【作者】 王沛；

【导师】 傅承新； 陈绍瑗；

【作者基本信息】 浙江大学 ， 植物学， 2006， 硕士

【摘要】 八角莲Dysosma versipellis(Hance)M.Cheng,隶属小檗科(Berberidaceae),药名鬼臼,为我国特有的濒危药用植物,其有效成分鬼臼毒素具有很强的抗肿瘤活性。而含鬼臼毒素的野生植物资源,由于人们多年滥挖乱采,资源濒于灭绝,难以满足医药领域的需要。本研究以八角莲无菌苗为材料,利用植物生理学、分子生物学、化学成分分析等研究方法与技术,建立了Ri质粒介导的八角莲毛状根的遗传转化体系,并对诱导转化形成的毛状根中的鬼臼毒素含量进行了分析,其主要研究内容及结果如下: (1)首次利用发根农杆菌15834建立起药用植物八角莲毛状根遗传转化体系。发根农杆菌15834感染八角莲茎段外植体30d后,从其切口处产生毛状根。在含NAA 1.0 mg/l和KT 0.2 mg/IMS培养基中预培养3d、无外源生长调节剂的MS培养基共培养3 d及添加100umol/l乙酰丁香酮,每天光照6h可得到较好的转化效果,毛状根的诱导率最高可达到30.9%。八角莲毛状根能在无外源激素的1/2MS固体培养基上快速自主生长。 (2)对八角莲毛状根基因组DNA,采用根据发根农杆菌Ri质粒基因组设计的引物,通过PCR检测、PCR—Southern杂交分析,结果表明:所获得的八角莲毛状根在564bp处有与rolC基因相同大小条带存在,发根农杆菌Ri质粒的rolC基因己整合到八角莲毛状根基因组并得到了表达。 (3)对在不同培养条件下形成的八角莲毛状根株系,以及由八角莲毛状根所形成的愈伤组织,采用化学方法提取其中的鬼臼毒素,应用HPLC分析了提取物中鬼臼毒素的含量,结果发现:八角莲毛状根中鬼臼毒素含量最高可达0.25%,高于组培根(离体培养)0.17%,接近于野生根0.29%。八角莲毛状根在每天6h光照培养条件下的鬼臼毒素含量(0.25%)要高于置于黑暗条件下培养的毛状根(0.11%);八角莲毛状根形成的愈伤组织中鬼臼毒素的含量要高于八角莲非转化根形成的愈伤组织,但低于由叶片、茎段形成的愈伤组织。更多还原

【Abstract】 Dysosma versipellis (Hance) M.Cheng is a local med herb that belongs to the Berberidaceae. Several natural chemical have been isolated from Dysosma Versioellis (Hance)M. Cheng. It is a source of highly valued podophyllotoxin which is currently being investigated for carinogenicity, cytotoxcity and antitumor activity.Based on these,we adopted techniques of plant physiology , molecular biology, pharmaceutical analysis interactively, established a system to transform hairy roots of Dysosma versipellis and gained the podophyllotoxin by the hairy roots culture. We also studied the content of podophyllotoxin, which would establish bedrock to obtain secondary metabolites of medicinal plants by hairy roots culture for industrialization.The main results were as follows: An efficient transformation system for the medicinal plant Dysosma versipelis was originally established with Agrobacterium rhizogenes 15834. Hairy roots could be obtained directly from the cut edges of stem explants 30 days after inoculation with the bacteria. The high frequency of transformation was obtained by three-day’s pre-culture on MS medium supplementeds with NAA 1.0 mg/1 and KT 0.2 mg/1 ,three-day’s co-culture on growth regulator free MS medium and addition AS(hydroxyacetosyringone) 100 umol/l, the inducing rate of hairy root was up to 30.9% . Hairy roots clones grew rapidly on solid growth regulator free 1/2 MS medium. The PCR, PCR-Southern analysis indicated that Ri plasmid left-hand T-DNA fragment(564bp) integrated into the genome of transformed roots.HPLC analysis showed that the contents of podophyllotoxin in hairy roots (0.25%) were higer than in vitro culture roots (0.17%) and close to wild roots (0.29%);the contents of podophyllotoxin in hairy roots under illumination was higer than hairy roots under gloominess(0.11%);that the contents of podophyllotoxin of callus from hairy roots(0.064%) was higer than callus from non-transformed roots and lower than callus from stems and leaves(0.17%).更多还原