雷公藤胚状体培养合成雷公藤甲素与总生物碱的初步研究

摘要

Tripterygium wilfordii Hook.f.is a kind of perennial woody vine plant,and it's period of growth is very long and the content of secondary metabolites is low.The natural resources can' t supply enough needs in medicine and agriculture.The way that modem cell engineering technology which produces the secondary metabolites can solve the natural resource shortage and protect the ecological environment for wild T.wilfordii.The leaves of one-year-old T.wilfordii were used to induce calli and suspension embryoid was established using the loose calli.The results showed that green embryoid could be easily induced from calli in 1/2MS+60 mL/L coconut juice liquid medium.The separate embryoid would grow and propagate in 1/2MS +0.5 mg/L NAA.The suitable condition for embryoid culture was 1/2MS basal medium with 1.0 mg/L NAA and 30 g/L sucrose,the initial inoculum was 1.5 g/100mL,pH 5.8,and the medium volume per flask was 120 mL/250 mL after screening inoculum size,pH,the medium volume per flask.The two culture systems were obtained according to the biomass accumulation after adding endophyte fungal elicitors.The triptolide was 1.07 and 1.44 times in embryoid than that in the leaves and in the natural root bark in 1/2MS+0.5 mg/L NAA+30 g/L sucrose+Y1(leaf endophytes),pH 5.8; the total alkaloids were 17.02 and 1.46 times in embryoid than that in the leaves and in the natural root bark in 1/2MS+0.5 mg/L NAA+30 g/L sucrose+G4(root endophytes),pH 5.8.The death rate of embryoid extracts was over 90％ after 72 h on Plutella xylostella L.3rd,the biological activity of embryoid extracts were the same as root bark.The content of triptolide and total alkaloid in embryoid which was induced from leaf calli matched the wild leaf and root bark,there was no significant difference of insecticidal activities between the embryoid extract and root bark.These results show that the culture of embryoid may be one of the pathways to solve the shortage of natural resource in T.wilfordii.%雷公藤(Tripterygium wilfordii Hook.f.)是一种多年生的木质藤本植物,生长周期长,次生代谢产物含量低,野生自然资源已经无法满足对其药用与农用的需求.为解决自然资源的短缺并保护雷公藤植物野生资源的生态环境,通过细胞工程技术生产雷公藤药用与农用活性物质是解决自然资源短缺,保护生态环境的途径之一.本研究中的雷公藤愈伤组织诱导自扦插一年生雷公藤叶片,将获得的愈伤组织建立细胞悬浮体系,诱导悬浮细胞得到生长稳定的胚状体培养体系.研究结果显示,添加60 mL/L椰汁的1/2 MS液体培养基可诱导愈伤组织形成浅绿色的胚状体,分离后的胚状体在1/2 MS添加0.5 mg/L a-萘乙酸(NAA)的液体培养基中可继续增殖分化.通过对接种量、培养基、装液量和pH值等培养条件的筛选和优化,在1/2MS培养基添加1.0 mg/L NAA和30g/L蔗糖,胚状体接种量为1.5 g/100 mL,pH值调至5.8,250 mL三角瓶中装液量为120 mL时最适合胚状体的培养生长；添加内生真菌诱导子,以生物量为指标,在1/2MS+0.5 mg/L NAA+30 g/L蔗糖+Y1(叶的内生菌),pH 5.8时雷公藤甲素在胚状体中的含量是叶中的1.07倍,是自然根皮粉中的1.44倍；在1/2MS+0.5 mg/L NAA+30 g/L蔗糖+G4(根的内生菌),pH 5.8时胚状体中总生物碱量17.02倍于叶中总生物碱量,1.46倍于自然根皮粉中总生物碱量.对3龄小菜蛾(Plutella xylostella)的毒杀试验表明,胚状体提取物与根皮粉提取物相似,100 mg处理浓度下,72 h时死亡率均在90％以上.通过雷公藤叶愈伤组织诱导的雷公藤胚状体培养体系,其次生代谢物(雷公藤甲素与雷公藤总生物碱)含量与野生雷公藤叶和根皮中的相当,胚状体提取物的杀虫活性与雷公藤根皮粉的杀虫活性无明显差异,说明雷公藤胚状体可作为解决雷公藤植物资源短缺的一条途径,为进一步研究和利用雷公藤这一植物资源提供基础资料.