【作者】 祁林林；

【导师】 丁兰；

【作者基本信息】 西北师范大学 ， 植物学， 2009， 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多个种,广泛分布于热带及亚热带地区。对映-贝壳杉烷二萜化合物是该属植物的主要次生代谢产物,目前已从该属植物中分离得到400余种该类化合物。在长期进化过程中形成的如此含量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有着特殊的意义,然而这些化合物是否在其自然生境中发挥着某些生态作用仍然不清楚。为了确定该类化合物的植物毒性,推断其是否有可能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,我们选取了4种含量丰富且具结构代表性的对映-贝壳杉烷二萜化合物,运用生物测定的方法研究了它们对常见受试植物莴苣的植物毒性,并探讨了具有较强植物毒性的化合物Leukamenin E可能的作用机制。研究结果如下:①4种二萜化合物Leukamenin E、Weisiensin B、Rabdosin B和Epinodosin在短期实验条件下对莴苣的种子萌发率、苗长、鲜重和干重等均没有表现出显著的影响,但是幼苗的根长和根毛发育却都受到了显著的影响。160μM的4种二萜化合物处理后,莴苣幼苗的根毛发育都受到了不同程度的抑制。除Rabdosin B以外,其余3种化合物在较高浓度(120-160μM)条件下都能够显著抑制幼苗的根长,化合物Rabdosin B在40-160μM的浓度范围内对莴苣幼苗根的生长表现出显著的促进作用。结果表明该类化合物能够特异影响莴苣幼苗根的生长及根毛发育,很有可能在其自然生境中发挥着重要的生态作用。②Leukamenin E在较低浓度(10-80μM)条件下就能够对莴苣幼苗的根毛发育过程产生显著的抑制作用,80μM的Leukamenin E就能够完全抑制根毛发育过程。而在较高浓度(50-200μM)条件下,根的净生长速率(NGR)也受到了显著的抑制。③100和200μM的Leukamenin E处理后莴苣幼苗的根系活力显著下降,说明高浓度的Leukamenin E处理后根系的代谢能力减弱,但是根系活力的降低可能只是高浓度条件下的一种综合反映,并不是根的生长受到抑制的直接原因。④Leukamenin E处理后根尖分生区的有丝分裂指数也受到了显著的抑制,而且与根的NGR变化趋势相一致,线性回归分析的结果表明根的NGR与有丝分裂指数具有显著的相关性,说明根的生长受到抑制的主要原因是由于根尖分生区的有丝分裂行为受到抑制的结果。有丝分裂指数的降低主要是由于前期细胞比率的下降,这种现象很有可能与细胞周期阻滞有关。⑤高浓度(100和200μM)的Leukamenin E处理后根尖分生区出现一些异常有丝分裂现象,主要与染色体受到损伤或者纺锤体微管受到影响有关系。纺锤体微管受到影响有可能阻止姐妹染色单体在后期的分离,从而增加了中期细胞相对于分裂期细胞的比率。⑥Leukamenin E和乙烯作用拮抗剂Ag+都能够显著抑制莴苣幼苗的根毛发育过程,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。Leukamenin E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Leukamenin E并没有影响乙烯的合成,而是和Ag+一样影响了乙烯的作用通路,但是Leukamenin E和Ag+抑制根毛发育的具体方式存在着差异。⑦Ag+对乙烯抑制根的生长这一反应同样具有拮抗特性,而Leukamenin E对这一乙烯反应不具有拮抗特性。可能的一种解释是,Leukamenin E是在乙烯通路中特异性调控根毛顶端生长的下游支路中起作用,而对于乙烯调控根的生长的支路则不起作用,该化合物在较高浓度条件下对根的生长的抑制作用主要是由于根尖分生区有丝分裂行为受到抑制所引起的;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,从而对乙烯调控的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-贝壳杉烷二萜化合物对莴苣幼苗根的生长及根毛发育过程具有较强的植物毒性。Leukamenin E在较低浓度条件下就能够抑制莴苣幼苗的根毛发育过程,其对根毛发育的抑制作用可能与影响乙烯作用通路有关系,而在较高浓度条件下对根的生长的抑制作用则主要是由于根尖分生区有丝分裂行为受到抑制所引起的。该类化合物较强的植物毒性预示着它们很有可能在自然生境中发挥着重要的生态作用,系统开展该类化合物化感作用的研究具有理论依据和重要的生态学意义。更多还原

