菘蓝（Isatis indigotica Fort.）离体培养系统的建立及其次生代谢产物的分析

【作者】 陈敏；

【导师】 孙敏；

【作者基本信息】 西南师范大学 ， 植物学， 2001， 硕士

【摘要】 本文以药用植物菘蓝（Isatis indigotica Fort.）为试材，建立了愈伤组织培养系统、细胞悬浮培养系统和转化毛状根培养系统；对主要次生代谢产物靛蓝、靛玉红产量进行了检测，以期为利用生物技术手段工业化生产具有活性的次生代谢产物奠定一定的基础。 建立了愈伤组织培养系统。植物激素的种类、浓度与相互间的组合，外植体的种类与生理状态对外植体愈伤组织的诱导有密切关系。应用正交设计方法对菘蓝愈伤组织诱导及生长条件进行了优化，获得了愈伤组织诱导最佳培养基MS+4 mg.L^-1 NAA+0.5 mg.L^-1 6-BA +1mg.l^-12.4-D和最优外植体胚轴，诱导率为10O％；筛选到愈伤组织生长最佳培养基MS+1 mg.L^-12,4-D+0.5 mg.L^-16-BA+4 mg.L^-1NAA。测定了菘蓝愈伤组织30天内的生长速率，生物量和靛蓝、靛玉红产量均在25天达到最大值，分别为21.3g.L^-1DW、29.14mg.L^-1和20.63mg.L^-1。 建立了菘蓝的细胞悬浮培养系统。探讨了接种量、培养时间、碳源和氮源等条件对菘蓝细胞生长和靛蓝、靛玉红积累的影响。接种量在55 g.L^-1FW、以蔗糖为碳源且浓度为50 g.L^-1、总氮浓度(NH₄NO₃+KNO₃)为MS培养基氮源的1.5倍且NH₄NO₃／KNO₃为1856.25 mg.L（-1）／3562.5mg.L^-1条件下，在第15d收获为培养的最佳条件。 对发根农杆菌（Agrobacterium rhizogenes）介导的菘蓝遗传转化条件进行了优化。发根农杆ZZ 四南师范人学硕十学位论文 藩蓝离体培养系统的建立及其次生代谢产物的分析I茵菌株A4在菌液光吸收值为0．7左右时，致根能力最强。利用不同外植体愈伤组织、真叶、叶 柄、子叶和胚轴等进行遗传转化，于叶的转化根诱导率最高。萌发dss天的幼茵所处的生理状 态最适宜转化。预培养2天、共培养》刁天转化根诱导率最高。so p．L’乙酚丁香酮（As） 同时加入转化菌液和培养基中转化根诱导率最高，50 mg．L人S加入培养基中与前者诱导率相 近，后者更为经济。通过对转化毛状根中冠痤碱的检测，证明Ri质粒TDNA转移并整合到蒋 蓝基因组中。 对转化的毛状根进行了离体培养。选取4个毛状根优良无性系进行培养，HR4毛状根系 的生长阻和靛蓝、靛玉红产量最高，分别为 16．68 g．L’DW、138．28 nlg．L4和 103．58 mg．LJ； 对基本培养基进行了选择，毛状根在＊MS培养基中生长最快，靛蓝、靛玉红产量也最高； 对毛状根生长速率进行了测定，在 20 d生物量和靛蓝、靛玉红产量达到最大，分别为 15．36 g．L‘DW、124．42 mg．L’和 gi．85 p．L’。 应用高效液相色谱法对毛状根、原植物、愈伤组织和悬浮细胞中主要次生代谢产物靛蓝、 靛玉红产量进行了检测。结果表明，毛状根中靛蓝、靛玉红的产量最高；原植物叶其次，根的 产量比较低：悬浮细胞和愈伤组织中产量最低。毛状根中靛蓝产量为8．36mg．宫‘＊W，是原植 物的叶5。29 p．g‘’DW的1．58倍，比原植物根3二imp．g勺W提高了3．60倍；毛状根中靛玉红 的产夏比原植物的根、叶都高，为 6．26mp．g‘DVI，是原植物叶 3 07 p．g勺W的 2．e倍，原植 物根1．96 ITlg．g‘Dw的3．N倍。毛状根中靛诽量是愈伤组织L58 p合‘DW的5．29倍，是悬 浮细胞2．16 mg．g勺W的3．87借；靛玉红是觎组织］．06 mg．g’DW的5．gi倍，是悬浮铡＊．24 mg｛’DW)的5刀5倍。 建立的离体培养系统中，愈伤组织和细砒悬浮培养能检测到次生代谢产物靛蓝、靛玉红， 但是产量比较低；遗传转化系统获得的毛状根具有代谢完整的表达通路，次生代谢产物靛蓝和 靛玉红的产量都大大地提高。虽然三者培养条件都可以人工控制，且生长快，可以进行离体培 养，但毛状根中主要药用成分高，特别适合于大规模培养。更多还原

