CN115948494A - 一种利用三七细胞合成齐墩果烷型皂苷的方法 - Google Patents
一种利用三七细胞合成齐墩果烷型皂苷的方法 Download PDFInfo
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Abstract
本发明公开了一种利用三七细胞合成齐墩果烷型皂苷的方法，该方法是在在三七细胞愈伤组织诱导培养基中添加二乙基二硫代氨基甲酸钠，在含有二乙基二硫代氨基甲酸钠的培养基上培养三七细胞，能促进三七中与齐墩果烷型皂苷合成相关的重要基因的表达，使三七细胞具备合成齐墩果烷型皂苷的能力，本发明操作简便，效果良好，具有应用到三七细胞大规模培养生产齐墩果烷型皂苷的潜力。
Description
技术领域
本发明涉及一种利用二乙基二硫代氨基甲酸钠促使三七细胞合成齐墩果烷型皂苷的方法。
背景技术
三七[
Panax notoginseng (Burk.) F. H. Chen]是五加科人参属多年生草本植物，为我国传统中药材，具有止血化瘀、预防脑血管疾病等功效。目前为止，已从三七的根、茎、叶和花中分离出200多种化学成分。其中，达玛烷型三萜皂苷为三七的主要活性成分。人参属药材中的三萜皂苷根据苷元结构的不同又可分为四环三萜达玛烷型皂苷和五环三萜齐墩果烷型皂苷。三七只含有达玛烷型皂苷，而珠子参、西洋参和人参同时含有齐墩果烷型皂苷和达玛烷型皂苷。但我们的前期研究表明，三七可能也具备合成齐墩果烷型皂苷的潜力。
二乙基二硫代氨基甲酸钠（DIECA）在植物中常用作茉莉酸（Jasmonic acid，JA）生物合成抑制剂，DIECA通过将13(S)-羟基过氧化亚麻酸分流到13-羟基亚麻酸，大幅减少合成JA的前体物质，导致JA的环化和最终合成量的减少。已有研究表明，施用DIECA可显著降低多种植物物种的JA水平，并降低一些抗性基因的表达，如
TaJRLL1和
PR3。此外，施用DIECA后，除了抑制JA外，也可能导致吲哚乙酸水平降低，油菜素类固醇增加，还观察到谷胱甘肽和活性氧的积累。
目前还未有任何通过添加外源化学物质使得三七细胞产齐墩果烷型皂苷的技术报道。
发明内容
本发明提供了一种利用二乙基二硫代氨基甲酸钠促使三七细胞合成齐墩果烷型皂苷的方法，本发明研究首次发现二乙基二硫代氨基甲酸钠（DIECA）可以激活三七细胞中齐墩果烷型皂苷合成通路，使三七细胞合成原本不具有的齐墩果烷型皂苷（竹节参皂苷Ⅳ、竹节参皂苷Ⅳa）。
本发明目的通过下述方案实现：
1、三七细胞获取
采用三七的茎或叶，使用添加2,4-二氯苯氧乙酸（2,4-D）1-3mg/L、6-糠基氨基嘌呤（KT）0.5-1.5mg/L的MS固体培养基，培养基pH 5.6，培养温度25±1℃，避光培养，培养周期4-5个月后，将获得的三七愈伤组织采用含2,4-D 1-3mg/L、KT 0.5-1.5mg/L的MS固体培养基继代培养10-20天，获得三七继代培养细胞；MS培养基的成分如表1所示；
2、齐墩果烷型皂苷的合成
将继代培养三七细胞接种在pH5.6的合成培养基（MS液体培养基+2,4-D 1-3mg/L+KT 0.5-1.5mg/L+20mmol/L-80 mmol/L DIECA）中暗培养，培养温度为25±1℃，齐墩果烷型皂苷合成阶段的细胞培养周期为12-20天。
表 
：MS培养基成分
3、齐墩果烷型皂苷合成酶基因表达量分析
提取步骤（2）DIECA处理的三七细胞中的RNA，并逆转录成cDNA；使用实时荧光定量PCR分析齐墩果烷型皂苷合成途径中关键基因的表达量差异；
4、检测DIECA处理的三七细胞中齐墩果烷型皂苷的含量
本发明方法操作简单，在含有20mmol/L-80mmol/L二乙基二硫代氨基甲酸钠的愈伤组织培养基上培养三七细胞，能促进三七中与齐墩果烷型皂苷合成相关的重要基因的表达，使三七细胞具备合成齐墩果烷型皂苷（竹节参皂苷Ⅳ、竹节参皂苷Ⅳa）的能力；本发明具有应用到三七细胞大规模培养生产齐墩果烷型皂苷的潜力，本发明方法适用于工业化生产和市场推广应用。
附图说明
图1 为实时荧光定量PCR检测三七细胞中基因相对表达量的检测结果，图中WT表示未经DIECA处理的细胞系，1是25mmol/L DIECA处理的；2是50mmol/L DIECA处理的；3是75mmol/L DIECA处理的；
图2为三七细胞系中皂苷含量图，图中WT表示未经DIECA处理的细胞系，1是25mmol/L DIECA处理的；2是50mmol/L DIECA处理的；3是75mmol/L DIECA处理的，竹节参皂苷Ⅳ和竹节参皂苷Ⅳa为齐墩果烷型皂苷。
