CN113621622A - 柑橘多甲氧基黄酮生物合成基因及其应用 - Google Patents
柑橘多甲氧基黄酮生物合成基因及其应用 Download PDFInfo
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- CN113621622A CN113621622A CN202110815150.8A CN202110815150A CN113621622A CN 113621622 A CN113621622 A CN 113621622A CN 202110815150 A CN202110815150 A CN 202110815150A CN 113621622 A CN113621622 A CN 113621622A
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Abstract
本发明公开了柑橘多甲氧基黄酮生物合成基因，核苷酸序列如SEQ ID No.1‑4所示。本发明所涉及的四个基因具有多甲氧基黄酮的生物合成功能，通过转基因手段，将这些基因在柑橘中过表达能够获得高产多甲氧基黄酮的转基因植物。本发明在提高柑橘果实品质、功能性柑橘育种及工厂化生产PMFs方面具有广泛的应用前景。
Description
技术领域
本发明属于基因工程技术领域，涉及柑橘多甲氧基黄酮(PolymethoxylatedFlavones, PMFS)生物合成基因。
背景技术
类黄酮广泛存在于人们的日常食物中，因其抗氧化、抗癌等特性常被用作各种保健品、药物和化妆品等，其在各种疾病防治疗中的作用已得到广泛研究。柑橘中大量积累多甲氧基黄酮，较其他类黄酮显示出更强的抗癌功能，受到国内外广泛关注。天然O-甲基化的PMFs 具有比其他普通类黄酮更优越的生物学活性，在植物生长发育和人类健康中发挥着重要作用。
本研究首先基于不同柑橘种质中聚甲氧基黄酮的评价、代表性柑橘种质不同组织/时期积累PMFs的特性分析、柑橘人工杂交群体PMFs积累表型的分离情况，筛选特异积累PMFs 的组织、时期和种质。随后，结合PMFs代谢组差异和转录组数据筛选PMFs生物合成相关基因和调控因子，通过体外和体内实验验证候选OMTs基因的生物学功能，同时根据柑橘人工杂交群体中PMFs的分布规律，通过BSA-seq筛选PMFs生物合成的主效基因。最后，通过不同种质间关键OMTs的序列变异和功能分化以及系统进化分析，阐述不同柑橘代表性种质间差异积累PMFs的分子机理。
发明内容
本发明的目的是提供一种柑橘多甲氧基黄酮生物合成基因及其应用。
申请人从柑橘果实黄皮层中提取总RNA并反转录成cDNA，然后设计四对引物分别对其进行PCR扩增，克隆出Ciclev10031951m.g(CrOMT3)、Ciclev10031949m.g(CrOMT4)、Ciclev10031952m.g(CrOMT5)、Ciclev10005276m.g(OMT7)的编码区序列，序列分别如 SEQID No.1-4所示。其中，Ciclev10031951m.g(CrOMT3)、Ciclev10031949m.g(CrOMT4)、Ciclev10031952m.g(CrOMT5)三个基因具有高度相似的序列，CDS长度均为1062bp，编码353个氨基酸，Ciclev10005276m.g(OMT7)的CDS长度为1071bp，编码356个氨基酸。
接着，申请人将转录组筛选到的4个候选OMTs均用酵母进行异源表达并通过添加含羟基的黄酮底物验证其对黄酮的O-甲基化催化功能。结果发现4个OMTs重组酶均具有 O-甲基化功能，是催化PMFs多个位点O-甲基化过程的关键基因，能直接形成柑橘特异性积累的PMFs。
利用原核表达体系重组上述4个候选基因的蛋白，通过体外添加含羟基的黄酮化合物，同样验证了4个基因对黄酮不同位点的O-甲基化修饰功能，可以合成多种黄酮O-甲基化衍生物。
