【作者】 景宏伟；

【导师】 丁兰；

【作者基本信息】 西北师范大学 ， 细胞生物学， 2010， 硕士

【摘要】 香茶菜属植物为唇形科多年生草本、灌木或亚灌木,全世界共有150多种,广泛分布于热带及亚热带地区。对映–贝壳杉烷型二萜是该属植物的主要次生代谢产物,在其嫩枝叶中含量丰富,目前已从该属植物中分离得到400余种对映–贝壳杉烷型二萜化合物。作为多年生落叶植物，其体内含量极为丰富的对映–贝壳杉烷型二萜化合物每年都会随着植株枯萎被释放到土壤中。在长期进化过程中形成含量如此丰富且种类繁多的次生代谢产物很有可能对物种自身的生存和演化有着重要的意义,然而这些化合物究竟在其自然生境中发挥着何种生态学作用仍然未知。为了研究该类化合物的植物毒性，推断其是否在自然生境中发挥重要的生态作用，为系统开展该类化合物化感作用的研究提供依据，我们选取甘肃产蓝萼香茶菜中含量丰富且具结构代表性的对映–贝壳杉烷型二萜化合物Rabdosin B，运用生物测定的方法研究了其对经典化感受试植物“莴苣”幼苗根生长和根毛发育的影响，并探讨了该化合物Rabdosin B的作用机制。研究结果如下：（1）对映–贝壳杉烷型二萜Rabdosin B作用于莴苣幼苗48 h后对根的生长表现出显著的“低促高抑”现象。低浓度的Rabdosin B（20μM，40μM，80μM）显著促进根的生长。相反，当浓度达到120-200μM时，又表现出极强的抑制作用。另外，所有测试的浓度（10μM，20μM，40μM）均显著的抑制根毛的发育，且存在浓度依赖性。（2）Rabdosin B处理莴苣幼苗48 h后根尖成熟区细胞的长度和根尖分生区细胞的有丝分裂指数均发生了显著的变化，且与根的NGR变化趋势相一致。线性回归分析表明：根的NGR与根尖成熟区细胞的长度和有丝分裂指数之间存在着显著的相关性。由此得出，低浓度的Rabdosin B（20μM，40μM，80μM）显著促进根的生长是通过增加成熟区细胞的长度并提高分生区细胞的分裂活性而导致的。（3）高浓度的Rabdosin B（120μM，160μM，200μM）对根生长的抑制作用主要是由于影响了根尖成熟区细胞的长度和分生区细胞的分裂活性。彗星电泳和流式细胞仪分析的结果表明，200μM Rabdosin B使根尖细胞DNA发生严重损伤，从而导致G2期和S期细胞的周期阻滞，进而降低根尖细胞有丝分裂活性。（4）Rabdosin B和Ag+（乙烯作用抑制剂）都能够显著的抑制莴苣幼苗根毛的发育，而且它们对于乙烯利促进根毛顶端生长的作用均具有拮抗特性。Rabdosin B对莴苣幼苗根毛发育的抑制作用并不能通过同时添加乙烯利而恢复，说明Rabdosin B并没有影响乙烯的合成，而是影响了乙烯信号通路。（5）Ag+对乙烯抑制根的生长同样具有拮抗特性，而Rabdosin B对这一乙烯反应不具有拮抗特性。可能的一种解释是，Rabdosin B是在乙烯信号通路中特异性调控根毛顶端生长的下游支路中起作用，而对于乙烯调控根生长的支路不起作用；而Ag+则可能与乙烯受体发生相互作用，即在乙烯通路中的上游位点起作用，从而对乙烯调控根毛顶端生长和根的生长均表现出拮抗特性。我们的研究首次证实了对映–贝壳杉烷型二萜化合物Rabdosin B对莴苣幼苗根的生长表现出显著的“低促高抑”现象，同时对根毛发育过程具有较强的植物毒性作用。Rabdosin B在较低浓度条件下对根生长的促进作用主要是通过增加根尖成熟区细胞的长度和提高分生区细胞的分裂活性而导致的。高浓度的抑制作用主要是由于同时影响了成熟区细胞的长度和分生区细胞的分裂活性。有丝分裂指数的降低是由于DNA损伤导致不同时期细胞的周期阻滞。Rabdosin B对莴苣幼苗根毛发育的抑制作用很有可能是通过拮抗乙烯信号通路而导致的。推测该化合物很有可能作为一种潜在的化感物质在自然生境中发挥着重要的生态学作用，为系统开展该类化合物化感作用的研究具有重要的理论依据和生态学意义。更多还原

