CN114634939A - 一种调节人参中茉莉酸甲酯合成的PgJMT1基因及其应用 - Google Patents
一种调节人参中茉莉酸甲酯合成的PgJMT1基因及其应用 Download PDFInfo
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- CN114634939A CN114634939A CN202210491395.4A CN202210491395A CN114634939A CN 114634939 A CN114634939 A CN 114634939A CN 202210491395 A CN202210491395 A CN 202210491395A CN 114634939 A CN114634939 A CN 114634939A
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- pgjmt1
- gene
- ginseng
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- methyl jasmonate
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Abstract
本发明提供一种调节人参中茉莉酸甲酯合成的PgJMT1基因及其应用，该调节人参中茉莉酸甲酯合成的PgJMT1基因来自人参，PgJMT1基因序列如SEQID No.1所示，PgJMT1基因编码蛋白氨基酸序列如SEQ ID No.2所示。该PgJMT1基因编码的蛋白具有明显的茉莉酸羧甲基转移酶特有功能基因序列和典型的S‑腺苷‑L‑蛋氨酸结合域，属于茉莉酸甲基转移酶(JMT)，是MeJA合成途径中催化JA合成MeJA的关键酶，控制着植物体内茉莉酸类物质的转化。将构建的PgJMT1基因超表达载体通过农杆菌介导转化人参叶片，适量表达PgJMT1基因的人参叶片中MeJA和人参皂苷的含量均显著提高。本发明在人参中利用PgJMT1基因提高人参皂苷产量和改善人参品质方面具有潜在的应用价值。
Description
技术领域
本发明涉及生物基因工程技术领域，具体涉及一种调节人参中茉莉酸甲酯合成的PgJMT1基因及其应用。
背景技术
人参为五加科人参属植物，是我国名贵的中药材，在许多亚洲国家已经有几千年的使用历史，人参中的次生代谢产物人参皂苷是其最主要的药效成分。茉莉酸及其衍生物如茉莉酸甲酯(methyl jasmonate，MeJA)和茉莉酸异亮氨酸复合物(jasmonoyl-L-isoleucine，JA-lle)等均属于茉莉酸类物质(jasmonates，JAs)，其中MeJA和JA-Ile是最具活性的信号分子。研究表明，MeJA作为环境信号分子对调控植物次生代谢的影响十分显著，可被当作外源性诱导剂调控植物特定的代谢途径，也可以通过调控植物次生代谢产物的方式响应防御反应。研究表明，MeJA由于其可以诱导三萜人参皂苷生物合成中一系列合成酶基因的表达，常被外源添加用于提高人参皂苷含量的研究。然而，由于植物生长代谢的复杂性，施加外源性MeJA的时机和量难以精确控制，极易对植物细胞的生长产生抑制，进而影响人参皂苷的产量。同时，外源施加MeJA等JAs不适用于大规模的田间种植。如何通过分子生物学水平调节人参内源性MeJA的合成，进而实现持久高效的人参皂苷合成与积累，以提高人参皂苷含量和人参的药用价值，对于精准调控人参皂苷的积累、规模化高效生产人参皂苷具有重要的应用价值。
发明内容
为解决上述技术问题，本发明提出了一种调节人参中茉莉酸甲酯合成的PgJMT1基因及其应用，其目的是通过筛选的PgJMT1基因转化人参组织并表达，从而促进人参细胞内源性MeJA含量增加，以内源性的MeJA调节人参皂苷的大量合成与积累，达到提高人参皂苷产量和提升人参品质的目的。
为了实现上述目的，本发明首先提供了一种调节人参中茉莉酸甲酯合成的PgJMT1基因，所述调节人参中茉莉酸甲酯合成的PgJMT1基因如SEQ ID No.1所示，所述调节人参中茉莉酸甲酯合成的PgJMT1基因来自人参。
作为优选，所述调节人参中茉莉酸甲酯合成的PgJMT1基因编码的蛋白氨基酸序列如SEQ ID No.2所示。
作为优选，所述调节人参中茉莉酸甲酯合成的PgJMT1基因的扩增引物序列如SEQID No.3和SEQ ID No.4所示。
作为优选，所述调节人参中茉莉酸甲酯合成的PgJMT1基因与表达载体组成重组载体。