在柑橘愈伤中超表达上述4个OMTs的悬浮系中添加黄酮底物，同样验证了其O-甲基化修饰功能，而在红橘幼苗叶片中瞬时干涉OMT3和OMT5后，发现3种及以上PMFs和 PMFs总含量显著降低，表明这些基因确实参与了PMFs的生物合成。
以上结果表明，本发明所涉及的四个基因具有多甲氧基黄酮的生物合成功能，能够通过转基因手段，将这些基因在柑橘愈伤组织中过表达，从而获得高产多甲氧基黄酮的转基因外植体。本发明在提高柑橘果实品质、功能性柑橘育种及工厂化生产PMFs方面应用前景广泛。
附图说明
图1：OMT3、OMT4和OMT5的氨基酸序列差异分析。
图2：HPLC分析酵母表达反应系统中OMTs候选基因催化O-甲基化产物，a图为酵母表达CrOMT3与芹菜素、芫花素和金合欢素反应产物HPLC色谱图；b图为CrOMT7与芹菜素反应产物HPLC色谱图；c图为酵母表达CrOMT3、CrOMT7催化芹菜素产生O-甲基化芹菜素衍生物的模型；图d、e和f分别为芫花素、金合欢素和7,4′-二甲氧基芹菜素标准品的二级质谱图。
图3：HPLC分析酵母表达反应系统提取物中CrOMT3、CrOMT4和CrOMT5催化产生的O-甲基化产物。
图4：SDS-PAGE分析大肠杆菌中表达候选基因的重组酶，M，蛋白marker；EV，空载。
图5：重组的CrOMT4和CrOMT5蛋白催化5-OH去甲川陈皮素、栀子黄素B和三裂尾草素，合成川陈皮素、橘黄酮和4′,5,6,7-四甲氧基黄酮。
图6：重组的CrOMT4和CrOMT5蛋白催化5,6-OH-4′,7-甲氧基黄酮，形成三裂尾草素和4′,5,6,7-四甲氧基黄酮。
图7：重组的CrOMT4和CrOMT5蛋白催化8-OH-六甲氧基黄酮，形成5,6,7,8,3′,4′-七甲氧基黄酮。
图8：重组的CrOMT4和CrOMT5蛋白催化半齿泽兰素-5-甲醚和去甲氧基矢车菊素后的产物。
图9：重组的CrOMT7蛋白催化芹菜素和芫花素的O-甲基化形成的产物。
图10：柑橘愈伤组织中超表达4个候选OMTs，图a为柑橘愈伤组织转基因过程；图b为明场与荧光显微镜下的柑橘愈伤；图c为柑橘愈伤转基因株系与野生型的表达量。
图11：柑橘愈伤中超表达候选OMTs悬浮体系添加芫花素和芹菜素的产物。图a为野生型伏令夏橙愈伤组织悬浮体系添加芫花素的产物；图b、c、d和e依次为为超表达OMT3、OMT7、OMT4和OMT5的愈伤组织悬浮体系添加芫花素的产物。
图12：柑橘愈伤中超表达候选OMTs悬浮体系添加5-OH-去甲川陈皮素和3′-OH-去甲川陈皮素的产物。图a、b和c分别为野生型伏令夏橙愈伤组织、超表达OMT4和超表达 OMT5愈伤悬浮体系添加5-OH-去甲川陈皮素和3′-OH-去甲川陈皮素的产物。
图13：瞬时干涉CrOMT3和CrOMT5的红橘叶片中OMTs表达量。
图14：瞬时干涉CrOMT3和CrOMT5的红橘叶片中PMFs的含量。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
1材料和方法
1.1实验材料
植物材料：盛花后120天的红橘的果实黄皮层。
试剂：底物饲喂实验中所用的类黄酮底物见表2和验证产物的标准品见表3，所有标准品均购置于上海源叶生物有限公司。
1.2实验方法
1.2.1 RNA提取和反转录
依照RN38-EASYspin Plus植物RNA试剂盒(Aidlab Biotech，中国)说明书从样品中提取总RNA。总RNA提取的样品为盛花后120天红橘的黄皮层。使用第一链cDNA合成试剂盒HiScript II 1st Stand cDNA Synthesis Kit(南京诺唯赞生物科技有限公司)，参照说明书将1μg总RNA进行反转录并合成cDNA，具体操作参照试剂盒说明书。
1.2.2基因克隆
依据克里曼丁橘基因组中OMTs的cDNA序列，设计扩增基因编码区(CDS)的PCR 引物，利用各种代表性柑橘种质黄皮层cDNA为模板进行PCR克隆，所用引物：
OMT3-F：GCAAAGCGTACGAGAAATTCAAG；
OMT3-R：GCATGGCCAAGCACTCTACTTAT。
OMT4-F：GCAAAGCGTAGGAGAAATTCAAGAAAC；
