【作者】 杨玲；

【导师】 丁兰；

【作者基本信息】 西北师范大学 ， 细胞生物学， 2013， 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。该属植物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极可能对物种自身的生存及其生态环境的调节作用有着特殊的意义。本研究选择了三种对映-贝壳杉烷型二萜化合物Leukamenin E、Rabdosin B和Epinodosin，以拟南芥幼苗为受试对象，采用主根和侧根的测定、DR5::uidA标记系的GUS组织染色、根尖淀粉粒观察、植物生长素添加及生长素运输阻断等方法研究了这三种化合物对拟南芥幼苗根系生长的调节作用，并探讨了Leukamenin E和Rabdosin B对主根生长及侧根发育的影响与根部生长素水平变化的相关性。最后通过qRT-PCR方法检测了Leukamenin E对生长素极性运输输出载体PIN家族基因的表达的影响。本研究取得如下主要结果：（1）10μM内Leukamenin E对拟南芥幼苗主根没有影响，20-80μM范围内Leukamenin E显著地抑制主根长并具有浓度依赖性；10μM Leukamenin E显著地提高侧根长度和侧根原基总数，20μM显著地提高侧根原基总数；80μMLeukamenin E极显著地抑制侧根长度及侧根原基总数；所有受试范围5-80μMLeukamenin E均极显著地促进侧根原基密度。DR5::uidA标记系的GUS染色结果显示，10μM和20μM Leukamenin E能显著提高拟南芥幼苗主根根尖生长素响应基因的表达；40μM和80μM处理组无显著性影响。（2）20μM Rabdosin B显著地促进主根长，80-120μM Rabdosin B显著地抑制其主根长；40-120μM Rabdosin B显著地促进侧根长度、侧根原基总数以及侧根原基密度。DR5::uidA标记系的GUS染色结果显示，40-120μM Rabdosin B能显著提高拟南芥幼苗主根根尖生长素响应基因的表达；5-20μM处理组无显著性影响。（3）40-120μM Epinodosin虽然显著地抑制主根长，但却能显著地促进侧根长度以及侧根原基密度；80-120μM Epinodosin显著地抑制侧根原基总数。DR5::uidA标记系的GUS染色结果显示，80μM和100μM Epinodosin能显著提高拟南芥幼苗主根根尖生长素响应基因的表达；5-40μM和120μM处理组无显著性影响。（4）生长素极性运输抑制剂NPA联合Leukamenin E和Rabdosin B处理拟南芥幼苗的实验表明，10μM Leukamenin E和80μM Rabdosin B均对NPA引起侧根长度减小没有逆转恢复作用，但对NPA引起侧根原基数减少有显著性恢复作用。进一步的DR5::uidA标记系的GUS染色结果显示，10μMLeukamenin E和80μMRabdosin B能恢复由NPA引起的根尖部生长素分布的减少，以及减弱由NPA引起的叶部生长素分布的积累增多，缓解NPA对生长素极性运输的抑制效应。（5）80μM Leukamenin E以及120μM Rabdosin B和120μM Epinodosin能够抑制根尖柱细胞中造粉体合成，影响了拟南芥根的向地性。（6）Leukamenin E对拟南芥根中生长素输出载体基因PIN1、PIN2、PIN3、PIN4和PIN7的表达具有及显著的抑制作用。（7）这3种化合物对拟南芥幼苗生长素极性运输的影响与生长素极性运输抑制剂NPA相比，其作用机制完全不同。前者通过抑制生长素输出载体基因表达使生长素积累在根尖部，对叶部生长素分布无影响，而NPA使生长素积累在叶部，对根部生长素分布无影响。综上，3种对映-贝壳杉烷型二萜化合物Leukamenin E、Rabdosin B和Epinodosin在一定浓度范围内显著影响拟南芥根中生长素的极性运输，通过显著地抑制拟南芥根中生长素输出载体基因PIN1、PIN2、PIN3、PIN4和PIN7的表达，导致根部生长素大量积累，从而引起对拟南芥主根长度的抑制效应及对侧根发育促进作用。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 approximately150species that arewidely distributed in tropical and subtropical regions. Ent-kaurane diterpenoids arethe main natural constituent in this genus and are especially abundant in freshbranches and leaves. To date, over400ent-kaurane diterpenoids have been isolatedfrom these plants. Such abundant and varied secondary metabolites formed during thelong evolutionary process may have special meaning to the survival of speciesthemselves.The effects of Leukamenin E、Rabdosin B and Epinodosin on root growth ofArabidopsis thaliana seedlings were evaluated by using the methods of primary rootand lateral root vitality,GUS staining of DR5:: uidA marker, starch grain in root tip,addition of auxin and interruption of auxin transport. In addition, the correlationamong the root growth and initiation of lateral root with the levels of auxin inArabidopsis thaliana roots of Leukamenin E and Rabdosin B were studied in ourresearch. Finally, we determined the PIN family genes expression treated withLeukamenin E.The results are shown as follows:(1) Leukamenin