作为优选，所述表达载体为pCAMBIA1302。
作为优选，所述重组载体的构建方式如下：以所述调节人参中茉莉酸甲酯合成的PgJMT1基因的开放读码框作为超表达序列，将该cDNA片段插入到植物表达载体pCAMBIA1302中制得。
基于一个总的发明构思，本发明还提供了一种调节人参中茉莉酸甲酯合成的PgJMT1基因在调节人参皂苷合成与积累中的应用。
作为优选，所述应用方式为利用农杆菌介导所述调节人参中茉莉酸甲酯合成的PgJMT1基因转化人参组织。
与现有技术相比，本发明具有以下有益效果：
本发明采用MeJA诱导的人参根转录组测序方法，初步筛选出人参JMT家族基因，再通过体外表达实验和发根农杆菌A4介导的转化人参叶片实验，从多个人参JMT基因中筛选到具有催化JA为MeJA的基因PgJMT1，PgJMT1基因编码的蛋白具有明显的茉莉酸羧甲基转移酶特有功能基序和典型的S-腺苷-L-蛋氨酸结合域，属于植物茉莉酸甲基转移酶JMT。人参PgJMT1基因编码蛋白能够催化JA合成MeJA，通过筛选的PgJMT1基因及其编码的蛋白调控人参内源性MeJA含量，以内源性的MeJA调节人参皂苷生物合成与积累，达到提高人参皂苷产量和提升人参品质的目的。该基因及其编码的蛋白是一种高效、特异性和切实可行的提高人参皂苷的方法。
本发明通过构建了PgJMT1基因的植物超表达载体，利用发根农杆菌A4介导PgJMT1基因转化人参叶片，通过PgJMT1基因在人参叶片中高水平表达，与对照人参叶片相比，获得的PgJMT1基因瞬时超表达人参叶片中人参总皂苷含量显著提高。由此说明，利用基因编辑或超表达PgJMT1基因能够获得人参总皂苷含量显著提高的人参组织或植株，为提高人参品质或提高人参总皂苷产量提供了一种高效的技术手段。
附图说明
图1是本发明实施例1中PgJMT1基因PCR产物电泳结果，图中，泳道1表示PCR扩增产物，M表示DNA标准分子量；
图2是本发明实验例1中荧光定量PCR(qRT-PCR)检测100μmol/L外源性MeJA处理不同时间后人参发根中PgJMT1基因的表达水平，以β-actin作为内参；
图3是本发明实验例2中PgJMT1基因原核表达载体表达框示意图；
图4是本发明实验例2中PgJMT1基因在大肠杆菌中表达的蛋白SDS-PAGE结果；
图5是本发明实验例3中PgJMT1重组蛋白及其催化活性测定结果，其中图5A和图5B为JA和MeJA标准品的UPLC-MS/MS分析结果；图5C和图5D为含有PgJMT1重组蛋白的粗提液与JA反应的产物，经UPLC-MS/MS分析后的结果；图5E和图5F为含有空载体的菌液提取的蛋白粗提液与JA反应产物的UPLC-MS/MS分析结果；
图6是本发明实验例4中PgJMT1基因植物超载体表达框示意图；
图7是本发明实验例4中qRT-PCR检测发根农杆菌A4介导转化人参叶片中PgJMT1基因表达水平；
图8是本发明实验例4中发根农杆菌A4介导转化PgJMT1基因的人参叶片中MeJA含量；
图9是本发明实验例4中发根农杆菌A4介导转化PgJMT1基因的人参叶片中人参皂苷含量。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
实施例1
PgJMT1基因的克隆
1、人参RNA提取及其反转录合成cDNA
(1)人参RNA提取
取1/2MS固体培养基中培养的人参发根，将其接种于1/2MS液体培养基中，在120rpm、25℃暗培养3周，然后在培养基中加入终浓度为100μmol/L的MeJA，再以同样的条件培养24h，诱导与MeJA有关的基因表达。
取MeJA诱导24h的人参发根，快速置于液氮预冷的研钵内，立即加入液氮后快速研磨成细粉。取40mg于1.5mL无RNA酶的离心管中，加入1mL TRIzol和40μLβ-巯基乙醇，快速混匀后置于室温放置5-10min；加入0.2mL氯仿，振摇l5s，室温放置10min；4℃，12000×g离心15min，收集上层置于于1.5mL离心管中，弃去沉淀；再加入0.4mL 3mol/L醋酸铵(pH5.2)和0.6mL异丙醇后混匀，室温放置10min，4℃、12000×g离心10min，弃去上清；加入1mL 75％的乙醇，混匀；4℃，10000×g离心5min，弃去上清液，再加入20μL DEPC水溶解RNA，备用。
(2)反转录合成cDNA
以oligod(T)18为引物，利用逆转录酶合成cDNA第一链，反转录反应体系见表1，按表1体系加入各组分后，轻柔搅拌混匀；室温放置10min后，移至恒温水浴箱，42℃反应1h；反应结束后，快速置于冰上冷却2min，最后置于-20℃备用。
