CN116891855B - 一种人参PgMYC24基因及其应用 - Google Patents
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Abstract
本发明公开了一种来自人参的基因，PgMYC24，及其在提高人参皂苷上的应用，所述人参PgMYC24基因的基因序列如SEQ ID NO.1所示。超量表达该基因可有效提高人参根部人参皂苷的含量，特别是二醇型人参皂苷，本发明共检测到6种单体皂苷，共有5个转基因阳性单根系的Rb2含量与转化空载的阴性对照相比显著上升，5个单根系的Rd含量显著上升，以上结果说明PgMYC24基因参与调控人参皂苷的合成，特别是对人参皂苷Rb2的合成具有显著促进作用。本发明超量表达PgMYC24基因可显著提高人参皂苷在科研上有着重要的研究价值，为开发高含量人参皂苷的人参种质资源提供有力的技术手段。
Description
技术领域
本发明属于生物技术领域，具体涉及一种提高人参单体皂苷含量的PgMYC24基因及其应用。
背景技术
人参(学名：Panax ginseng C.A.Meyer)，属五加科人参属植物，是著名的药用植物，主要分布于中国东北、韩国和日本，在世界范围内广泛使用。人参中含皂苷、多糖、聚炔、黄酮等多种化学成分，其中人参皂苷是人参的次级代谢产物，是人参的主要生物活性成分，有广泛的生理和药理活性，包括免疫系统调节、抗应激、降血糖、抗炎、抗氧化及抗癌作用，在心血管系统有抗心律失常、抗心肌肥厚、抗心肌缺血、抗心肌细胞凋亡等作用，在抗肿瘤方面具有诱导细胞凋亡、抑制肿瘤细胞增殖、调控信号通路、调节免疫功能等作用，具有较好的临床应用价值，已应用于多种疾病的治疗和预防。人参皂苷按照苷元类型可分为达玛烷型(原人参二醇型人参皂苷、原人参三醇型人参皂苷)和齐墩果酸型人参皂苷，其生物合成受到多基因调控。
MYC转录因子属于bHLH类转录因子家族成员，广泛存在于动植物中，是茉莉酸信号途径的重要调控因子。MYC蛋白包含家族特有的保守螺旋-环-螺旋结构和典型的保守结构域b HLH-MYC_N。其中，HLH结构域位于蛋白的C端，约由55个氨基酸构成，包含2个双亲性α螺旋，行使同源或异源二聚体的功能。b HLH-MYC_N包含1个JAZ相互作用结构域和1个与中介复合物的亚基MED25互作的MYC转录激活域，该结构域位于MYC蛋白的N端，与DNA结合有关。已有研究发现该家族成员在植物信号转导中起到重要的调节作用，其中At MYC2、At MYC3和At MYC4已证实参与茉莉酸信号的调控。茉莉酸(JA)是植物次生代谢的保守激发子。JA信号能够触发广泛的转录重新编程，导致整个代谢途径的协同激活。At MYC2作为茉莉酸信号的转录因子是通过N端保守的bHLH-MYC_N结构域与茉莉酸的转录抑制因子JAZ相互作用来调节茉莉酸信号转导。研究表明，在人参不定根悬浮培养的过程中，外源施加茉莉酸能够特异性地促进原人参二醇人参皂苷(Rb)的积累，而原人参三醇人参皂苷(Rg)的积累不显著。但其调控机制尚不清晰。
人参发状根具有遗传背景清晰、培养周期短、培养不受季节限制、品质均一、可产人参皂苷等优点。因此，以人参发状根为材料，结合分子生物学的手段，能够快速验证目的基因是否调控人参皂苷的生物合成。
发明内容
本发明提供了一种能提高人参单体皂苷的PgMYC24基因及其应用，为开发高含量人参皂苷的人参种质资源提供有力的技术手段。
为了达到上述目标，本发明提供的技术方案如下：
本发明提供了一种人参PgMYC24基因，所述人参PgMYC24基因的基因序列如SEQ IDNO.1所示。
本发明还提供了含有上述人参PgMYC24基因的载体。
本发明进一步提供了上述人参PgMYC24基因在提高人参单体皂苷含量中的应用。
本发明具有如下有益效果：
