广藿香原生质体融合与茉莉酸甲酯参与调控的百秋李醇生物合成机制初步研究
发布时间：2018-10-30 20:08
【摘要】：广藿香(Pogostemon cablin(Balnco)Benth.)为唇形科刺蕊草属植物,具有芳香化浊,开胃止呕,解暑的功效,为“十大广药”之一,其中以“石牌”品种最优。为解决抗性下降、资源短缺的现状,考虑以广藿香原生质体对称融合的多倍体育种方法改良品种,可以在提高石牌广藿香有效成分及产量的基础上避免化学诱导多倍体带来的嵌合体缺陷;此外,在原有研究基础上通过添加MeJA诱导子的广藿香悬浮细胞与空白对照的转录组数据分析,建立原始数据,挖掘相关转录因子,更深入地探究茉莉酸信号参与调控的百秋李醇生物合成机制,为提升广藿香药源品质及其可持续开发利用提供新的思路和技术手段。主要研究结果如下:(1)广藿香悬浮细胞原生质体分离在原有实验基础上优化了酶解分离方案,考察了预处理方法、酶液pH、酶解温度对广藿香原生质体分离的影响。最终确定选取培养9-15 d处于对数生长期的广藿香悬浮细胞为原材料,混合酶液选用纤维素酶、半纤维素酶和果胶酶,质量体积比依次为1.5%、0.5%、0.8%,酶液渗透势以0.4 moL/L甘露醇调节,pH设为5.8,温度25℃,以50rpm避光振荡酶解12 h,最终产量为13.2×105个/g,活力为81.8%。(2)广藿香悬浮细胞原生质体培养原生质体培养主要考察了培养方法、培养密度、培养基激素配比、铵盐含量、碳源、酸水解酪蛋白含量。最终确定以铵盐减半的MS1培养基进行海藻酸钠包埋,激素选用0.2 mg/L NAA、2.0 mg/L 6-BA,培养密度为2.0×105个/mL,蔗糖添加量1.0%,酸水解酪蛋白500mg/L。在培养第6天观察到细胞第一次分裂,14天左右细胞开始大量分裂,分裂频率为6.91%,第16天形成细胞团,植板率为1.83%,培养第63天左右观察到肉眼可见愈伤组织。(3)广藿香悬浮细胞原生质体融合本实验以测定融合产物直径变化范围的物理选择法来进行产物筛选,经测量与统计,确立融合产物筛选范围是69.33-87.35μm。采取高pH高钙法对称融合广藿香原生质体,考察了PEG浓度、融合时间、融合密度、融合液加入量对融合的影响。最终确定选用40%的PEG 6000进行化学促融30 min,融合液加入量为原生质体悬浮液体积的0.5倍,融合密度2.0×105个/mL,聚合率57.19%,融合产物经海藻酸钠包埋培育2个月,观察到再生愈伤组织,将愈伤组织转移至诱导丛生芽培养基,暂未观察到再生植株,培育方法有待进一步优化。(4)茉莉酸甲酯(MeJA)诱导后广藿香悬浮细胞转录组测序分别对添加了MeJA诱导子的广藿香悬浮细胞与空白对照组进行了Illumina转录组测序,共获得了662270条Unigenes,注释率为74.04%;获得KOG功能注释Unig ene共有328994条,涉及25个功能大类;GO库中注释到的Unigene共有607785条,有339437条Unigenes归入生物学过程,129556条Unigenes归入细胞组分,138792条Unigenes归入分子功能;KEGG库注释到163979条Unigenes,归属到345条代谢通路,与萜类相关的通路有8条,涉及943条Unigenes,萜类骨架生物合成MVA途径中共注释到5个相关酶基因,涉及44条Unigenes,萜类骨架生物合成DXP途径中共注释到7个相关酶基因,涉及20条Unigenes,倍半萜合成途径共注释到6个相关酶基因,涉及83条Unigenes,分别为法呢基二磷酸法呢基转移酶(farnesyl-diphosph ate farnesyltransferase)、角鲨烯单加氧酶(squalene monooxygenase)、NAD+依赖性法呢醇脱氢酶(NAD+-dependent farnesol dehydrogenase)、(E,E)-α-法呢烯合酶(alp ha-fa-rnesene synthase)、(-)-大根香叶烯合酶[(-)-germacrene D synthase]、丙二烯加氧酶(pre-mnaspirodiene oxygenase);此外鉴定出185221个SSR位点,其中最多的是单核苷酸基元,占比64.04%,其次是二核苷酸基元,占比23.21%。以上为深入探讨茉莉酸信号参与调控的百秋李醇生物合成机制及广藿香的遗传育种、基因改良研究提供了一定的参考信息。
[Abstract]:Pogostemon cablin (Balnco) Benth.) It is one of the ten wide medicinal herbs, which is one of the ten wide medicinal herbs, and is characterized by that the Shipai variety is optimal. In order to solve the problem that the resistance is reduced and the resource is short, considering the polyploid breeding method and the variety which are symmetrically fused with the protoplast, the chimera defects caused by the chemical induced polyploids can be avoided on the basis of improving the active ingredients and the yield of the stone cards, and moreover, On the basis of the original research, by adding MeJA inducing submandibular suspension cells and blank control group data analysis, establishing raw data, digging relevant transcription factors, and deeply exploring the biosynthesis mechanism of Baiqiu Li alcohol in the regulation of jasmonate signal, To provide new ideas and technical means for improving the quality and sustainable development and utilization of medicine source. The main results were as follows: (1) The protoplast isolation of suspended cells was optimized by the original experiment, and the effects of pretreatment, pH and temperature of enzyme on the protoplast isolation were investigated. In the end, it was determined that the suspension cells cultured for 9 to 15 days were in logarithmic growth phase were the raw materials, the mixed enzyme solution was cellulase, hemicellulase and pectinase, the mass volume ratio was 1.5%, 0.5%, 0.8% in turn, the osmotic potential of the enzyme solution was adjusted with 0.4 moL/ L mannitol, and the pH was 5. 8. At the temperature of 25 鈩，
本文编号：2301044
[Abstract]:Pogostemon cablin (Balnco) Benth.) It is one of the ten wide medicinal herbs, which is one of the ten wide medicinal herbs, and is characterized by that the Shipai variety is optimal. In order to solve the problem that the resistance is reduced and the resource is short, considering the polyploid breeding method and the variety which are symmetrically fused with the protoplast, the chimera defects caused by the chemical induced polyploids can be avoided on the basis of improving the active ingredients and the yield of the stone cards, and moreover, On the basis of the original research, by adding MeJA inducing submandibular suspension cells and blank control group data analysis, establishing raw data, digging relevant transcription factors, and deeply exploring the biosynthesis mechanism of Baiqiu Li alcohol in the regulation of jasmonate signal, To provide new ideas and technical means for improving the quality and sustainable development and utilization of medicine source. The main results were as follows: (1) The protoplast isolation of suspended cells was optimized by the original experiment, and the effects of pretreatment, pH and temperature of enzyme on the protoplast isolation were investigated. In the end, it was determined that the suspension cells cultured for 9 to 15 days were in logarithmic growth phase were the raw materials, the mixed enzyme solution was cellulase, hemicellulase and pectinase, the mass volume ratio was 1.5%, 0.5%, 0.8% in turn, the osmotic potential of the enzyme solution was adjusted with 0.4 moL/ L mannitol, and the pH was 5. 8. At the temperature of 25 鈩，
本文编号：2301044
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