表1反转录反应体系
| 组分 | 加样量 |
| Template mRNA(200ng/μL) | 10μL |
| 5×1st strand synthesis buffer | 4μL |
| dNTP mixture(10mmol/L) | 1μL |
| RNase inhibitor | 1μL |
| oligo(dT)(50μmol/L) | 2μL |
| M-MLV(200U/μL) | 1μL |
| RNase-free H<sub>2</sub>O | 1μL |
| 总体积 | 20μL |
2、PCR扩增PgJMT1基因
根据MeJA诱导的人参转录组测序所获得的候选PgJMT1基因序列信息，以PrimerPremier 5软件设计PCR扩增引物，PgJMT1基因PCR扩增引物如下。
PgJMT1-F：5′-ATGGATTCTGAGAAAGTTTCTGC-3′；(SEQ ID No.3)
PgJMT1-R：5′-TCATAATTTCCTAAGAAC-3′；(SEQ ID No.4)
PCR扩增PgJMT1基因的反应条件见表2。PCR产物以琼脂糖凝胶电泳进行分析。
表2 PCR反应条件
图1为PgJMT1基因PCR产物经1％琼脂糖凝胶电泳后的结果，图1中泳道1表示PCR扩增产物，M表示DNA标准分子量，结果显示PCR产物大小约为1119bp，与预期的理论大小一致。
3、PgJMT1基因的亚克隆及其测序分析
将上述PCR产物中大小约为1119bp的电泳条带胶回收，再将胶回收产物连接至pGEM-T Easy亚克隆载体，转化感受态大肠杆菌DH5α，经筛选后，提取阳性菌落的质粒，对质粒进行测序，测序结果在NCBI中Blast进行比对分析，结果显示该基因编码的蛋白具有明显的茉莉酸羧甲基转移酶特有功能基序和典型的S-腺苷-L-蛋氨酸结合域，属于植物茉莉酸甲基转移酶JMT，通过分子对接分析也证明获得了PgJMT1基因。
实验例1
荧光定量PCR(qRT-PCR)分析PgJMT1基因的表达水平
1、RNA提取及反转录
取1/2MS固体培养基中25℃暗培养3周后的发根，接种于1/2MS液体培养基中25℃、110rpm暗培养21d，再加入100μmol/L MeJA进行诱导处理，处理不同时间后分别取出发根，用于提取RNA，提取方法同实施例1。上述RNA以oligod(T)18为引物，以逆转录酶合成cDNA。β-actin和PgJMT1基因的qRT-PCR分析引物如下：
β-actin荧光定量引物F：5′-TGCCCCAGAAGAGCACCCTGT-3′；(SEQ ID No.5)
β-actin荧光定量引物R：5′-AGCATACAGGGAAAGATCGGCTTGA-3′；(SEQ ID No.6)
PgJMT1荧光定量引物F：5′-TGGCTCAGGACACAACACTT-3′；(SEQ ID No.7)
PgJMT1荧光定量引物R：5′-TCGTTTAGGTACACGCGGAA-3′；(SEQ ID No.8)
2、qRT-PCR分析PgJMT1基因表达水平
以CFX Connect荧光定量PCR仪对PgJMT1基因表达水平进行分析检测，按照SYBRPremix Ex Taq荧光定量PCR试剂盒进行扩增，qRT-PCR反应体系见表3，反应条件见表4。
表3 qRT-PCR反应体系
表4 qRT-PCR反应条件
每个样品三次重复。反应结束后确认扩增曲线和溶解曲线，采用2-ΔΔCt方法计算PgJMT1基因的表达水平差异。图2是qRT-PCR检测100μmol/L外源性MeJA处理不同时间后人参发根中PgJMT1基因的表达水平结果，以β-actin作为内参。结果显示人参发根中PgJMT1基因的表达水平受MeJA诱导后会显著提升，当MeJA处理48h后，PgJMT1基因表达水平最高，是对照人参发根中表达水平的2.48倍，随后PgJMT1基因的表达水平下降，表明PgJMT1基因与MeJA介导的信号通路有关。
实验例2
PgJMT1基因原核表达载体构建及其诱导表达
1、PgJMT1基因原核表达载体构建(载体表达框示意图如图3所示)
PCR扩增PgJMT1，回收PCR产物，用同源重组方式将PgJMT1基因连接至pET32a载体。将连接产物转化E.coli DH5α，涂布在含氨苄青霉素的LB固体培养基上37℃培养过夜。挑取阳性克隆进行菌落PCR检测，并测序验证。
2、PgJMT1重组蛋白诱导表达