OMT4-R：TGAAATAATTTTTCTGCATCCTTTATTCAGAT。
OMT5-F：ATGGATTCTATAGTTGATGGAGAAAGAGAC；
OMT5-R：TTACAGAAGCAGAGGAAACAAAAGAATTTT。
OMT7-F：GATCAATCAGGCAAGCTTAATTT；
OMT7-R：GACTTGCGGGAATACGGTTACTC。
对PCR产物胶检并回收相应片段，用凝胶DNA回收试剂盒(Simgen，杭州)回收产物，并将其进行质粒连接、转化和测序。测序正确的阳性菌液，使用AxyPrep质粒DNA 小量试剂盒(Axygen，美国)提取质粒，用于载体构建。
1.2.3酵母表达系统和底物添加实验：根据pESC-URA载体的序列，选择ClaI酶切位点(ACTAGT)和SpeI酶(ATCGAT)，设计包含目的基因CDS区F/R端添加酶切位点与载体序列的引物进行PCR扩增，用ClaI酶和SpeI酶(Biolabs公司的产品)线性化pESC-URA 载体(Agilent，美国)，将PCR胶回收产物和双酶切回收产物进行一步法重组(诺唯赞，南京)反应，37℃反应30min，将反应产物用热激方法转化大肠杆菌DH5α，培养12h后，挑取单克隆，用GAL10F/R引物进行阳性检测，测序正确的菌液，提取质粒，即酵母表达载体构建完成。
OMT3/4/5共用引物：
正向引物OMT3/4/5-URA-F CTAAAGGGCGGCCGCACTAGTATGGATTCTATAGTTGA TG；
反向引物OMT3/4/5-URA-R：TCATCCTTGTAATCCATCGATCTACTTATAGAACTCCA TAA
OMT7正向引物：OMT7-URA-F CTAAAGGGCGGCCGCACTAGTATGGATGCGAATCAAGATCTAGG；
OMT7反向引物OMT7-URA-R：TCATCCTTGTAATCCATCGATTTATGGATAGACTTCAATGAGGGA；
阳性检测正向引物GAL10-F GGTGGTAATGCCATGTAATATG；反向引物 GAL10-R：GGCAAGGTAGACAAGCCGACAAC。通过小量法转化酵母，酵母体系中的底物饲喂实验参考Berim等(2018)方法，在原方法的提取类黄酮部分进行优化。
1.2.4原核表达和底物添加实验：根据pMAL-c2x载体序列，选择BamHI酶(GGATCC)和PstI酶(CTGCAG)，设计包含目的基因CDS区F/R端添加酶切位点与载体序列的引物进行PCR扩增，用BamHI酶和PstI酶(Biolabs)线性化pMAL-c2x载体，将PCR胶回收产物和双酶切回收产物进行一步法重组(诺唯赞，南京)反应，37℃反应30min，将反应产物用热激方法转化大肠杆菌DH5α，培养12h后，挑取单克隆，用P3′和P5′引物进行阳性检测，测序正确的菌液，提取质粒，将测序正确构建成功的质粒，以及未插入片段的空载质粒转入表达菌株(BL21(DE3)大肠杆菌感受态)中，用热激法转化，涂皿后37℃过夜培养，再挑取单克隆进行阳性检测。
正向引物pMal-c2X-OMT3/4/5/6-BamH GAAGGATTTCAGAATTCGGATCCATGGATTCTATAGTTGATG；
反向引物pMal-c2X-OMT3/4/5/6-PstI-R CCAGTGCCAAGCTTGCCTGCAGCTACTTATAGAACTCCATAA。
正向引物pMal-c2X-OMT7-BamH:GAAGGATTTCAGAATTCGGATCCATGGATGCGAATCAAGATCTA；
反向引物pMal-c2X-OMT7-PstI-R:CCAGTGCCAAGCTTGCCTGCAGTTATGGATAGACTTCAAT。
将测序正确的菌液质粒转入大肠杆菌BL21(DE3)表达菌株进行扩大培养，经IPTG诱导后，将菌液进行细胞破碎、蛋白纯化和蛋白浓缩，获得纯化后的目的蛋白用于底物添加实验。
1.2.5柑橘愈伤组织遗传转化
