【摘要】：廣藿香(Pogostemon cablin(Balnco)Benth.)為唇形科刺蕊草屬植物,具有芳香化濁,開胃止嘔,解暑的功效,為“十大廣藥”之一,其中以“石牌”品種最優(yōu)。為解決抗性下降、資源短缺的現(xiàn)狀,考慮以廣藿香原生質(zhì)體對稱融合的多倍體育種方法改良品種,可以在提高石牌廣藿香有效成分及產(chǎn)量的基礎(chǔ)上避免化學(xué)誘導(dǎo)多倍體帶來的嵌合體缺陷;此外,在原有研究基礎(chǔ)上通過添加MeJA誘導(dǎo)子的廣藿香懸浮細(xì)胞與空白對照的轉(zhuǎn)錄組數(shù)據(jù)分析,建立原始數(shù)據(jù),挖掘相關(guān)轉(zhuǎn)錄因子,更深入地探究茉莉酸信號參與調(diào)控的百秋李醇生物合成機制,為提升廣藿香藥源品質(zhì)及其可持續(xù)開發(fā)利用提供新的思路和技術(shù)手段。主要研究結(jié)果如下:(1)廣藿香懸浮細(xì)胞原生質(zhì)體分離在原有實驗基礎(chǔ)上優(yōu)化了酶解分離方案,考察了預(yù)處理方法、酶液pH、酶解溫度對廣藿香原生質(zhì)體分離的影響。最終確定選取培養(yǎng)9-15 d處于對數(shù)生長期的廣藿香懸浮細(xì)胞為原材料,混合酶液選用纖維素酶、半纖維素酶和果膠酶,質(zhì)量體積比依次為1.5%、0.5%、0.8%,酶液滲透勢以0.4 moL/L甘露醇調(diào)節(jié),pH設(shè)為5.8,溫度25℃,以50rpm避光振蕩酶解12 h,最終產(chǎn)量為13.2×105個/g,活力為81.8%。(2)廣藿香懸浮細(xì)胞原生質(zhì)體培養(yǎng)原生質(zhì)體培養(yǎng)主要考察了培養(yǎng)方法、培養(yǎng)密度、培養(yǎng)基激素配比、銨鹽含量、碳源、酸水解酪蛋白含量。最終確定以銨鹽減半的MS1培養(yǎng)基進行海藻酸鈉包埋,激素選用0.2 mg/L NAA、2.0 mg/L 6-BA,培養(yǎng)密度為2.0×105個/mL,蔗糖添加量1.0%,酸水解酪蛋白500mg/L。在培養(yǎng)第6天觀察到細(xì)胞第一次分裂,14天左右細(xì)胞開始大量分裂,分裂頻率為6.91%,第16天形成細(xì)胞團,植板率為1.83%,培養(yǎng)第63天左右觀察到肉眼可見愈傷組織。(3)廣藿香懸浮細(xì)胞原生質(zhì)體融合本實驗以測定融合產(chǎn)物直徑變化范圍的物理選擇法來進行產(chǎn)物篩選,經(jīng)測量與統(tǒng)計,確立融合產(chǎn)物篩選范圍是69.33-87.35μm。采取高pH高鈣法對稱融合廣藿香原生質(zhì)體,考察了PEG濃度、融合時間、融合密度、融合液加入量對融合的影響。最終確定選用40%的PEG 6000進行化學(xué)促融30 min,融合液加入量為原生質(zhì)體懸浮液體積的0.5倍,融合密度2.0×105個/mL,聚合率57.19%,融合產(chǎn)物經(jīng)海藻酸鈉包埋培育2個月,觀察到再生愈傷組織,將愈傷組織轉(zhuǎn)移至誘導(dǎo)叢生芽培養(yǎng)基,暫未觀察到再生植株,培育方法有待進一步優(yōu)化。(4)茉莉酸甲酯(MeJA)誘導(dǎo)后廣藿香懸浮細(xì)胞轉(zhuǎn)錄組測序分別對添加了MeJA誘導(dǎo)子的廣藿香懸浮細(xì)胞與空白對照組進行了Illumina轉(zhuǎn)錄組測序,共獲得了662270條Unigenes,注釋率為74.04%;獲得KOG功能注釋Unig ene共有328994條,涉及25個功能大類;GO庫中注釋到的Unigene共有607785條,有339437條Unigenes歸入生物學(xué)過程,129556條Unigenes歸入細(xì)胞組分,138792條Unigenes歸入分子功能;KEGG庫注釋到163979條Unigenes,歸屬到345條代謝通路,與萜類相關(guān)的通路有8條,涉及943條Unigenes,萜類骨架生物合成MVA途徑中共注釋到5個相關(guān)酶基因,涉及44條Unigenes,萜類骨架生物合成DXP途徑中共注釋到7個相關(guān)酶基因,涉及20條Unigenes,倍半萜合成途徑共注釋到6個相關(guān)酶基因,涉及83條Unigenes,分別為法呢基二磷酸法呢基轉(zhuǎn)移酶(farnesyl-diphosph ate farnesyltransferase)、角鯊烯單加氧酶(squalene monooxygenase)、NAD+依賴性法呢醇脫氫酶(NAD+-dependent farnesol dehydrogenase)、(E,E)-α-法呢烯合酶(alp ha-fa-rnesene synthase)、(-)-大根香葉烯合酶[(-)-germacrene D synthase]、丙二烯加氧酶(pre-mnaspirodiene oxygenase);此外鑒定出185221個SSR位點,其中最多的是單核苷酸基元,占比64.04%,其次是二核苷酸基元,占比23.21%。以上為深入探討茉莉酸信號參與調(diào)控的百秋李醇生物合成機制及廣藿香的遺傳育種、基因改良研究提供了一定的參考信息。
[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，

本文編號：2301044