(1)挑取含PgJMT1基因的阳性重组菌的单菌落，接种于2mL含氨苄青霉素的LB液体培养基中，180rpm、37℃活化过夜后，按照1∶100比例接种到100mL LB培养基中，180rpm、37℃培养使OD600至0.6-0.8，加入IPTG使其终浓度为0.4mmol/L，并在20℃条件下诱导表达，然后每诱导2h取样一次，从而确定获得重组蛋白表达的最佳诱导时间。
(2)诱导表达结束后，取1mL菌液，4℃、12000rpm离心5min，收集菌体；用去离子水洗涤两次；加入200μL细胞裂解液，4℃反应10min，然后放入冰箱-20℃放置30min，37℃热激10min，待溶液呈澄清，12000rpm离心30min，取上清。
(3)将上清液加入2×SDS上样缓冲液，煮沸10min；4℃、12000rpm离心1min；取上清10μL上样。配制10％的分离胶和5％的浓缩胶，进行SDS-PAGE凝胶电泳检测分析。
图4是IPTG诱导后的PgJMT1蛋白表达情况电泳图，1-9泳道表示终浓度为0.4mmol/L IPTG诱导0、2、4、6、8、10、12、14和16h后的菌液提取的总蛋白，泳道10为转空载体的作为对照菌提取的总蛋白，M表示蛋白质标准分子量；结果显示诱导时间对PgJMT1蛋白的表达有较为明显的影响，在4h时该蛋白已有很明显的表达，其表达量随着时间增加而呈现逐渐增加的趋势，在12h时蛋白的表达量达到较高水平。
实验例3
1、PgJMT1重组蛋白活性测定
(1)取IPTG诱导14h后的重组菌提取蛋白，以转化空载体的大肠杆菌作为实验对照组(pET32a-BL21)，分别以JA作为候选底物进行反应。分别加入MgCl2、S-腺苷甲硫氨酸(S-adenosyl-L-methionine，SAM)、JA、ATP、DDT和适量的粗蛋白提取液，反应体系详见表5。
表5 PgJMT1重组蛋白酶活力测定反应体系
(2)将上述各组分混合后，25℃反应12h后，加入100μL无水乙醇终止反应，反应液冻干，以实施例5中的方法提取可能的产物MeJA，再用100μL质谱级甲醇复溶，超声处理10min，以0.22μm滤膜过滤，UPLC-MS/MS检测MeJA。
图5是PgJMT1重组蛋白活性测定结果，其中图5A和图5B为JA和MeJA标准品的UPLC-MS/MS分析结果；图5C和图5D为含有PgJMT1重组蛋白的粗提液与JA反应的产物，经UPLC-MS/MS分析后的结果，结果显示底物JA含量显著降低，MeJA明显增加；图5E和图5F为含有空载体的菌液提取的蛋白粗提液与JA反应的产物，经UPLC-MS/MS分析后的结果，结果显示底物JA含量没有变化，目标产物中也未见MeJA的产生；结果表明重组PgJMT1蛋白可以将JA催化为MeJA。
实验例4
PgJMT1基因植物超表达载体构建及其在人参叶片瞬时超表达
1、PgJMT1基因植物超表达载体构建，其超表达载体表达框如图6所示。
(1)PCR扩增用于同源重组的PgJMT1基因片段
根据PgJMT1基因序列设计同源重组引物以扩展其cDNA全长，同源重组扩增引物如PgJMT1-F1(SEQ ID No.9)和PgJMT1-R1(SEQ ID No.10)。并以Bgl II限制性核酸内切酶酶切pCAMBIA1302制备线性化载体，将PCR扩增产物和线性化的载体胶回收。
PgJMT1-F1：
5′-GGACTCTTGACCATGGATTCTGAGAAAGTTTCTGC-3′；(SEQ ID No.9)
PgJMT1-R1：
5′-TCGCCTTTGGAAGTTGAATGCCTCATAATTTCCTAAGAAC-3′；(SEQ ID No.10)
PCR反应条件为见表6。
表6 PCR反应条件
(2)pCAMBIA1302-PgJMT1表达载体构建及转化发根农杆菌A4
以In-Fusion HD Cloning Kit将PgJMT1基因重组至pCAMBIA1302载体，经PCR和测序鉴定重组载体连接正确后，将构建的重组表达载体命名为pCAMBIA1302-PgJMT1。以冻融法将构建好的pCAMBIA1302-PgJMT1载体转化发根农杆菌A4，转化后阳性克隆以PCR进行鉴定。再经测序鉴定成功后获得含PgJMT1基因超表达载体的农杆菌。
2、农杆菌介导PgJMT1基因转化人参叶片
(1)含pCAMBIA1302-PgJMT1的发根农杆菌培养