以红橘果皮黄皮层cDNA为模板，扩增得到候选基因的编码区全长序列，通过Gateway 方法构建带GFP标签的pH7WG2D超量表达载体干涉载体后，采用根癌农杆菌(EHA105) 侵染法转入柑橘愈伤组织中，筛选得到超量表达的转基因单系。设置对照为空白载体转基因株系。
载体正向接头引物Adapter-attB1：GGGGACAAGTTTGTACAAAAAAGCAGGCTCC；
反向接头引物Adapter attB1：GGGGACCACTTTGTACAAGAAAGCTGGGTT。
OMT3/4/5共用的正向接头引物attB-OMT3-F：AAAAAGCAGGCTCCATGGATTCTATAGTTGATGGAGAAAGAGAC；
反向接头引物attB-OMT3-R：AGAAAGCTGGGTTCTACTTATAGAACTCCATAACCCAGAAATTACC。
OMT7正向接头引物attB-OMT7-F：AAAAAGCAGGCTCCATGGATGCGAATCAAGAT CTA；
反向接头引物attB-OMT7-R：AGAAAGCTGGGTTTTATGGATAGACTTCAATGAGGG A。
参照曹洪波(2012)的方法，侵染含CrOMT3、CrOMT4、CrOMT5和CrOMT7超表达载体和空载的伏令夏橙愈伤组织，在加头孢霉素和潮霉素的MT培养基上持续培养40-50 d，待新的阳性愈伤组织长出，并经阳性鉴定后，即可大量扩繁，并收集转基因愈伤组织用于基因定量和代谢物检测分析。柑橘愈伤组织悬浮体系建立和饲喂黄酮底物：参考烟草B Y2悬浮体系(陈嘉景，2017)。
1.2.6红橘幼苗叶片瞬时表达
通过PCR克隆得到目的基因编码区400bp到500bp长的保守序列，经Gateway反应体系转入pK7GWIWG2D(ii)二元载体中，该载体包含CAMV-35S启动子区域和一个GFP 报告基因。重组载体转入到GV3101-pSoup-P19系列根癌农杆菌中。
OMT3、OMT4和OMT5共用正向接头引物attB-RNAi-OMT3/4/5-F：GGGGACAAGTT TGTACAAAAAAGCAGGCTCCGCTTCAGATATTGCAGCCCA；
反向接头引物attB-RNAi-OMT3/4/5-R：GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGGTAGTGATGGCTTGAAGAGT。
参照石梅艳博士毕业论文(2020)瞬时注射柑橘果实的方法注射红橘幼苗叶片。
2结果与分析
2.1柑橘中PMFs合成相关OMTs的克隆
以红橘果实黄皮层cDNA为材料，克隆出Ciclev10031951m.g(CrOMT3)、Ciclev10031949m.g(CrOMT4)、Ciclev10031952m.g(CrOMT5)的编码区序列，均与克里曼丁基因组中参考序列一致，有意思的是这三个基因具有高度相似的序列，氨基酸同一性超过92％(表 1)，CDS长度均为1062bp，3个基因序列全长与克里曼丁橘参考基因组中参考序列一致，编码353个氨基酸，将上述4个基因的氨基酸进行比较(图1)，发现CrOMT4、CrOMT5 与CrOMT3分别有27和20个氨基酸的差异，氨基酸同一性高达91％(表2)。CrOMT7 的CDS长度为1071bp，编码356个氨基酸。
表1柑橘中3种OMT的氨基酸同一性百分比
基因Ciclev10031951m.g(CrOMT3)、Ciclev10031949m.g(CrOMT4)、Ciclev10031952 m.g(CrOMT5)、Ciclev10005276m.g(OMT7)的核苷酸酸序列分别如SEQ IDNo.1-4所示，基因编码的氨基酸序列分别如SEQ ID No.5-8所示。
2.2PMFs生物合成关键基因生物学功能验证
2.2.1酵母表达与底物饲喂体系初步验证候选基因O-甲基化活性