人参中有169个bHLH类转录因子，MYC转录因子属于bHLH类转录因子家族成员之一，广泛存在于动植物中。本发明公开了一种来自人参的基因，PgMYC24，及其在提高人参皂苷上的应用。超量表达该基因可有效提高人参根部人参皂苷的含量，特别是二醇型人参皂苷，本发明共检测到6种单体皂苷，共有5个转基因阳性单根系的Rb2含量与转化空载的阴性对照相比显著上升，5个单根系的Rd含量显著上升，以上结果说明PgMYC24基因参与调控人参皂苷的合成，特别是对人参皂苷Rb2的合成具有显著促进作用。本发明超量表达PgMYC24基因可显著提高人参皂苷有着重要的研究价值，为开发高含量人参皂苷的人参种质资源提供有力的技术手段。
附图说明
图1：人参总RNA电泳图。
图2：PgMYC24基因PCR扩增电泳图(M：DL15000；泳道1-2：PCR产物)。图3：PgMYC24基因表达载体双酶切图(M：DL2000；泳道1：目的片段表达载体酶切；泳道2：载体质粒酶切)。
图4：超量表达PgMYC24基因人参发状根的诱导(A：不定根预培养；B：侵染共培养；C：发状根培养；D：发状根固体培养；E：固体扩繁阳性发状根单根系；F：液体扩繁阳性发状根单根系；G-H：阳性发状根取样)。
图5：dPCR技术扩增阳性发状根植株中PgMYC24基因(S01-S06:6株不同的阳性发状根)。
图6：超量表达PgMYC24基因人参发状根中人参皂苷含量检测。
具体实施方式
材料来源
人参不定根材料由吉林农业大学人参生物技术研究与应用科研团队提供。
主要内容
利用生物信息学的方法，在吉林人参转录组数据库中对MYC转录因子家族进行鉴定，系统发育分析，GO功能注释，表达模式分析，及与人参皂苷生物合成的相关性分析。
1、人参总RNA的提取与cDNA的合成
采用Trizol法提取人参不定根组织中的总RNA(图1)。经反转录试剂盒反转录为cDNA。
2、PgMYC24基因的克隆
目的基因全长1450bp，ORF：16bp-1362bp，448aa，基因序列如SEQ ID NO.1所示。
根据上述序列，利用Primer Premier 5设计关于目的基因的PCR扩增引物。引物序列如下：PgMYC24-F：碱基序列如SEQ ID NO.2所示，PgMYC24-R：碱基序列如SEQ ID NO.3所示。根据上述引物，以cDNA为模板对目的基因进行扩增，扩增条件：预变性94℃，5min；变性94℃，30s、退火53℃，15s、延伸72℃，61s，35个循环；延伸72℃，10min；4℃保存。经琼脂糖凝胶电泳对目的条带大小进行验证，结果如图2所示，目的条带大小正确。对以上电泳结果进行琼脂糖凝胶回收。对pBI121载体进行线性化双酶切，限制性内切酶分别为Sma I和Sac I。利用琼脂糖凝胶对线性化载体进行凝胶回收。随后设计pBI121载体的同源臂引物：T-MYC24-F：碱基序列如SEQ ID NO.4所示T-MYC24-R：碱基序列如SEQ ID NO.5所示。根据同源臂引物，以琼脂糖回收的目的片段为模板，对目的基因进行扩增，扩增条件：预变性94℃，5min；变性94℃，30s、退火53℃，15s、延伸72℃，61s，35个循环；延伸72℃，10min；4℃保存，对PCR产物进行琼脂糖凝胶回收。
3、PgMYC24基因植物超量表达载体的构建
将回收片段通过无缝克隆的方式与pBI121线性化载体片段进行连接。通过热击法转入大肠杆菌DH5α感受态细胞中，利用含有50μg/mL卡那霉素的抗性平板进行筛选，对其中的抗性克隆进行PCR验证(图3)，将阳性克隆送至测序公司测序。
4、超量表达PgMYC24基因遗传转化工程菌的构建
利用热击法将重组的PgMYC24基因植物超量表达载体转入农杆菌C58C1感受态细胞中，利用含有50μg/mL卡那霉素与25mg/mL利福平的抗性平板进行筛选，对抗性克隆进行PCR验证，结果显示条带大小符合目的基因长度，因此超量表达PgMYC24基因遗传转化工程菌的构建成功，保存备用。
5、农杆菌介导法转化PgMYC24基因
(1)预培养
选取长势粗壮的不定根用刀斜切成小段至于MS固体培养基中，在温度为22℃，光处理16h，暗处理8h的培养室中，对材料进行预培养(2天)。
(2)工程菌活化
挑取阳性单菌落在加入卡那霉素，利福平，链霉素的三抗YEP培养基中进行扩大培养(培养条件为28℃，170rpm)，至OD600＝0.6。将活化好的菌液转移至50ml离心管进行离心收集菌体。用20μM乙酰丁香酮(AS)和液体1/2MS培养基重悬菌体，28℃，放置1h备用。