挑取含有pCAMBIA1302-PgJMT1载体的农杆菌和对照农杆菌(含有空载体pCAMBIA1302)单菌落分别接种于10mL加有相应抗生素的YEB液体培养基中培养16-24h。取1mL菌液转接到100mL加有相应抗生素的YEB液体培养基中，并加入10μL 100mmol/L乙酰丁香酮(acetosyringone，AS；母液用DMSO配制，使其终浓度为20μmol/L)。28℃培养过夜。培养液于5000rpm、4℃离心10min，收集菌体，用1/2MS培养基清洗3次后，用1/2MS+AS(终浓度为20μmol/L)培养基将菌液稀释至OD600为0.8左右，作为转化人参叶片的侵染液。
(2)发根农杆菌A4介导PgJMT1基因转化人参叶片
用1mL无针头的无菌注射器吸取农杆菌侵染液，从人参叶片下表皮注射到人参叶片内；注射3天后，剪下人参叶片，分析PgJMT1基因表达水平、MeJA和人参皂苷含量。
3、qRT-PCR分析人参叶片中PgJMT1基因的瞬时表达水平
取发根农杆菌A4侵染3天后的人参叶片，采用实施例2方法中qRT-PCR分析人参叶片中PgJMT1基因的表达水平，结果如图7所示。
图7是荧光定量PCR(qRT-PCR)检测发根农杆菌A4介导转化PgJMT1基因3天后人参叶片中PgJMT1基因的表达水平；以β-actin作为内参；图中对照表示转化空载体的人参叶片，T5、T9、T12和T16分别表示转化含PgJMT1基因的人参叶片；结果显示转化PgJMT1基因的人参叶片中PgJMT1基因的表达水平均上调，T5、T9、T12和T16叶片中PgJMT1基因的表达水平分别是对照的1.26、1.81、2.45和1.83倍。表明PgJMT1基因在瞬时超表达的人参叶片中表达。
4、瞬时超表达PgJMT1基因的人参叶片中MeJA和人参皂苷含量测定
(1)MeJA提取及含量测定
取发根农杆菌A4侵染3天后的人参叶片，在液氮中研磨成细粉，称取适量人参叶片细粉，加入异丙醇-水-盐酸混合提取液，添加8μL 1μg/mL的内标溶液，4℃振荡30min；加入二氯甲烷，低温振荡30min；4℃13000×g离心5min，取下层有机相；避光，以氮气吹干有机相，以甲醇(0.1％甲酸)复溶，4℃13000×g离心10min，取上清液过0.22μm滤膜，UPLC-MS/MS检测MeJA，结果如图8所示。
图8是发根农杆菌A4介导转化PgJMT1基因3天后的人参叶片中MeJA含量，图中对照表示转化空载体的人参叶片，T5、T9、T12和T16分别表示转化含PgJMT1基因的人参叶片；结果显示转化PgJMT1基因的人参叶片中MeJA含量明显上调，T5、T9、T12和T16叶片中MeJA含量分别是对照的2.91、2.21、3.32和1.98倍
(2)人参皂苷提取及含量测定
取发根农杆菌A4侵染3天后的人参叶片，自来水清洗2min后用双蒸水清洗两遍，60℃干燥至恒重。将其研磨成细粉，用80％甲醇60℃浸提(1g：40mL)，超声波处理3次，每次15min；60℃水浴蒸干甲醇，水洗超声溶解，乙醚萃取两次，取水相，用水饱和正丁醇萃取，收集正丁醇层。60℃水浴蒸干正丁醇，即得到人参总皂苷，用适量的甲醇超声波溶解，定容至刻度，过0.45μm的微孔滤膜，得到样品溶液。采用液相色谱检测人参皂苷的含量：以人参总皂苷Rb1、Rb2、Rc、Rd、Re、Rg1和Rg3作为标准品分别测定样品中的各皂苷单体的含量，再以各皂苷单体含量之和代表人参细胞中总皂苷的含量，结果如图9所示。
图9是发根农杆菌A4介导转化PgJMT1基因3天后的人参叶片中人参皂苷含量，图中对照表示转化空载体的人参叶片，T5、T9、T12和T16分别表示转化含PgJMT1基因的人参叶片；结果显示转化PgJMT1基因的人参叶片中人参总皂苷含量明显上调，T5、T9、T12和T16叶片中人参皂苷分别是对照的3.11、2.03、2.58和1.72倍。尽管T12叶片中MeJA含量最高，但MeJA含量相对较低的T5叶片中人参皂苷含量最高，人参皂苷的含量是对照的3.11倍。上述结果表明PgJMT1基因可以有效的促进人参细胞中MeJA的合成，合适浓度的内源性MeJA能促进人参皂苷的合成与积累。
序列表
<110> 湖南工程学院
<120> 一种调节人参中茉莉酸甲酯合成的PgJMT1基因及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1119
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggattctg agaaagtttc tgctaacacc atggattctg agaaagtttt ccacatggct 60