基于以上优化后的酵母表达系统，本研究中将转录组筛选到的4个候选OMTs均用酵母进行异源表达并通过添加5-、6-和4′-位含3个羟基的芹菜素初步验证其对黄酮的O-甲基化催化功能。结果发现，酵母表达CrOMT3(Ciclev10031951m.g)能催化底物芹菜素形成3个新的色谱峰P2、P3和P4，经LC-MS二级碎片离子推测为芫花素、金合欢素和7,4′-二甲氧基芹菜素(图2d，e，f)，并用标准品进行了验证；酵母表达CrOMT3也能催化芫花素和金合欢素O-甲基化形成7,4′-二甲氧基芹菜素(图2a)。以上结果初步表明CrOMT3对黄酮7-,4′-位点具有O-甲基化催化活性。
酵母表达CrOMT7同样能催化芹菜素7-,4′-O-甲基化形成芫花素和7,4′-二甲氧基芹菜素，而不能形成金合欢素(图2b)，表明CrOMT7对黄酮7-,4′-位O-甲基化具有先后顺序或具有偏好性。
对CrOMT4(Ciclev10031949m.g)和CrOMT5(Ciclev10031952m.g)也运用酵母进行了异源表达和底物饲喂，以添加底物石吊兰素为例，表达CrOMT3的酵母菌还能催化石吊兰素7-位O-甲基化形成栀子黄素B，却不能继续催化栀子黄素B的5-位进一步的O-甲基化(图3)，而表达CrOMT4和CrOMT5的酵母菌则能将石吊兰素5-位和7-位均O-甲基化形成柑橘中特异积累的橘黄酮和栀子黄素B(图3)。
综上所述，本研究通过酵母异源表达OMTs初步验证了4个OMTs：CrOMT3、CrOMT4、CrOMT5和CrOMT7重组酶的O-甲基化功能，尤其是CrOMT4、CrOMT5对石吊兰素5- 位和7-位的O-甲基化功能催化功能，能直接形成2种柑橘特异性积累的PMFs，表明其可能是催化PMFs多个位点O-甲基化过程的基因，可作为关键基因做下一步验证。
2.2.2原核表达重组候选OMTs基因蛋白和底物孵育实验初步构建红橘PMFs生物合成途径
因酵母反应体系产物较少，且所需底物量较大，因此本研究中利用原核表达体系重组上述4个已验证对黄酮具有O-甲基化功能的候选基因的蛋白：CrOMT3、CrOMT4、CrOMT5、CrOMT7。本研究成功利用pMal-C2x载体构建了以上四个基因的原核表达载体，经原核表达在破碎菌体的上清溶液中表达出这4个蛋白，且经纯化后获得了浓度和纯度较高的重组蛋白(图4)。通过体外添加含羟基的黄酮化合物(表2)，经甲醇提取后HPLC检测同样验证其中4个基因对黄酮不同位点的O-甲基化修饰功能，可以合成多种黄酮O-甲基化衍生物。
表2底物饲喂实验中的类黄酮化合物的结构
当添加柑橘中特异积累的3种5-OH黄酮底物：5-OH去甲川陈皮素、栀子黄素B和三裂尾草素，重组的CrOMT4和CrOMT5蛋白催化黄酮5-位点的O-甲基化功能，能一步合成三种柑橘特异性PMFs：川陈皮素、橘黄酮和4′,5,6,7-四甲氧基黄酮(图5)。当添加 5,6-OH-4′,7-甲氧基黄酮，重组的CrOMT4和CrOMT5蛋白能催化黄酮5-，6-位的O-甲基化，催化形成柑橘中的PMFs：三裂尾草素和4′,5,6,7-四甲氧基黄酮(图6)。当添加柑橘中特异积累的8-OH-六甲氧基黄酮后，重组的CrOMT4和CrOMT5蛋白能催化黄酮8-位的 O-甲基化，能催化形成柑橘特异积累的5,6,7,8,3′,4′-七甲氧基黄酮(图7)。当分别添加3′- 位含-OH的半齿泽兰素-5-甲醚后和5-,7-,3′-位含3个-OH的去甲氧基矢车菊素黄酮后， CrOMT4和CrOMT5重组蛋白能催化半齿泽兰素-5-甲醚形成柑橘特异积累的甜橙黄酮，而去甲氧基矢车菊素黄酮则被催化产生5个新的色谱峰，产物之一为甜橙黄酮(图8)，推测其他产物为不同组合的去甲氧基矢车菊素黄酮的O-甲基化衍生物。
当添加芹菜素，重组的CrOMT7蛋白能催化其4′-位的O-甲基化形成芫花素。当添加芫花素，重组的CrOMT7蛋白能催化其7-位的O-甲基化形成7,4′-二甲氧基芹菜素(图9)，表明CrOMT7蛋白对黄酮具有7-和4′-位的O-甲基化催化功能。
当添加更多位点含有-OH的黄酮后，检测其产物发现CrOMT4和CrOMT5重组蛋白对黄酮5-、6-、7-、8-、3′-和4′-位点具有O-甲基化功能，催化羟基化黄酮合成相应的O-甲基化衍生物(表3)。
表3重组OMT4和OMT5对各种类黄酮底物的催化产物
注：-表示无产物
