C端连接标签得到的融合蛋白质。
2.与权利要求1所述的蛋白相关的生物材料，为下述A1)至A8)中的任一种：
A1)编码权利要求1所述蛋白的核酸分子；
A2)含有A1)所述核酸分子的表达盒；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述表达盒的重组载体；
A5)含有A1)所述核酸分子的重组微生物；
A6)含有A2)所述表达盒的重组微生物；
A7)含有A3)所述重组载体的重组微生物；
A8)含有A4)所述重组载体的重组微生物。
3.根据权利要求2所述的相关生物材料，其特征在于：A1)所述核酸分子的序列如SEQID NO.2所示。
4.权利要求1所述的蛋白或权利要求2或3所述的生物材料在植物育种中的用途。
5.根据权利要求4所述的用途，其特征在于：所述育种为培育苦味增加、或苦味降低、或无苦味的植物。
6.权利要求1所述的蛋白或权利要求2或3所述的生物材料在调控葫芦素E合成或转运中的用途。
7.权利要求1所述的蛋白或权利要求2或3所述的生物材料在调控葫芦素E合成基因Bi的表达水平中的用途。
8.一种调控植物苦味的方法，其特征在于：包括调控植物中权利要求1所述的蛋白的表达量和/或活性的步骤。
9.根据权利要求6或7所述的用途、或权利要求8所述的方法，其特征在于：所述调控为降低或提高。
10.一种抑制植物病原菌的方法，其特征在于，包括采用权利要求1所述的蛋白或权利要求2或3所述的生物材料将植物细胞内合成的葫芦素E运输到细胞外的步骤；所述植物病原菌为细菌病原菌或真菌病原菌，且存在于根际、或叶际、或果实，且可被葫芦素E抑制。
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