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1、河南大学 硕士学位论文 盐胁迫下拟南芥乙烯合成突变体生理生化表型 姓名：徐俊凯 申请学位级别：硕士 专业：植物学 指导教师：江静 2011-06 I 摘摘 要要 植物激素乙烯调节各种植物生长发育和胁迫反应过程。 但是有关盐胁迫条件下乙烯 生物合成及信号转导途径尚需深入探讨。 乙烯合成酶(ACC synthase, ACS)基因家族中共有 12 个基因，本论文选取其中一个基 因，命名为 AtESTH (Arabidopsis thaliana gene for Ethylene Synthesis factor)，研究其在 NaCl 胁迫下的生理表型及相关分子机制。萌发和根生长实验表明，与野生型
2、(wild type,WT)相比， Atesth 幼苗对 NaCl 具有一定的抗性， 表现在： 150mM NaCl 处理条件下， WT 萌发率约为 7%， 而 Atesth 萌发率为 15%， 其萌发率是 WT 的 2 倍多； 在 150mM NaCl 处理条件下生长 10 天的幼苗，WT 平均主根根长平均为 0.35 (0.06) cm，Atesth 根长为 0.95 ( 0.03) cm，后者是前者的 3 倍左右。半定量实验结果显示，在 150mM NaCl 处理 下，AtESTH 表达量与 control 相比明显上升，证明在转录水平上 AtESTH 参与拟南芥对 NaCl 胁迫应答。
3、 H+-ATP 酶活性对维持植物细胞内的离子平衡具有重要意义。实验结果显示，在对 照条件下， WT 和 Atesth 植株的 H+-ATP 酶活性相差不大； 150mM NaCl 处理 72 小时后， Atesth 突变体中该酶活性明显上升，说明 H+-ATP 酶活性变化参与 AtESTH 对 NaCl 胁迫 调节。150mM NaCl 处理后，激光共聚焦扫描结果显示，WT 中 H+探针的荧光强度提高 了 11%，Atesth 中荧光强度提高了 33%。这说明盐胁迫下 AtESTH 通过对 H+-ATP 酶活 性调节，影响了拟南芥对 NaCl 胁迫的抗性。 正常情况下，植物细胞内维持一定水平的
4、活性氧(Reactive oxygen species，ROS)， 但在胁迫条件下，植物细胞内 ROS 水平会迅速上升，过量的 ROS 会对细胞造成伤害。 DAB 染色结果显示，150mM NaCl 处理 4 小时后，WT 和 Atesth 的根部着色都变深，但 是 Atesth 比 WT 处理前和处理后染色要浅； 同时通过激光共聚焦实验， 发现 WT 和 Atesth 在 150 mM NaCl 处理前后， ROS 水平 WT 植株有明显上升， 约为处理前的 4 倍； 而 Atesth 根部的荧光强度变化不明显；测定 CAT 活性发现，NaCl 处理后，WT 幼苗体内 CAT 活 性提高了
5、51%，Atesth 幼苗体内 CAT 活性提高了 114%，这说明 ROS 代谢可能参与了 AtESTH 对盐胁迫应答。 II 利用 Ca2+探针和激光共聚焦技术监视突变体中胞质自由 Ca2+浓度变化，实验结果显 示，野生型的 Ca2+增加 70%，而突变体为 100%，同时它们都会在较短的时间内升高， 突变体相对应 WT 上升快。说明 AtESTH 可以促使细胞释放 Ca2+而减少 Na+吸收，以此 应对盐胁迫。 综上所述，AtESTH 通过对细胞内 ROS、 H+和 Ca2+浓度的调节影响拟南芥对盐胁 迫的应答，来提高植物对盐的适应性。 关键字：ROS，盐胁迫，乙烯，ACS III AB
6、STRACT Ethylene plays an important role in plant growth and development and in responses to various stresses. However, it remains to clarify whether and how ethylene biosynthesis and signal transduction are invovled in response to salt stress. Ethylene synthase (ACC synthase, ACS) gene family have 1
7、2 member, we selected one of them,which named AtESTH (Arabidopsis thaliana gene for Ethylene Synthesis factor, AtESTH) to study wether response to salt stress. 150mM NaCl treatment for 5 days，germination rate of WT is about 7% , and Atesth germination rate is 15%, which is 2-times as much as WT; 150
8、mM NaCl treatment for 10 days, the average root length of WT is 0.35 0.06 cm, and the average root length of Atesth is 0.95 0.03 cm, which the latter is nearly 3-times as much as the former. The RT-PCR results show that Atesth expression significantly increased compared with the control under the tr
9、eatment of 150mM NaCl, indicating that AtESTH is involved in the response of Arabidopsis to salt stress at the transcriptional level. H+-ATP play an important role in keeping ion balance of plant cells. Experiment results show that the H+-ATP enzyme activity is similar between WT and Atesth under th
10、e control conditions. But treatment with 150mM NaCl for 72 hours, H+-ATP enzyme activity of Atesth significantly increased comparing to WT, indicating that H +-ATP activity changes in the Atesth plants salt stress of the adjustment process. 150 mM NaCl induce the fluorescence intensity of pH probe i
11、ncreased in Atesth mutant root cells comparing to that in WT by Confocal recording with the result of WT is raise 11% so the Atesth is 33%. This shows that AtESTH affect Arabidopsis tolerence to salt stress by regulating H+-ATPase activity and cellular pH. Under normal circumstances, plants maintain
12、 a certain level of intracellular ROS (Reactive oxygen species, ROS). But ROS levels in plant cells increased rapidly in response to stresses. By DAB staining and the treatment with 150mM NaCl for 4 hours, ROS levels of Atesth mutants did not significantly change comparing to that of WT. The Confoca
13、l recording show that WT is 4-times than it not treat by 150 mM NaCl,and the Atesth isnot change. Moreover, NaCl treatment increased CAT activity in WT（51%)or Atesth （114% ）plants, indicating that AtESTH participate in ROS metabolism in response to salt stress. Ca2+ increased in a short time with th
14、e result of that WT increased 70% and Atesth increased 100%.suggesting that AtESTH might promote cytoplasma free Ca2+ to decrease absorb of the Na+ to IV response to salt stress. In summary, AtESTH might improve the adaptability of plants to salt stresss by adjusting the H+-ATPase activity and ROS m
15、etabolism . KEY WORDS：ROS, Salt stress, Ethylene, ACS VII 缩写词缩写词 （Abbreviations） 英文缩写英文缩写 英文全称英文全称 中文名称中文名称 ABRC Arabidopsis biological resource center拟南芥生物资源中心 ACC 1-aminocyclopropane-1-carboxylate oxidase 1 -氨基环丙烷-1-羧酸氧化酶 ACO ACC oxidase ACC 氧化酶 ACS ACC synthase ACC 合成酶 AdoMet S-adenosylmethionine
16、 S-腺苷甲硫氨酸 CAT catalase 过氧化氢酶 DEPC Diethyl pyrocarbonate 焦碳酸二乙酯 DMSO Dimethyl sulfoxide 二甲基亚砜 dNTP Deoxynucleotide triphosphates (usually a mixture of dATP/dTTP/dCTP/dGTP) 脱氧腺苷三磷酸 (dATP/dTTP/dCTP/dGTP 的混 合物） EB Ethidium bromide 溴化乙锭 EGTA ethylene glycol tetraacetic acid 乙二醇二乙醚二胺四乙酸 H2O2 Hydrogen peroxide 过氧化氢 LSCM Lase scanning confocal microscope 激光扫描共聚焦显微镜 MAPK