Note，-represents no product
2.2.3利用柑橘愈伤组织悬浮体系底物饲喂实验验证OMTs基因功能
基于体外验证功能的4个候选OMTs，本研究利用实验室保存的伏令夏橙愈伤组织为材料，柑橘PMFs代谢谱中已测定该愈伤组织中不积累PMFs，且前期经qRT-PCR验证其中4个候选OMTs在柑橘愈伤组织中表达量较低。因此将连接GFP报告基因的CrOMT3、 CrOMT4、CrOMT5、CrOMT7基因在伏令夏橙愈伤中超表达(图10a)。经GFP荧光筛选和qRT-PCR验证(图10b)，已经获得CrOMT3、CrOMT4、CrOMT5和CrOMT7三个基因阳性的转基因系，各个转基因愈伤阳性系中基因超表达倍数达十几倍甚至100倍以上(图 10c)，随后进行扩大培养，以获得大量材料用于随后的类黄酮检测和柑橘悬浮体系的构建。
首先，本研究对野生型(伏令夏橙)和4个超表达系愈伤组织中类黄酮进行了提取，因愈伤组织中类黄酮含量较低，因此采取浓缩提取的方法以此提高类黄酮代谢物的检测范围，结果发现野生型和超表达株系中均未产生新的类黄酮代谢物，推测可能是柑橘愈伤组织中缺少多种中间体类黄酮物质而无法进行类黄酮修饰过程中比较靠后的O-甲基化修饰反应。因此，借鉴烟草BY2悬浮体系和饲喂类黄酮底物的方法(陈嘉景，2017)建立柑橘悬浮体系结合饲喂多种含-OH的黄酮底物的反应体系。因柑橘愈伤组织生长较烟草愈伤组织慢，因此将初悬浮10d后的愈伤组织分成相同质量的小瓶继续进行悬浮培养，在小瓶悬浮 4d后添加底物，继续悬浮6d后进行HPLC检测。
在CrOMT5-OX柑橘悬浮系中饲喂芹菜素后，检测产物仅发现少量芫花素产生。前人研究表明柑橘愈伤中主要积累黄酮醇类(刘朝阳2016)，且在柑橘愈伤中黄酮7-位易发生糖基化和酰基化(陈嘉景2017)，结合以上柑橘愈伤超表达OMT3悬浮系统中添加芹菜素的结果，因此推测饲喂的黄酮底物大多流向糖基化和酰基化的修饰过程中，而导致仅极少量底物能被O-甲基化。基于CrOMT3体外的酶功能，推测CrOMT3-OX柑橘悬浮系中产生的是芹菜素4′-位O-甲基化衍生物。
考虑到以上实验结果中产生的黄酮O-甲基化产物的不确定性且产量较低，而黄酮7- 位相较于其他位点更容易O-甲基化，因此本研究尝试饲喂黄酮7-位点具有-OCH3的底物，将该位点保护起来，进而验证黄酮其他位点的O-甲基化功能。结果发现：在CrOMT3-OX、CrOMT4-OX、CrOMT5-OX和CrOMT7-OX柑橘悬浮系中添加芫花素后，均能产生7,4′-二甲氧基芹菜素(图11a，b，c，d)，从而在柑橘愈伤组织中验证了CrOMT3、CrOMT4、CrOMT5 和CrOMT7的4′位O-甲基化功能。
在CrOMT5-OX和CrOMT4-OX柑橘悬浮系中饲喂5-OH-去甲川陈皮素和3′-OH-去甲川陈皮素后，均检测到新的色谱峰产生，经标准品验证为川陈皮素(图12)，即在柑橘愈伤体内验证了CrOMT4和CrOMT5对黄酮3′-和5-位的O-甲基化功能，尤其是柑橘 CrOMT5-OX悬浮系能大量生产柑橘果实中积累最高的川陈皮素。
2.2.4柑橘幼苗叶片瞬时表达OMTs
选取能积累PMFs的30d苗龄的红橘实生幼苗作为材料。红橘成年态实生苗叶片中能检测到7种主要积累的PMFs，但在30d和60d红橘叶片中主要能检测到6种PMFs。将构建的CrOMT3和CrOMT5瞬时干涉表达载体，经根癌农杆菌侵染注射进柑橘叶片，待生长7d后取样，采用HPLC检测叶片中PMFs的含量，并同时检测叶片中对应的2个OMTs 的表达量。
由于CrOMT4和CrOMT5序列高度相似，无法设计特异引物，因此设计了一对能同时定量CrOMT4和CrOMT5基因表达量的引物。结果发现在CrOMT3和CrOMT5的瞬时干涉系叶片中，两个OMTs表达量显著降低(图13)，均有3种或以上的PMFs含量显著降低且 PMFs的总含量显著低于对照(图14)，表明CrOMT3和CrOMT5参与了PMFs的生物合成。
参考文献
1.曹洪波.转基因调控柑橘类胡萝卜素积累的细胞学和代谢研究.武汉：华中农业大学图书馆，2012.