(3)侵染与共培养
将预培养的不定根材料，切成小段，置于备用的菌液中侵染15min。随后，倒出菌液，将侵染的后的不定根取出，用滤纸吸干表面的水分，置于含有20μMAS的固体1/2MS培养基中，在黑暗条件下共培养48h。
(4)除菌
将共培养后的材料取出，吸干表面菌液，转移至加入头孢霉素的固体1/2MS培养基中，15-30天后，诱导长出发状根。将得到的阳性根系，在250ml摇瓶中进行扩大培养(图4)。
6、阳性植株dPCR检测
本研究采用765个分子和12个通道的微流体芯片，按照实验步骤对6株阳性发状根的转基因拷贝数进行测定，5株阳性发状根样品的通道中均扩增PgMYC24基因，而转空载体的对照的通道没有荧光信号，测定结果如图5所示。对阳性发状根基因的拷贝数进行分析，首先计算出样品的拷贝数浓度，计算公式如下：Ccopies＝d×M/V＝d/N×Vp×log(1-H/N)/log(1-1/N)(Ccopies-基因的拷贝数浓度copies/μL，d-样品稀释因子，M-分配到每个通道的DNA模板分子数，N-每个通道的总分子数，H-每个通道得到有效扩增而检测的阳性分子数，Vp-每个分子的体积)。最后再根据公式：基因拷贝数＝基因拷贝数浓度/内标准基因拷贝数浓度，本研究中内标基因拷贝数浓度为4536.65copies/μL，最终计算出PgMYC24基因的平均拷贝数为2.13。具体结果见表1。
表1dPCR PgMYC24基因拷贝数分析结果
7、超量表达PgMYC24基因阳性发状根中皂苷含量的检测
利用索氏提取法对人参发状根进行人参皂苷提取，利用高效液相色谱法检测其中的人参单体皂苷组成和含量。
将扩大培养的阳性发状根置于37℃的烘箱进行烘干，称取1.0g人参粉末用滤纸包裹，置于100ml三角瓶中，50ml无水甲醇浸泡12h。将三角瓶放入超声清洗器中，60℃超声30min。
将浸泡人参粉末的滤纸包与液体全部倒入索氏提取器中，加入无水甲醇至总体积为100ml。温度设置为90℃，回流时间设置为36h。
将提取后的甲醇倒入圆底烧瓶中，利用旋转蒸发仪，进行减压旋蒸至干燥，加入20mL纯净水回溶样品，用等体积的乙酸乙酯萃取三次除杂，留下水相，将水相继续用等体积水饱和正丁醇萃取三次，留下正丁醇相；
将正丁醇相倒入圆底烧瓶，减压旋干，用5mL色谱甲醇回溶，并用0.22μm有机系滤膜过滤，除去大颗粒杂质，得到人参皂苷样品。
8、高效液相色谱法检测人参皂苷
采用的色谱柱为Waters C18柱(4.6×250mm，5μm)，流动相为水和有机相乙腈，采用梯度洗脱的方法(梯度洗脱条件如表2所示)，流速为1.0mL/min，柱温设为35℃，进样体积为30μL，样品检测波长为203nm。
表2梯度洗脱条件
| 时间(min) | 乙腈(％) | 水(％) |
| 0 | 18 | 82 |
| 40 | 21 | 79 |
| 42 | 26 | 74 |
| 46 | 32 | 68 |
| 66 | 33.5 | 66.5 |
| 71 | 38 | 62 |
| 86 | 65 | 35 |
| 91 | 65 | 35 |
| 96 | 85 | 15 |
| 103 | 85 | 15 |
| 120 | 18 | 82 |
| 125 | 18 | 82 |
待样品出峰后，计算各个单体皂苷的含量，计算公式：标准品浓度/标准品峰面积＝样品浓度/样品峰面积。
结果共检测到六种单体皂苷。不同阳性发根的单体皂苷含量结果如图6所示，共有5个转基因阳性单根系的Rb2含量与转化空载的阴性对照相比显著上升，5个单根系的Rd含量显著上升，以上结果说明PgMYC24基因参与调控人参皂苷的合成，特别是对人参皂苷Rb2的合成具有显著促进作用。
Claims (3)
1.一种人参PgMYC24基因，其特征在于：所述人参PgMYC24基因的基因序列如SEQ IDNO.1所示。
2.含如权利要求1所述人参PgMYC24基因的载体。
3.含如权利要求1所述人参PgMYC24基因在提高人参单体皂苷含量中的应用。
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