【Abstract】 Isatis indigotica Fort. was taken as the material in this dissertation. The callus culture system, suspension cell culture system and transgenic baby-root culture system were established and the yield of secondary metabolites, indigotin and indinibin, were detected in onier to do some basic work for production active secondai-y metabolites industrially by the biotechnological methods. Callus culture system was established. Different sorts, concentrations and combinatione of plant growth regulators were very important to induction of callus as well as parts and physiological status of ex-plants. Conditions of I. indigotica callus induction were optimized through the orthogonal test of [³⁴?The best medium was MS+4 mg.L?NAA+O.5 mg.L?6-BA +1 mg.L?2,4-D, and the best part of the ex-plants was hypocotyl. Under this condition, rate of the callus induction was 1000/0. The best medium for callus growth was screened out as MS+l mg.L?2,4-D+ 0.5 mg.L?6-BA+ 4 mg.L?NAA. The growth rate of callus was measured within 30 days every five days. The curve of the growth shaped ?? About 25 days later. bio-mass yield of the callus was 21.3 g.L扗W and the yeilds of indigotin was 29.14 mg.L) and indirubin ,20. 63 mg.L? The suspension cell culture system was established and inoculum densities, caiton sources and nitrogen sources were discusseii The yield of bio-mass and yield of indigotin and indirubin were highest in following conditions. The inoculum density was 55 g.L扚W; the caibon source added in the MS medium was sucrose and its concentration was 50 g.L? total nitrogen concentration in the medium was 1.5 times as much as that in MS medium and the ratio of ammonium and nitrate NI-I4 N03/KNO3 3 was 1856.25 mg.LP/3562.5 mg.L? and harvested at 15k?day. Conditions of transferred gene via Agrobacferiwn rhiwgenes were optimized Compared ^th strain R1000, A4 had stronger ability to induce haiiy roots when it抯 0D6(0 was about 0.7. Among Different parts of ex-plants, such as calli, leaves, petioles, cotyledons and hypocotyls, cotyledons had the highest inducing rate as 65.63%. Ex-plants sprouted out for 4桽 days, pre-cultured for 2? days and co- cultw^d with Agrobacterium rhizogenes for 2? days had the highest inducing rate. Added SOmg.U? AS in the medium also promoted the transferring rate. I-Iaiiy roots were identified by detection of opine. It indicated that T-DNA in Ri plasmid was transferred and integrated into I indigotica genome successfully. The transferred haiiy roots were cultured in vitm. Four hairy root clones were cultured and the result showed that clone FIR4 grew fastest and the yield of indigotin and indirubin were highest. The bio-mass yield of the clone HR4 was 16.68 gJJ1DW, and the yield of indigotin and indirubin were 138.28 mg.L?and 103.58 rng.L?respectively. Hairy roots grew fastest.in 1/2 MS medium without plant growth regulators among different basic media. And growth rate of the hairy root was detected In 20th day, the bio-mass ,yield of indigoiin and indirubin were highest as 15.36 g.L扗W 124.42 mg.L? and 91.85 mg.L?respectively. lndigotin and indirubin in different tissues, such as hairy roots, calli, suapension cells, roots and leaves of normal plants had been detected by I-IPLC. The result showed that the yield of indigotin and indin.tbin in hairy roots were highest. Yield of indigotin and indirubin in normal leaves were lower than that in hairy roots but higher than that in normal roots.更多还原