2.陈嘉景.柑橘中类黄酮新橘皮糖苷代谢关键基因分离和功能分析.武汉：华中农业大学图书馆，2017.
3.刘朝阳.甜橙CsMYBF1基因的功能鉴定和调控机理研究.[博士学位论文].武汉：华中农业大学图书馆，2016.
4.石梅艳.CgMYB58对华农红柚汁胞木质素和类胡萝卜素生物合成的调控机理.[博士学位论文].武汉：华中农业大学图书馆，2020.
5.Berim A&Gang DR.Production of methoxylated flavonoids in yeastusing ring A hydroxylases and flavonoid O-methyltransferases from sweetbasil.Applied microbiology and biotechnology,2018,102(13),5585-5598.
6.Itoh N,Iwata C&Toda H.Molecular cloning and characterization of aflavonoid-O-methyltransferase with broad substrate specificity andregioselectivity from Citrus depressa.BMC plant biology,2016,16(1),1-13.
7.Zhao X,Xing T,Li YF,Jiao B&Jiang D.Efficient analysis ofphytochemical constituents in the peel of Chinese wild citrus Mangshanju(Citrus reticulata Blanco)by ultra high performance liquid chromatography-quadrupole time-of-flight-mass spectrometry.Journal of separation science,2018,41(9),1947-1959。
序列表
<110> 华中农业大学
<120> 柑橘多甲氧基黄酮生物合成基因及其应用
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Ser Val Val Glu Cys Ser Leu Asp Ala Ser Gly Ala Arg Arg Leu Tyr
85 90 95
Ser Leu Asn Ser Val Ser Lys Tyr Tyr Val Pro Asn Lys Asp Gly Val
100 105 110
Leu Leu Gly Pro Leu Leu Gln Met Asn Gln Asp Lys Val Leu Leu Glu
115 120 125
Ser Trp Ser Gln Leu Lys Asp Ala Ile Leu Glu Gly Gly Ile Pro Phe
130 135 140
Asn Arg Ala His Gly Val His Val Phe Glu Tyr Ala Gly Leu Asp Pro
145 150 155 160
Arg Phe Asn Lys His Phe Asn Thr Ala Met Tyr Asn Tyr Thr Ser Leu
165 170 175
Val Met Ser Asn Ile Leu Glu Ser Tyr Lys Gly Phe Asp Asn Ile Lys
180 185 190
Gln Leu Val Asp Val Gly Gly Ser Leu Gly Val Thr Leu Gln Ala Ile
195 200 205
Thr Thr Lys Tyr Pro Tyr Ile Lys Gly Ile Asn Phe Asp Gln Pro His
210 215 220
Val Ile Asp His Ala Pro Ser His Pro Arg Ile Glu His Val Arg Gly
225 230 235 240
Asp Met Phe Gln Ser Val Pro Lys Gly Asp Ala Ile Phe Met Lys Ser
245 250 255
Val Leu His Asp Trp Asn Asp Glu His Cys Leu Lys Leu Leu Lys Asn
260 265 270
Cys Tyr Lys Ser Ile Pro Glu Asp Gly Lys Val Ile Val Val Glu Ser
275 280 285
Met Leu Pro Glu Val Pro Asn Thr Ser Ile Glu Ser Lys Ser Asn Ser
290 295 300
His Leu Asp Val Leu Met Met Ile Gln Ser Pro Gly Gly Lys Glu Arg
305 310 315 320
Thr Arg His Glu Phe Met Thr Leu Ala Thr Gly Ala Gly Phe Gly Gly
325 330 335
Ile Ser Cys Glu Leu Ala Ile Gly Asn Leu Trp Val Met Glu Phe Tyr
340 345 350
Lys
<210> 8
<211> 356
<212> PRT
<213> 柑橘(Citrus reticulata Blanco)
<400> 8
Met Asp Ala Asn Gln Asp Leu Gly Ala Lys Glu Leu Phe Gln Gly Gln
1 5 10 15
Ala Gln Leu Tyr Lys Leu Met Phe Asn His Leu Ser Ser Met Ser Leu
20 25 30