具体实施方式
下面通过实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1：三七细胞的培养
1、采集三七茎叶，使用MS培养基+ 2,4-D 2mg/L + KT 1mg/L，培养基pH5.6，培养条件为固体培养，温度25±1℃，避光培养，培养周期4-5个月，获得的三七愈伤组织；
2、将获得的三七愈伤组织采用含2,4-D 1-3mg/L、KT 0.5-1.5mg/L的MS固体培养基继代培养15天，获得三七继代培养细胞；
3、配制MS液体培养基+ 2,4-D 2mg/L + KT 1mg/L +DIECA 50mmol/L的液体合成培养基，培养基pH5.6，将步骤2获得的三七细胞接种装有50mL液体合成培养基的锥形瓶中进行培养，培养温度为25±1℃，暗培养，细胞培养周期为15天，同时做了DIECA 为25mmol/L、75mmol/L的实验，并以不添加DIECA的液体合成培养基为对照（WT）。
实施例2：DIECA对三七齐墩果烷型皂苷合成酶基因
β-AS、CYP716A52v2、
OAGT、
PjmUGT1和
PjmUGT2表达量的影响
收集实施例1中生长状态良好的三七细胞，采用改良的异硫氰酸胍法提取三七细胞总RNA，操作如下：将研钵、研钵棒进行去RNA酶-高温干热灭菌处理后，冷却至室温，称取适量三七愈伤组织细胞置于处理过的研砵中，利用液氮将三七愈伤组织研磨成粉末，加入10 %（w/v）预冷的 RNA 提取缓冲液和1.0 %（w/v）的β-巯基乙醇，充分研磨。取1.0mL 研磨液转移至2mL 的离心管中，加入500μL RNA提取酚、100μL氯仿以及1/10体积的2mol/L醋酸钠溶液（pH为4.0），剧烈振荡混匀，冰上静置5 min 后于4℃、12500g离心15min；缓慢吸取上清液转移至新的2mL离心管中，加入1:1等体积的RNA提取酚/氯仿，剧烈震荡混匀，冰上静置5min后，4 ℃、12500g离心15min（重复此步骤至中间层几乎消失）。缓慢吸取上清液转移至新的2mL 离心管中，加入等体积的氯仿，剧烈震荡混匀，冰上静置5min，4℃、12500 g离心15min，缓慢吸取上清液转移至新的1.5mL的离心管中，加入1/10体积的3 M醋酸钠溶液（pH为5.2），再加入等体积异丙醇，缓慢颠倒混匀，于-20℃静置1.5h充分沉淀后，4℃、12500g离心25min，弃上清液，用75%的乙醇溶液吹打洗涤沉淀，两次吹打洗涤后，使用移液枪吸掉液体并置于超净工作台进行风干，至乙醇挥发彻底后，加入15-30μL无RNaes的水溶解 RNA沉淀，琼脂糖凝胶电泳检测所提三七愈伤组织细胞总 RNA 完整性，然后用紫外分光光度计检测所提 RNA 的浓度和纯度。
按照GoScriptTMReverseTranscriptase System试剂盒操作说明，将提取的RNA逆转录成cDNA，反应体系和操作过程为：在离心管中加入5μg总RNA、1μL Random Primer和1μLOligo(dT)15，用Nuclease-free Water补齐至10μL，混匀，进行70℃、5min预变性，完成后立即置于冰上冰浴5min。随后将离心管在离心机中短暂离心，使反应液收集于管底，再向其中加入4µL GoScriptTM 5×Reaction Buffer、2µL MgCl2 (25mM)、1µL PCR Nucleotide Mix(10mM)、0.5µL RecombinantRNasinRibonucleaseInhibitor和1µL GoScriptTM ReverseTranscriptase，混匀，瞬时离心，25℃退火5 min，42℃延伸1.5 h，最后，放置70℃水浴锅中15min，使逆转录酶失活，结束反应，短暂离心，-20℃保存备用。
将逆转录合成的cDNA稀释5倍，即将20.0µL cDNA稀释至100.0µL。以稀释后的cDNA为模板，根据GoTaq® 2-Step RT-qPCR System试剂盒说明书以及三七18S rRNA基因（登录号：D85171.1）、β-香树脂醇合成酶（β-amysyrinsynthase ，β-AS）基因（登录号：KP658156）、齐墩果酸合酶（Oleanic acid synthase，CYP716A52v2）基因（登录号：JX036032.1）、齐墩果酸葡萄糖醛酸基转移酶（oleanolic acid glucuronosyltransferase,OAGT）基因（登录号：MH819287.1）、