【Abstract】 Isodon plants are labiatae perennial herbs, shrubs, or sub-shrubs. The genus is composed of approximately 150 species that are widely distributed in tropical and subtropical regions. Ent-kaurene diterpenoids are the main natural products in this genus from which more than 400 have been extracted. Such abundant and varied secondary metabolites formed during the long evolutionary process may have special meaning to the survival of species themselves. However, whether these diterpenoids possess certain ecological role in natural surroundings is still unknown.In order to evaluate the phytotoxicity of ent-kaurene diterpenoids, determine whether they may play important ecological roles in natural surroundings and provide foundations for the further systematic allelopathic research of these compounds, four abundant and structurally representative ent-kaurene diterpenoids were adopted and their phytotoxicity on lettuce, a commonly used test organism, was investigated by bioassay method. Possible modes of action of the most phytotoxic compound leukamenin E was also explored in this paper. The results are given as follows:①All of the four diterpenes including Leukamenin E, Weisiensin B, Rabdosin B and Epinodosin did not affect germination rate, hypocotyl length, fresh weight and dry weight of lettuce in the short term experiment, but both primary root length and root hair development were prominently affected. Root hair development could be blocked to different degrees when treated with either of the four diterpenes at the concentration of 160μM. All of the four diterpenes except Rabdosin B inhibited the root growth significantly at the higher concentrations (120-160μM). Rabdosin B at the concentrations of 40-160μM stimulated the root growth significantly. The results indicated that the compounds can specifically affect the root growth and the root hair development of lettuce seedlings, possibly playing important ecological role in natural surroundings.②Root hair development of lettuce seedlings was strongly inhibited even at the lower concentrations (10-80μM) of Leukamenin E and 80μM Leukamenin E could completely block it. At the higher concentrations (50-200μM), the NGR of primary root was also significantly inhibited.③Root vitality of lettuce seedlings decreased significantly when treated with 100 and 200μM Leukamenin E, indicating that the higher concentrations of Leukamenin E reduced the root metabolic ability. However, reduction of root vitality might be a comprehensive response at the higher concentrations instead of the direct reason of root growth inhibition.④Mitotic index of root apical meristem was significantly inhibited in a pattern similar to the NGR after Leukamenin E treatments. Linear regression analysis revealed a positive correlation between NGR and mitotic index, suggesting that inhibition of root growth may result from suppressed mitotic activity. Reduction of MI was mainly due to the decreased percentage of prophase cells, which may be related to cell cycle arrest.⑤Some aberrant mitosis occurred in the root apical meristem treated with higher concentrations (100 and 200μM) of Leukamenin E, some of which may be related to chromosome damage and alterations in spindle microtubules. It was supposed that altered spindle microtubles might delay chromosome segregation and thus increase the percentageof metaphases relative to the mitotic cells.⑥Both Leukamenin E and an ethylene antagonist Ag+ inhibited root hair development of lettuce seedlings and showed antagonistic activity to ethephon enhanced root hair length. Seedlings with suppressed root hairs that were treated with Leukamenin E could not be reversed by exogenous application of ethephon, suggesting that Leukamenin E may interfere with the ethylene response pathway instead of ethylene biosynthesis as with Ag+. Differences between the interfering mechanism of Ag+ and Leukamenin E are apparent.⑦Addition of Leukamenin E did not overcome the inhibitory effect on root growth caused by ethephon as Ag+ did. It is possible that Leukamenin E interferes with the downstream portion of the ethylene pathway, thus, specifically controlling root hair development instead of affecting root growth. Its inhibitory effects on root growth at the higher concentrations are largely attributed to the decreased mitotic index in the root apical meristem, while 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 for the first time the strong phytotoxic effects of ent-kaurene diterpenoids on root growth and root hair development in lettuce seedlings. Leukamenin E at the lower concentrations could inhibit root hair development possibly through interfering with ethylene response pathway, while its inhibitory effects on root growth at the higher concentrations were largely due to the suppressed mitotic activity in root apical meristems. The strong phytotoxicity of these compounds suggested their possible important ecological role in natural surroundings. Systematic research into the allelopathic effects of these compounds are of theoretical foundations and important ecological meanings.更多还原