Lys Cys Ala Ile Glu Leu Gly Ile Ala Asp Ile Ile His Ser His Gly
35 40 45
Arg Ala Ile Thr Leu Ser Glu Leu Val Ser Ala Leu Asp Ile Gln Pro
50 55 60
Thr Lys Thr Thr Gly Leu Phe Arg Leu Met Arg Leu Leu Val His Ser
65 70 75 80
Gly Cys Phe Asn Lys Thr Lys Val Asn Gly Gln Glu Glu Ala Tyr Gly
85 90 95
Leu Thr Ala Ala Ser Thr Leu Leu Ile Lys Asp Lys Pro Tyr Cys Leu
100 105 110
Ser Pro Thr Val Ser Val Phe Leu Asp Pro Cys Phe Val Ala Ala Phe
115 120 125
Gln Ser Leu Gly Ser Trp Phe Lys Gly Thr Glu Leu Thr Leu Trp Glu
130 135 140
Thr Val His Gly Ile Lys Phe Trp Glu Phe Met Asn Gln Asn Pro Gly
145 150 155 160
Ile Asn Gln Arg Phe Asn Glu Ala Met Ala Ser Asp Thr Glu Ile Leu
165 170 175
Thr Ser Phe Val Val Lys Ala Glu Cys Lys Gln Ile Phe Glu Gly Leu
180 185 190
Gly Ser Leu Val Asp Val Gly Gly Gly Asn Gly Ser Leu Ser Arg Ile
195 200 205
Ile Ser Glu Ala Phe Pro Gly Ile Lys Cys Thr Val Leu Asp Leu Pro
210 215 220
His Val Val Ala Asn Leu Pro Glu Ala Asp Asn Leu Lys Tyr Ile Ala
225 230 235 240
Gly Asp Met Phe Gln Phe Ile Pro Pro Ala Asp Ala Phe Leu Phe Lys
245 250 255
Leu Ile Phe His Gly Leu Gly Asp Glu Asp Gly Leu Lys Ile Leu Lys
260 265 270
Lys Arg Arg Glu Ala Ile Ala Ser Asn Gly Lys Arg Gly Lys Val Ile
275 280 285
Ile Ile Asp Ile Val Ile Asn Ala Glu Glu Glu Glu Pro Glu Leu Thr
290 295 300
Glu Thr Lys Phe Leu Phe Asp Ile Leu Met Ser Val Asn Ala Asn Gly
305 310 315 320
Lys Glu Arg Thr Glu Ser Glu Trp Ala Lys Leu Phe Ser Asp Ala Gly
325 330 335
Phe Ser His Tyr Lys Ile Thr Pro Ile Phe Gly Met Arg Ser Leu Ile
340 345 350
Glu Val Tyr Pro
355
Claims (6)
1.柑橘多甲氧基黄酮生物合成基因，核苷酸序列如SEQ ID No.1或SEQ ID No.2或SEQID No.3或SEQ ID No.4所示。
2.权利要求1所述基因编码的蛋白，氨基酸序列分别如SEQ ID No.5-8所示。
3.含有权利要求1所述基因的表达载体。
4.含有权利要求1所述基因的宿主菌。
5.权利要求1所述的基因、权利要求2所述的蛋白、权利要求3所述的表达载体、权利要求4所述的宿主菌在柑橘多甲氧基黄酮生物合成中的应用。
6.一种柑橘多甲氧基黄酮生物合成方法，其特征在于：通过转基因手段，将权利要求1所述的基因在柑橘愈伤组织中过表达，获得转基因外植体。
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|---|---|---|---|---|
| CN107099559A (zh) * | 2016-02-22 | 2017-08-29 | 西姆莱斯有限公司 | 生物技术制备甲基化的肉桂酸和肉桂酸酯、甲基化的苯乙胺和其偶联产物的方法 |
| CN109321543A (zh) * | 2018-11-02 | 2019-02-12 | 浙江大学 | 参与柑橘果皮黄酮合成的氧甲基转移酶及其编码基因与应用 |
| CN110699399A (zh) * | 2019-10-22 | 2020-01-17 | 浙江大学 | 柑橘氧甲基转移酶CitOMT2的体外酶活应用 |
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| CN107099559A (zh) * | 2016-02-22 | 2017-08-29 | 西姆莱斯有限公司 | 生物技术制备甲基化的肉桂酸和肉桂酸酯、甲基化的苯乙胺和其偶联产物的方法 |
| CN109321543A (zh) * | 2018-11-02 | 2019-02-12 | 浙江大学 | 参与柑橘果皮黄酮合成的氧甲基转移酶及其编码基因与应用 |
| CN110699399A (zh) * | 2019-10-22 | 2020-01-17 | 浙江大学 | 柑橘氧甲基转移酶CitOMT2的体外酶活应用 |
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