PjmUGT1基因和
PjmUGT2基因设计引物，进行荧光定量PCR，引物序列如下：
18S-F：5’-GGGGAGTATGGTCGCAAGG-3’，
18S-R：5’-CAGAACATCTAAGGGCATCACAG-3’
β-AS-F：5’-GTATTCCCTGTAGAGCATCGCAT-3’，
β-AS-R：5’-GGCACAGGCGTTGTTTTCAC-3’；
CYP716A52v2-F：5’-AGGAGCAAATGGAGATAGTGA-3’，
CYP716A52v2-R：5’-GATTGAGAAACCGTTGTAGG-3’；
OAGT-F：5’-GCATAATCTCGGACAAGTAC-3’，
OAGT -R：5’-AAAGGTTGGGAGTCTGAAGT-3’；
PjmUGT1-F：5’-TCACATAAATCCGATGGTCC-3’，
PjmUGT1-R：5’-AGAAATCCCTGAAATCCTCC-3’；
PjmUGT2-F：5’-GCATTCTCCCTTTGTTTCAG-3’，
PjmUGT2-R：5’-CGACTTGCCTCACTCTTCCT-3’；
具体反应体系和操作过程为：在PCR管中加入20ng cDNA、25µLGoTaq®qPCR MasterMix (2×)和0.2µL qPCRPrimers(18S-F/18S-R，β-AS-F/β-AS-R，CYP716A52v2-F/CYP716A52v2-R、OAGT-F/OAGT-R、PjmUGT1-F/PjmUGT1-R和PjmUGT2-F/PjmUGT2-R 10mmol/L)，用Nuclease-Free Water补齐至50µL。将反应体系漩涡混匀后，离心将其收集到管底，随后将其置于荧光定量PCR仪中进行反应，采用两步法进行荧光定量PCR，反应参数如下：热启动 95℃2min；变性95℃15s，退火/延伸60℃ 1min，共45个循环。每个样品对应的每个基因重复检测2次。
结果如图1所示，用含DIECA的培养基培养的三七细胞中
β-AS、CYP716A52v2、
OAGT、
PjmUGT1和
PjmUGT2的表达量要比未经DIECA处理的三七细胞高（图1），说明DIECA处理三七细胞，能够促进三七齐墩果烷型皂苷合成酶基因
β-AS、CYP716A52v2、
OAGT、
PjmUGT1和
PjmUGT2的表达。
实施例3：DIECA对三七齐墩果烷型皂苷合成量的影响
收集实施例1中生长状态良好的三七细胞系，分别置于55℃烘箱烘至恒重，充分研磨成粉末后过100目筛。称取三七细胞粉末各0.5g，分别置于含有50mL甲醇洗净的三角瓶中过夜浸泡。在超声处理1.5h（60W超声4s，间歇2s），常温条件下4000rpm离心30min，收集上清液，即得三七总皂苷溶液，并于4℃冰箱保存备用。
精确吸取样品150μL上述已制备好的各三七细胞株系对应的总皂苷溶液于带塞子的试管中（每个样品设置3个平行），于55℃条件下挥发掉溶剂，用10mL蒸馏水进行溶解，并用等体积的水饱合正丁醇萃取3次，收集萃取液，并将萃取液放置于50~55℃烘箱中进行烘干。烘干后用适量甲醇溶液进行溶解并定容至25mL，经0.45μM的微孔滤膜过滤后即为皂苷溶液。利用HPLC检测三七细胞中皂苷的种类及其含量，HPLC检测条件如下：色谱柱为Waters-XTerra-MS-C18（5μm，250 mm×4.6 mm，USA）。流动相为水和乙腈。梯度洗脱：0-20min，20%乙腈；20-30min，20%-35%乙腈；30-40min，35%乙腈； 40-50min，35-40%乙腈；50-60min，40-100%乙腈。流速为1.0mL/min，柱温 30℃。检测波长设置为203nm，结果如图2所示，用含DIECA的培养基培养的三七细胞中产生了齐墩果烷型皂苷（竹节参皂苷Ⅳ、竹节参皂苷IVa），并且达玛烷型皂苷（人参皂苷Rd、人参皂苷Rb1）含量略微低于未经DIECA处理的三七细胞含量。
Claims (2)
1.一种利用三七细胞合成齐墩果烷型皂苷的方法，其特征在于：在三七细胞愈伤组织诱导培养基中添加二乙基二硫代氨基甲酸钠。
2.根据权利要求1所述的利用三七细胞合成齐墩果烷型皂苷的方法，其特征在于：二乙基二硫代氨基甲酸钠的添加浓度为20mmol/L-80mmol/L。
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