WU Shun,SUN Jianchun,ZHOU Kai,et al.Induction of Hairy Root of Uncaria rhynchophylla and Content Determination of Rhynchophylline[J].Northern Horticulture,2019,43(15):49-54.[doi:10.11937/bfyy.20190144]
钩藤毛状根的诱导及其钩藤碱含量的测定
- Title:
- Induction of Hairy Root of Uncaria rhynchophylla and Content Determination of Rhynchophylline
- Keywords:
- Uncaria rhynchophylla; AS; Agrobacterium rhizogenes; hairy root; rhynchophylline
- 文献标志码:
- A
- 摘要:
- 为建立钩藤毛状根的培养体系,以钩藤组培无菌苗为试材，利用发根农杆菌A4、ATCC15834、R16011侵染钩藤外植体，探讨了不同钩藤外植体、菌液浓度、AS浓度、干燥处理时间对毛状根诱导率的影响。结果表明：以钩藤叶片作为外植体,预培养3 d，以OD600=0.6的A4菌株浸泡30 min后，干燥处理1 h，转入含100 μmol?L-1乙酰丁香酮无激素WPM培养基中共培养3 d，在含600 mg?L-1头孢克肟筛选培养基上能获得较高的毛状根诱导率，毛状根诱导率达53.66%。PCR扩增鉴定表明，钩藤细胞核基因组中已成功整合发根农杆菌的发根基因。钩藤毛状根在无激素1/2MS液体培养基中培养20 d后，部分毛状根中钩藤碱含量明显提高，最高含量是带钩枝条中的2.06倍，达1.211 mg?g-1。
- Abstract:
- In order to found the culture system of hairy root for Uncaria rhynchophylla,the effect of different explant,concentration of bacterium,AS concentration,drying time on induction of hairy root was studied by using the sterile seedlings as a material and making Agrobacterium rhizogenes(A4,ATCC15834,R16011) infect these explants.The results showed that the induction rate of hairy root was higher when sterile leaves were used as the explant,preincubated for 3 days,soaked for 30 minutes by OD600=0.6 A4 solution,dried for 1 hour,cultured in hormone-free WPM medium with 100 μmol?L-1 AS,selected on screening medium with 600 mg?L-1 cefminox.The induction rate of hairy root was 53.66%.It was confirmed by PCR analysis that the rooting gene in Agrobacterium rhizogenes was integrated into the nuclear genome for Uncaria rhynchophylla.The content of rhynchophylline increased in some hairy roots after hairy roots were cultured in hormone-free 1/2MS liquid medium for 20 days,in which the highest content of rhynchophylline reached a level of 1.211 mg?g-1 DW and was 2.06 times as much as hook branch.
参考文献/References:
[1]罗小金,王春梅.异钩藤碱对阿尔茨海默病及其它中枢神经系统疾病的药理作用研究进展[J].医学研究生学报,2015,28(10):1112-1115.[2]石京山,余俊先,陈修平,等.钩藤总碱、钩藤碱和异钩藤碱的药理作用[J].Acta Pharmacologica Sinica,2003(2):97-101.[3]何昱,洪筱坤,王智华.钩藤及其有效成分的药理研究进展[J].上海中医药杂志,2003(11):57-60.[4]黄华,丁伯平.钩藤生物碱对中枢神经系统的药理作用研究进展[J].现代药物与临床,2013,28(5):806-810.[5]李娜,丁伯平,黄华,等.钩藤碱阻滞钙离子通道的药理作用研究进展[J].现代药物与临床,2014,29(9):1073-1076.[6]宋欣濛,薛睿,季宇彬.钩藤中吲哚类生物碱化学成分及药理活性研究进展[J].亚太传统医药,2014,10(5):64-68.[7]吴伟明,李志峰,欧阳辉,等.钩藤化学成分分析[J].中国实验方剂学杂志,2015,21(18):56-58.[8]焦扬,王模强,华丹,等.中药钩藤化学成分研究[J].天津医科大学学报,2013,19(2):107-109.[9]李金玲,赵致,龙安林,等.贵州野生钩藤生长环境调查研究[J].中国野生植物资源,2013,32(4):58-60.[10]谈荣慧,张金家,赵淑娟.丹参毛状根的诱导及培养条件的优化[J].中国中药杂志,2014,39(16):3048-3053.[11]黄伟剑.广藿香毛状根培养及细胞悬浮系建系的研究[D].广州:广东药学院,2015.[12]孙晶,徐洁森,赵立子,等.北柴胡毛状根诱导及其植株再生体系的建立[J].药学学报,2013,48(9):1491-1497.[13]吕政,张淑丽,路放,等.关苍术毛状根培养体系建立及其多糖含量测定[J].中国药学杂志,2014,49(16):1386-1392.[14]张伦,张熙,吴建军,等.银杏毛状根的诱导和培养[J].贵州科学,1999(2):132-134.[15]毛堂芬,刘作易,贺定祥,等.钩藤的组织培养与植株再生[J].植物生理学通讯,2006(6):1127-1128.[16]黄瑞松,覃冬杰,张鹏,等.广西不同产地和不同采收期大叶钩藤中钩藤碱定量分析[J].中草药,2012,43(1):178-181.[17]盛立柱,宋冰,戴月婷,等.农杆菌介导的刺芹侧耳遗传转化体系的建立[J].菌物学报,2015,34(4):653-661.[18]陈翠娜,冯德玉,张新,等.乙酰丁香酮及共培养基pH对棉花遗传转化的影响[J].上海交通大学学报(农业科学版),2013,31(1):18-22.
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备注/Memo
第一作者简介：吴顺(1976-),男，博士,副教授,现主要从事药用植物资源学及其基因工程育种等研究工作。E-mail：2568662083@qq.com.基金项目：湖南省教育厅科学研究资助项目(16C1677);中南林业科技大学2018年大学生研究性学习与创新性实验计划资助项目。收稿日期：2019-03-05