【作者】 陈亚琼；

【导师】 李名扬； 赵德修；

【作者基本信息】 西南农业大学 ， 细胞生物学， 2003， 硕士

【摘要】 水母雪莲(Saussurea medusa．Maxi m)属菊科(Compositae)、菜蓟(Trib．Cynareae Less)、凤毛菊属(Saussurea DC．)、雪兔子属亚属(Subgen Eriocoryne (DC) Hook．f．)，是一种多年生株高8～15cm的丛生草本高山植物。主要生长于青海、甘肃、四川、云南、西藏等地4750～5600m的高山流石滩或高山草甸。其自然生长环境的土壤偏酸性，有机质含量极低，气候多变，最高月平均温度3～5℃，最低月平均温-19～-21℃，年降水量800 mm，无霜期仅有50d左右。 作为多年生一次开花结实植物，水母雪莲种子在0℃发芽，3～5℃生长，幼苗能在-21℃的严寒下生长5～6年后进入繁殖期。复头状花序于7月开花，花期30d左右，果期8～9月，成熟的线形瘦果由冠毛在风力作用下脱离母体而散播，随之植株枯黄死亡。 水母雪莲性苦、温。具有清热解毒、抗炎镇痛、祛风除湿，通经活络，壮阳补血、消肿、利痰、敛伤的功效。用于炭疽病、中风、风湿关节炎、崩漏带下、痛经、胎衣不下、肾虚腰痛、遗精阳痿等病症。近年来又发掘出如延缓衰老、抗肿瘤、抗辐射等更有价值的效用。 作为苯丙氨酸合成途径的一个分枝，类黄酮的生物合成途径是研究最早且最深入的次生代谢途径之一，其中关键酶有PAL(苯丙氨酸裂解酶)、4CL(4-香豆酸CoA连接酶)、CHS(查尔酮合成酶)、CHI(查尔酮异构酶)以及F3H和DFR等，它们相继在不同的物种中被克隆出来。现在发现转录因子P基因可以通过控制CHI、CHS和DFR的合成而影响类黄酮的生物合成。这些成果为利用分子克隆技术人工调控水母雪莲的类黄酮代谢奠定了理论基础。 由于长期的过量开采，已经被列为国家二级濒危植物的现状使雪莲单方和复方产品难以满足市场对天然药品的需求。植物学家对如何协调保护珍稀物种和大量获得其特有成分的关系给予了极大的关注，目前利用现代生物技术可以大规模地获得野生雪莲的有效药用成分—雪莲总黄酮，如人工栽培、诱导筛选高含量突变细胞系、大规模培养悬浮细胞，并开展了诱导转基因发状根、克隆转化和高量表达关键酶基因、反义基因抑制衍生代谢支路等研究工作。但是人工栽培受地域限制，发酵培养仍不能克服细胞退化和难以达到20L的规模，所以目前通过培养悬浮细胞大规模获得水母雪莲类黄酮还不成熟。通过培养水母雪莲的发状根将是获得雪莲类黄酮极有前途的方法，目前还是一项待填补的空白。 发根农杆菌(Agrobacterium rhizogenes)是一种革兰氏阴性土壤细菌，细胞内有200kb左右的双链闭环DNA—Ri质粒(root inducing plasmid)。根据转化细胞合成的冠瘿碱(opine)的类型不同，Ri质粒分为3种类型：甘露碱型(mannopine type)、黄瓜碱型(cucumopine type)和农杆碱型(agropine type)。T-DNA上携带的基因tms1,tms2与生长素自主合成有关，基因rolA，rolB，rolC，rolD，rolE与受体部位表现毛状根的现象有关，其中最重要的基因是rolB。 发状根(hairy root)是发根农杆菌感染大多数双子叶植物后产生的一种“病态”现象，其细胞整和有来自Ri质粒的外源T-DNA，具有单细胞起源、激素生长自主、多分枝、多根毛、纤细、向地性消失、生长迅速、次生代谢物含量较高甚至合成新成分等特点。通过毛状根得到的再生植株表现出矮化、早熟、叶皱缩、节间短、叶幅宽、世代期缩短、根系异常发达、产生大量的不定根和遗传稳定等特点。发根农杆菌遗传转化体系的这一特点在作物育种、植物基因工程、次生代谢物工厂化生产上有广阔的应用前景。 为了获得水母雪莲的毛状根，以筛选黄酮高含量转基因细胞系并研究水母雪莲的毛状根液体培养条件。本实验建立了水母雪莲的高频再生体系和发根农杆菌介导的遗传转化体系：确立了毛状根的组织化学和PCR检测方案；获得高产黄酮转基因毛状根细胞系；并优化了在转基因发状根中合成类黄酮的培养条件。为筛选水母雪莲代谢突变体和转基因研究奠定了一定的基础， 陈亚琼：m质粒介导水母雪莲的遗传转化及毛状根中决删合成的调节P义摘要也为研究类黄酮代谢途径的关键酶基因的转化、高效表达及作用机理提供了理想的实验体系。主要的实验结果如下： 1 水母雪莲高频再生体系的建立 通过体细胞胚胎发生途径和器官发生途径，水母雪莲（SQussurea medusa Maxim）可以在常温下获得再生植株。愈伤组织在诱导体细胞胚胎培养基（MSS）MS＋BA（4．0 ms／L）＋NAA（1，0 mg／L）＋GA。（2．0 mg／L）上培养 30 d后经过体细胞胚胎发生途径产生大量的丛生苗；子叶培养在诱导子叶产生丛生苗培养基（MSOC）MS＋BA（2．0。g／L）＋NAA（0．01 mg／L）＋甘氨酸（20～50。g／L）上，培养10d后以器官发生的方式产生丛生鼠 叶片完成这一过程需要在诱导叶片产生丛生苗培养基（MSOL）MS＋BA（1．omg／L）＋NAA（0．2 mg／L）＋甘氨酸（20～50mg／L）上培养 15d左右。实验证明：①高山植物水母雪莲的再生过程可以无需冷处理。②比较两种再生系统，器宫发生系统有周期短、均匀度高同步性好、平均成苗数高的优势。③添加甘氨酸 10～50 mg／L和活性炭 0二～0．5％利于再生。④再生苗总黄酮含量达 4％，是生药含量的 4～8倍。更多还原