ggaggagttg gagagactag ctatgccaaa aattccttga ttcagaaaaa ggcatctgat 120
atggccaaga acataaccct ccaaaccatt caacaagttt ataatctcca tacaacaact 180
tcaaagagtt ttggcatagc tgacttgggg tgtggctcag gacacaacac tttatcaatc 240
atcaaacaaa tgcttgaagc atgcagttat gatgaatatg atgacaacat taacaatgag 300
ttccgcgtgt acctaaacga ccttccaaac aacgatttca acgcaatctt caaggtgttg 360
ccggatttct acacagagtt gaggagggat gggaagggaa agtttgatca tatatatata 420
ggtgcttatc ctggctcttt ctatggaaga ctttttccag aaaagtgctt gcacttcatt 480
tattccaaca acagcttgca ttggctttct aaggtaccac catccattta tgacaagcaa 540
aacaattcta cgaacaaggg caacatttac ataacagaat caagccccct agaggtgtct 600
caggcatact ttaggcagtt ccaagaggac ctctggctgt ttctacggtc ccgatctgaa 660
gaacttgttg ccggaggacg tatggtgttg atcgtgtcgg gcagaagtgg ccggaatcat 720
gacgacagag gcattacatt tttatgggca cttctttcta aatcactcgc aattttagtt 780
tctcagggac tagttgaaga ggaaaagctt gatgggtaca atgttcagtt ttatgcacca 840
tcagaagatg aaataaaaga tgaagtaata agagagggat ctttccaaat ggaccgtttt 900
gaaatgtttg aaatagacaa ggttgttgac ggtggtgcaa gctacggaac ggcggtggca 960
aagacggcta aggcggtgca aggaccgatg atatgccaac attttggcga tggagttcta 1020
gacagtgtgt ttgagaacta tggaagatta gttgatgaaa acatggctgt agaggagata 1080
aggcctatat attttgtttt tgttcttagg aaattatga 1119
<210> 2
<211> 372
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Asp Ser Glu Lys Val Ser Ala Asn Thr Met Asp Ser Glu Lys Val
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Phe His Met Ala Gly Gly Val Gly Glu Thr Ser Tyr Ala Lys Asn Ser
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Leu Ile Gln Lys Lys Ala Ser Asp Met Ala Lys Asn Ile Thr Leu Gln
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Thr Ile Gln Gln Val Tyr Asn Leu His Thr Thr Thr Ser Lys Ser Phe
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Gly Ile Ala Asp Leu Gly Cys Gly Ser Gly His Asn Thr Leu Ser Ile
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Ile Lys Gln Met Leu Glu Ala Cys Ser Tyr Asp Glu Tyr Asp Asp Asn