【Abstract】 Isodon plants are important genus of the Labiatae family and are perennial herbs,shrubs, or sub-shrubs. The genus is composed of approximately 150 species thatare widely distributed in tropical and subtropical regions. Ent-kauranediterpenoids are the main natural constituent in this genus and are especiallyabundant in fresh branches and leaves. To date, over 400 ent-kaurane diterpenoidshave been isolated from these plants. Being abundant and varied secondarymetabolites formed during the long evolutionary process, may have specialmeaning to the survival of species themselves. However, whether thesediterpenoids mediate interactions in natural surroundings is still unknown.In order to evaluate the phytotoxicity of the ent-kaurane diterpenoids, determinewhether they may play important ecological roles in natural surroundings andprovide foundations for the further systematic allelopathic research, rabdosin B,one of the abundant and structurally representative ent-kaurane diterpenoids fromIsodon japonica var. galaucocalyx obtained from Gansu, was adopted and theeffects on root growth and on root hair development were investigated in lettuceseedlings by bioassay method. Possible action mode of rabdosin B was alsoexplored in our research. The results are given as follows:(1) Rabdosin B, an ent-kaurane diterpenoid purified from the air-dried aerialparts of Isodon japonica var. galaucocalyx, showed a biphasic, dose-dependenteffect on root growth and a strong inhibitory effect on root hair development inlettuce seedlings (Lactuca sativa L.). Lower concentrations of rabdosin B (20μM,40μM, 80μM) significantly promoted the root growth, but its higher levels at120-200μM, by contrast, had inhibitory effects. Additionally, all of the testedconcentrations (10μM, 20μM, 40μM) inhibited root hair development of lettuceseedlings in a dose-dependent manner.(2) Different concentrations of rabdosin B affected both the size of mature cellsat the root-hair-forming regions and the mitotic activity of meristematic cells inlettuce seedlings’root tips. Regression analysis revealed a positive correlationbetween NGR and cell length and the mitotic index. Based on the results, weconcluded that the promotion effect of rabdosin B at the lower concentrationsresulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in lettuce seedlings’root tips.(3) Rabdosin B at higher concentrations inhibited the root growth by stronglyaffecting both the cell length in the mature region and the division of meristematiccells. Comet assay and cell cycle analysis demonstrated that the decrease ofmitotic activity of root meristematic cells was due to DNA damage induced cellcycle retardation of G2 phase and S phase at different times.(4) Both rabdosin B and Ag+ (an ethylene antagonist) inhibited root hairdevelopment of lettuce seedlings and showed antagonistic activity to ethephonenhanced root hair length. Seedlings with suppressed root hairs that were treatedwith rabdosin B could not be reversed by exogenous application of ethephon,suggesting that rabdosin B may interfere with the ethylene response pathwayinstead of ethylene biosynthesis.(5) Addition of rabdosin B did not overcome the inhibitory effect on rootgrowth caused by ethephon as Ag+ did. It is possible that rabdosin B interfereswith the downstream portion of the ethylene pathway, thus, specificallycontrolling root hair development instead of affecting root growth. However, Ag+may interact with an ethylene receptor at a point upstream in the ethylene pathway,thus, both affecting root hair development and root growth.Our research demonstrated the dual stimulatory and inhibitory effects ofrabdosin B on root growth and inhibitory effects on root hair development inlettuce seedlings. Further investigations on the underlying mechanism revealedthat the promotion of root growth resulted from enhanced cell length and increasedmitotic activity. In contrast, rabdosin B at higher concentrations inhibited rootgrowth by affecting both the cell length and cell division. Also, rabdosin B at 200μM could induce DNA damage in lettuce root tips, which resulted in the cell cycleretardation, and thus decreased the mitotic activity. In addition, we presumed thatrabdosin B inhibited root hair development through the mechanism of action as apotential ethylene action antagonist. Further studies need to be carried out toconfirm the role of rabdosin B in mediating the ecological interactions in naturalsurroundings.更多还原