【Abstract】 As a kind of Chinese traditional medical herb, Sattssurea medusa Maxism belongs to Compositae familia, Saussurea DC.geneus, Subgen Eriocoryne (DC) Hook.f. This perennial herb grow in gravelly soil or meadom at altitude of 4750-5600m in Qinhai ,Gansu ,Sicuan ,Yunnan and Tibet.The poor nutritional condition and killing freeze climate are the features of natural growing environment of Saussurea medusa Maxism. It can germinate at 0癈 and develop at 3-5 C even suffer from chilliness of -21 C.At the fifth or sixth year, it ablooms at July, fruits at August or September. When the seeds are scattered by wind, the plant turn to death.Saussurea medusa Maxism was used by several minorities in China, because it have some special pharmaceutical effects such as detoxification, detumescence, abirritation, antitumor, anti-radiation and anti-ageing etc.But Saussurea medusa Maxism was on the edge of annihilation for being exploited excessively at a long time.still the natural resource of it is hard to meet the increasing need of market .So ths botanical scientist paid more attention to the relationship between protecting this endangered species and producing it’s pharmaceutical ingredient. Presently, the scientist have made out some successes by using biotechnology and molecuology, for example, planting out regeneration shoots, culture suspended cell in ferment tank, screening mutant tissue with high content of flavonoids, cloning and sup-expression of key enzyme, restraining derivative pathway by anti-DNA or anti-RNA and inducing genetic transformated hairy root.Agrobacterium rhizogenes (Rhizobiacease) is a kind of Gram-negative bacterium, which contains a big Ri plasmid (200kb)-root inducing plasmid. There are three types of Ri plasmid, mannopine type, cucumopine type and agropine type, according to the type of opine synthesized in transgenic cell. The gene rolA, rolB, rolC and rolD from Ri T-DNA play an important role in hairy root induction in transformed plants, characterized by an abundant proliferation of adventitious roots from the site of infection.The hairy root is originated from single cell and grows quickly without auxin. More interesting, the content of secondary metabolite always increased markedly in hairy root. So the transgenic system by Ri plasmid would be wildly used in plant breeding, plant gene engineering and pharmaceutical element production.In order to establish the genetic transformation system of Saussurea medusa Maxism byAgrobacterium rhizogenes, some work were done .The main results were following:1 establishment of regeneration systemsTwo systems of regeneration from Saussurea medusa Maxim were established without cold treatment.The somatic embryos were induced from callus cultured in MISE for 35 days. The shoots were induced from cotyledon after cultured in MISC 20 days, and from leave which were cultured in MISL .The experiment showed that the carbon and glycine in the medium could help to increase the regeneration rate to 95%. Compared with the regeneration from somatic embryos, the organic regeneration system had some characters such as short period, high efficiency and low abnormal shoot rate. More importently, the content uf total flavonoids in shoot was about 4%, which was 3-4 times of wild type.2 establishment of genetic transformation system by Agrobacterium rhizogenesThe different types of explants (hypocotyls, cotyledon, leaf blade, petiole) were infected by strain R1601 of Agrobacterium rhizogenes. Pre-cultured in the medium MS + 2,4 -D( 1.0 mg/L)+BA (0.2 mg/L) for 2-4d, the plant material were pre-treated in sterile sucrose solution (80-100 g/L) for 1 hour, then quickly dipped into R1601 which had been induced by As (50 mol/L) and proline (250 mg/L) for 30 min. The explants were co-cultured on the same medium for 3-4d in dark after dried by sterile paper carefully. When the co-culture was over ,the materials were washed by sterile water 5-7 times ,then disgermed in liquid MS containing Cef (500 mg/L) for 30min. After cultured in inducing medium MS更多还原