E have no effect on primary root growth of Arabidopsis thalianaat concentration of10μM, while primary root is inhibited significantly with higherconcentration of Leukamenin E (20-80μM) and show a dose-dependent manner. Thelength and number of lateral root was promoted significantly at10μM, and thenumber of root primordium was promoted significantly at20μM, but its higher levelsat80μM, by contrast, had inhibitory effects. Additionally, all of the testedconcentrations (5-80μM) promoted Lateral root primordia density significantly ofArabidopsis thaliana. Histochemical GUS staining patterns of DR5::uidA indicated that DR5expressionat root tip was improved at lower concentrations of Leukamenin E (10μM,20μM)，and higher levels at40-80μM was no effect on it.(2) Primary root was promoted significantly at20μM, while inhibited significantlyat higher concentrations(80-120μM) of Rabdosin B. Additionally, the length andnumber of lateral root and Lateral root primordia density were all promotedsignificantly at tested concentrat-ions (40-120μM).Histochemical GUS staining patterns of DR5::uidA indicated that DR5expression at root tip was improved at concentrations of40-120μM of Rabdosin B.(3)Although Epinodosin inhibited primary root significantly at concentrations of40-120μM, the Lateral root primordia and length was promoted significantly.Thenumber of lateral root primordia was inhibited at concentrations of80-120μM.Histochemical GUS staining patterns of DR5::uidA indicated that DR5expressionat root tip was improved at concentrations of80and120μM of Epinodosin, othertreated had no effect.(4) The experiment of the interruption of auxin transport by NPA indicated that10μM Leukamenin E and80μM Rabdosin B could not recover the inhibitory effect oflateral root length by NPA, but in contrast, could significantly recover the number oflateral root primordium.Histochemical GUS staining patterns of DR5::uidA indicated that10μMLeukamenin E and80μM Rabdosin B could reduce DR5expression at cotyledoncompared with treatd use NPAalone, and improved expression at root tip.(5)80μM Leukamenin E,120μM Rabdosin B and120μM Epinodosin could affectThe root of geotropism of Arabidopsis thaliana.(6)The gens expression of PIN1、PIN2、PIN3、PIN4and PIN7was inhibited byLeukamenin E.(7)The influence mechanism of this three kinds of compound on polar auxintransport of Arabidopsis thaliana is quite another thing compered with NPA. The former lead to auxin accumulation in root tip by inhibiting the auxin output carriergene expression, but NPA is lead to auxin accumulation in cotyledon not influence onroot.In conclusion, the mechanism of Leukamenin E, Rabdosin B and Epinodosininhibit primary root and promote lateral root by the way of affecting polar auxintransport of Arabidopsis thaliana by inhibiting the auxin output carrier PIN familygens expression and lead to auxin accumulation in root tip.更多还原