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Ile Asn Asn Glu Phe Arg Val Tyr Leu Asn Asp Leu Pro Asn Asn Asp
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Phe Asn Ala Ile Phe Lys Val Leu Pro Asp Phe Tyr Thr Glu Leu Arg
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Arg Asp Gly Lys Gly Lys Phe Asp His Ile Tyr Ile Gly Ala Tyr Pro
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Gly Ser Phe Tyr Gly Arg Leu Phe Pro Glu Lys Cys Leu His Phe Ile
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Tyr Ser Asn Asn Ser Leu His Trp Leu Ser Lys Val Pro Pro Ser Ile
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Tyr Asp Lys Gln Asn Asn Ser Thr Asn Lys Gly Asn Ile Tyr Ile Thr
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Glu Ser Ser Pro Leu Glu Val Ser Gln Ala Tyr Phe Arg Gln Phe Gln
195 200 205
Glu Asp Leu Trp Leu Phe Leu Arg Ser Arg Ser Glu Glu Leu Val Ala
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Gly Gly Arg Met Val Leu Ile Val Ser Gly Arg Ser Gly Arg Asn His
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Asp Asp Arg Gly Ile Thr Phe Leu Trp Ala Leu Leu Ser Lys Ser Leu
245 250 255
Ala Ile Leu Val Ser Gln Gly Leu Val Glu Glu Glu Lys Leu Asp Gly
260 265 270
Tyr Asn Val Gln Phe Tyr Ala Pro Ser Glu Asp Glu Ile Lys Asp Glu
275 280 285
Val Ile Arg Glu Gly Ser Phe Gln Met Asp Arg Phe Glu Met Phe Glu
290 295 300
Ile Asp Lys Val Val Asp Gly Gly Ala Ser Tyr Gly Thr Ala Val Ala
305 310 315 320
Lys Thr Ala Lys Ala Val Gln Gly Pro Met Ile Cys Gln His Phe Gly
325 330 335
Asp Gly Val Leu Asp Ser Val Phe Glu Asn Tyr Gly Arg Leu Val Asp
340 345 350
Glu Asn Met Ala Val Glu Glu Ile Arg Pro Ile Tyr Phe Val Phe Val
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Leu Arg Lys Leu
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<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggattctg agaaagtttc tgc 23
<210> 4
