[关键词]
[摘要]
目的 获得1,4-丁二胺-氮-甲基转移酶(putrescine N-methyltransferase,PMT)和莨菪碱-6β-羟化酶(hyoscyamine6β-hydroxylase,H6H)双基因共转化的颠茄发根的再生植株。方法 选用PMT和H6H双基因共转化的东莨菪碱高产发根单克隆T4,用1/2MS+1.0mg/LNAA固体培养基培养1、5、10d后,转入1/2MS固体培养基中诱导不定芽和不定根,培养条件均为(25±1)℃,55μmol/(m2.s),12h/d。采用PCR扩增检测再生植株的PMT、H6H和NPT-。结果 发根在1/2MS+1.0mg/LNAA固体培养基上培养5d后,转入1/2MS固体培养基上培养,60%的外植体能自发长出不定芽,1/2MS+0.1mg/LIBA固体培养基中诱导生根较1/2MS固体培养基强,15d产生的条数平均为9条,形成了完整的再生植株。PCR检测表明所有再生植株均为颠茄的转基因再生植株。结论 建立了颠茄转化发根再生体系,为实现颠茄托品烷类生物碱的代谢工程及开展分子育种奠定了基础。
[Key word]
[Abstract]
Objective To establish a regeneration protocol for hairy root lines of Atropa belladonna by co-transformed putrescine N-methyltransferase(PMT) and hyoscyamine 6β-hydroxylase(H6H) genes.Methods To transfer the transgenic hairy root line of T4 to 1/2 MS solid medium + 1.0 mg/L NAA and incubated 1,5,and 10 d at(25.0±1.0) ℃ with photoperiod(55 μmol/m2·s) 12 h/d so as to induce callus,then transfer the explants to hormone-free 1/2 MS solid medium and incubated at the same conditions to induce adventitious buds and roots,and then extract DNA from regeneration plantlets leaves so as to detect PMT,H6 H,and NPT-Ⅱ as well.Results After being cultured for 5 d on 1/2 MS solid medium + 1.0 mg/L NAA,the hairy root was transferred to hormone-free 1/2 MS solid medium,then adventitious buds ermerged from 60% explant and rooted spontaneously on 1/2 MS solid medium,but the rooting percentage was improved better at the presence in 1/2 MS + 0.1 mg/L solid sedium IBA than that in 1/2 MS solid medium.The average twigs were nine produced during 15 d and the complete regeneration plantlets were formed.PCR detection demonstrated that PMT,H6 H,and NPT-Ⅱ had inserted into the genome of the regeneration plantlets of A.belladonna.Conclusion The results presented here provide a protocol of transgenic hairy root regeneration of A.belladonna,which could be helpful in tropane alkaloids metabolic engineering and molecuar breeding.
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[基金项目]
国家自然科学基金资助项目(30500303);重庆市自然科学基金资助项目(CSTC2006BB5307)