三叶青毛状根的诱导及其液体培养体系的研究
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摘要
为建立三叶青毛状根的诱导和液体培养体系,采用发根农杆菌株系C58C1和ATCC15834进行毛状根的诱导,通过无菌三叶青叶片预培养、浸染、共培养、筛选、形态和PCR鉴定等过程获得三叶青毛状根根系,并对三叶青毛状根和两年生块根总黄酮进行比较分析。试验结果表明,发根农杆菌C58C1能从三叶青叶片诱导出毛状根,三叶青毛状根在不添加生长素的1/2MS液体培养基中生长状态良好,其总黄酮含量约是2年生三叶青块根的1/5。
In order to establishment of hairy root induction and liquid culture systems of Tetrastigma hemsleyanum. Agrobacterium rhizogenes C58 C1 and ATCC15834 strains were used here. The candidate hairy roots of Sanyeqing were obtained by pre-culture, infection, co-cultivation and selection. Only the C58 C1 strain could successfully induce the hairy roots in our lab, and the hairy roots grew well in 1/2 MS liquid medium without adding auxin. The total content of flavonoid of hairy roots was about twenty percent of the 2-year old tuberous roots.
In order to establishment of hairy root induction and liquid culture systems of Tetrastigma hemsleyanum. Agrobacterium rhizogenes C58 C1 and ATCC15834 strains were used here. The candidate hairy roots of Sanyeqing were obtained by pre-culture, infection, co-cultivation and selection. Only the C58 C1 strain could successfully induce the hairy roots in our lab, and the hairy roots grew well in 1/2 MS liquid medium without adding auxin. The total content of flavonoid of hairy roots was about twenty percent of the 2-year old tuberous roots.
引文
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[22]王静,郭万里,孔亚泽,等.珍稀药用植物三叶青的高效微繁体系研究[J].浙江理工大学学报,2016,35(7):636-642.
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[24]Sadiye H,Aynur G,Ismail H A,et al.Induction of Gentiana cruciata hairy roots and their secondary metabolites[J].Biologia,2011,6(4):618-625.
[25]田思迪,李云芳,张宗申,等.影响金铁锁毛状根生长及皂苷积累的因素[J].时珍国医国药,2012,23(4):824-826.
[26]孙彬贤,杨光孝,汪沁琳,等.人参毛状根合成人参皂苷培养条件的优化[J].中成药,2003,25(9):746-748.
[2]南京中医药大学.中药大辞典,下册[M].上海:上海科学技术出版社,1977.
[3]张胜强,张洪艳,黄建伟,等.Notch1下调对三叶青黄酮抑制食管癌ec9706细胞迁移和侵袭的影响[J].中国实验方剂学杂志,2017,23(5):162-167.
[4]Lin Z,Chen L,Qiu Q,et al.Isolation and identification of antiproliferative compounds from the roots of Tetrastigma hemsleyanum against MDA-MB-435S cell lines[J].Pakistan Journal of Pharmaceutical Sciences,2016,29(4):1 171.
[5]丁小鸽,张春椿,秦海燕,等.三叶青乙酸乙酯提取物对人胰腺癌PANC-1细胞凋亡的影响及机制研究[J].毒理学杂志,2017,31(3):182-186.
[6]袁雨,朱炳喜,刘军权,等.三叶青水提取物对nk细胞杀伤胃癌bgc-823细胞的影响[J].胃肠病学和肝病学杂志,2016,25(6):633-637.
[7]张祺箐,冯正权.三叶青黄酮干预荷癌小鼠treg细胞生成及其cd152表达的研究[J].新中医,2017,49(11):5-8.
[8]Zhong L R,Chen X,Wei K M.Radix Tetrastigma hemsleyanum flavone induces apoptosis in human lung carcinoma a549 cells by modulating the mapk pathway[J].Asian Pacific Journal of Cancer Prevention,2013,14(10):5 983-5 987.
[9]魏克民,丁刚强,浦锦宝,等.中草药三叶青抗肿瘤作用机制实验研究和临床应用[J].医学研究杂志,2007,11:41-43.
[10]陈红,廖淑彬,陈丹,等.闽产三叶青地上部分抗炎提取物的急性毒性研究[J].福建医科大学学报,2017,51(5):287-290.
[11]朱美晓,鄢连和.浙江省珍稀濒危畲药金线吊葫芦资源调查[J].中药材,2014,37(5):766-770.
[12]Dai Y J,Shen Z G,Liu Y,et al.Effects of shade treatments on the photosynthetic capacity,chlorophyll fluorescence,and chlorophyll content of Tetrastigma hemsleyanum Diels et.Gilg[J].Environmental&Experimental Botany,2009,65(2):177-182.
[13]王静,郭万里,杨东风,等.珍稀药用植物三叶青的研究进展.植物学研究,2015,4:39-46.
[14]彭昕,张剑,何军邀,等.三叶青松散型和致密型愈伤组织悬浮培养及黄酮积累的比较研究[J].中草药,2012,43(3):577-580.
[15]刘东,杨峻山.中国特有植物三叶青化学成分的研究[J].中国中药杂志,1999(10):35-36+62.
[16]李瑛琦,陆文超,于治国.三叶青的化学成分研究[J].中草药,2003,34(11):982-983.
[17]曾美玲,沈耐涛,吴赛伟,等.基于UPLC-Triple-TOF/MS方法的三叶青化学成分分析[J].中草药,2017,48(5):874-883.
[18]Liu H X,He L,Huang R M,et al.Chemical constituents of the rhizomes of Tetrastigma hemsleyanum[J].Chemistry of Natural Compounds,2015,51(6):1 077-1 079.
[19]Ye C L,Liu X G.Extraction of flavonoids from Tetrastigma hemsleyanum Diels et.Gilg and their antioxidant activity[J].Journal of Food Processing&Preservation,2015,39(6):2 197-2 205.
[20]Abraham J,Thomas T D.Hairy root culture for the production of useful secondary metabolites[M].Biotechnology and Production of Anti-Cancer Compounds.2017.
[21]Du S,Xiang T,Song Y,et al.Transgenic hairy roots of Tetrastigma hemsleyanum:Induction,propagation,genetic characteristics and medicinal components[J].Plant Cell Tissue&Organ Culture,2015,122(2):373-382.
[22]王静,郭万里,孔亚泽,等.珍稀药用植物三叶青的高效微繁体系研究[J].浙江理工大学学报,2016,35(7):636-642.
[23]Murashige T,Skoog F.A revised medium for rapid growth and bio assays with tobacco tissue cultures[J].Physiol Plant,2010,15(3):473-497.
[24]Sadiye H,Aynur G,Ismail H A,et al.Induction of Gentiana cruciata hairy roots and their secondary metabolites[J].Biologia,2011,6(4):618-625.
[25]田思迪,李云芳,张宗申,等.影响金铁锁毛状根生长及皂苷积累的因素[J].时珍国医国药,2012,23(4):824-826.
[26]孙彬贤,杨光孝,汪沁琳,等.人参毛状根合成人参皂苷培养条件的优化[J].中成药,2003,25(9):746-748.
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