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tcataatttc ctaagaac 18
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tgccccagaa gagcaccctg t 21
<210> 6
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agcatacagg gaaagatcgg cttga 25
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tggctcagga cacaacactt 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tcgtttaggt acacgcggaa 20
<210> 9
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggactcttga ccatggattc tgagaaagtt tctgc 35
<210> 10
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcgcctttgg aagttgaatg cctcataatt tcctaagaac 40
Claims (8)
1.一种调节人参中茉莉酸甲酯合成的PgJMT1基因，其特征在于，所述调节人参中茉莉酸甲酯合成的PgJMT1基因如SEQ ID No.1所示。
2.根据权利要求1所述的一种调节人参中茉莉酸甲酯合成的PgJMT1基因，其特征在于，所述调节人参中茉莉酸甲酯合成的PgJMT1基因编码的蛋白氨基酸序列如SEQ ID No.2所示。
3.根据权利要求1所述的一种调节人参中茉莉酸甲酯合成的PgJMT1基因，其特征在于，所述调节人参中茉莉酸甲酯合成的PgJMT1基因的扩增引物序列如SEQ ID No.3和SEQ IDNo.4所示。
4.根据权利要求1所述的一种调节人参中茉莉酸甲酯合成的PgJMT1基因，其特征在于，所述调节人参中茉莉酸甲酯合成的PgJMT1基因与表达载体组成重组载体。
5.根据权利要求4所述的一种调节人参中茉莉酸甲酯合成的PgJMT1基因，其特征在于，所述表达载体为pCAMBIA1302。
6.根据权利要求4所述的一种调节人参中茉莉酸甲酯合成的PgJMT1基因，其特征在于，所述重组载体的构建方式如下：以所述调节人参中茉莉酸甲酯合成的PgJMT1基因的开放读码框作为超表达序列，将该cDNA片段插入到植物表达载体pCAMBIA1302中制得。
7.一种如权利要求1-6任一项所述的一种调节人参中茉莉酸甲酯合成的PgJMT1基因在调节人参皂苷合成与积累中的应用。
8.根据权利要求7所述的应用，其特征在于，所述应用方式为利用农杆菌介导所述调节人参中茉莉酸甲酯合成的PgJMT1基因转化人参组织。
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| CN117535316A (zh) * | 2024-01-04 | 2024-02-09 | 湖南工程学院 | 一种人参PgJOX4基因及其在调节人参皂苷生物合成中的应用 |
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| CN117511970A (zh) * | 2024-01-04 | 2024-02-06 | 湖南工程学院 | 一种受冠菌素诱导的人参PgJOX2基因及其应用 |
| CN117535316A (zh) * | 2024-01-04 | 2024-02-09 | 湖南工程学院 | 一种人参PgJOX4基因及其在调节人参皂苷生物合成中的应用 |
| CN117511970B (zh) * | 2024-01-04 | 2024-03-29 | 湖南工程学院 | 一种受冠菌素诱导的人参PgJOX2基因及其应用 |
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| CN118556551A (zh) * | 2024-08-01 | 2024-08-30 | 中华全国供销合作总社昆明食用菌研究所 | 利用茉莉酸提高羊肚菌菌丝体中腺苷含量的方法 |
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