【作者】 张博；

【导师】 沈文飚；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 钴是植物的有益元素,作为根瘤菌中若干种酶的金属辅基,是植物固氮过程中必不可少的元素。一氧化碳(carbon monoxide, CO)由血红素加氧酶(heme oxygenase, HO, EC 1.14.99.3)催化血红素(heme)分解产生,是动物炎症反应中的重要信号分子,并介导了生长素诱导的黄瓜不定根发生过程。为了了解钴(Ⅱ)对番茄幼苗侧根发生的影响以及HO/CO系统在钴(Ⅱ)诱导的侧根发生过程中的作用,本文研究了氯化钴(CoCl2)、诱导型HO专一性抑制剂锌原卟啉(zinc ProtoporphyrinⅨ, ZnPPⅨ)、HO催化产物CO等处理对番茄侧根发生的影响及HO的变化。研究发现,CoCl2不仅能以浓度依赖方式促进番茄幼苗侧根发生,而且能以浓度依赖和时间进程方式促进番茄侧根发生区域LeHO-1/LeHO-2的转录水平、HO一1蛋白表达及HO活性。为了进一步验证CoCl2诱导的番茄侧根发生由HO/CO系统介导的实验假设,本文使用ZnPPⅨ以及CO水溶液处理番茄幼苗,并对侧根产生情况进行了分析。实验发现,200μM ZnPPⅨ能使明显抑制CoCl2诱导的侧根生成,而添加50%饱和度的CO水溶液则能使幼苗侧根数目达到CoCl2单独处理的水平。进一步研究表明,ZnPPⅨ能显著抑制CoCl2对HO蛋白表达和活性的诱导,50%饱和度的CO水溶液能消除ZnPPⅨ对HO活性的抑制,这与ZnPPⅨ和CO对CoCl2诱导的侧根发生的影响是一致的。上述结果说明HO可能介导了CoCl2诱导的番茄侧根发生,而且HO的功能可能是通过其催化产物C0实现的。进一步对CoCl2及HO/CO系统诱导番茄侧根发生的分子机理进行了研究,发现CoCl2能够不同程度上调细胞周期基因LeCYCA2;1、LeCYCA3;1、LeCYCD3;1和LeCDKA1的表达；ZnPPⅨ可以抑制CoCl2对细胞周期基因的上调,而CO则能够缓解ZnPPⅨ的抑制效果,暗示细胞周期基因与CoCl2对番茄幼苗侧根的诱导相关。综上所述,HO/CO信号系统可能通过影响细胞周期基因的表达参与CoCl2诱导的番茄幼苗侧根发生。更多还原

【Abstract】 Cobalt is supposed to be a beneficial element of plant, which plays an essential role in nitrogen-fixation as the metal component of several enzymes in rhizobia. Carbon monoxide (CO), the side-production of heme degradation catalyzed by heme oxygenase (HO, EC 1.14.99.3), acts as an important signal molecular in inflammatory reactions, and was newly proved to be involved in the auxin-induced cucumber adventitious rooting process.In order to decode the relation among divalent cobalt ion, HO/CO system and lateral root development, morphological characters of root system and transcription and activity change of HO were investigated after various treatments. We found that CoCl2 not only induced lateral root formation in a dose-dependent manner, but also elevated the transcription of LeHO-1/LeHO-2, protein expression and activity of HO enzyme in a dose-dependent and time-course manner in lateral root initiation zoon of tomato seedlings. To testify the hypothesis that HO/CO system was involved in cobalt-induced lateral rooting process, the specific inhibitor of HO-1, zinc protoporphyrinⅨ(ZnPPⅨ) and CO aqueous solution were applied to the experimental system. We observed decreased lateral root formation in ZnPPⅨcombined CoCl2 treated seedlings; by contract, the inhibition was restored when 50% CO was added to the former mentioned system. Meanwhile, similar change was found in HO enzyme activity and protein expression. These results suggested HO’s involvement in the CoCl2-induced lateral root development and the possibility that CO might be the executer of HO in this process.Furthermore, we investigated the molecular mechanism of CoCl2-induced, HO/CO-dependent lateral root formation. We found CoCl2 differentially up-regulated cell cycle genes, such as LeCYCA2;1, LeCYCA3;1, LeCYCD3;1 and LeCDKA1; the application of ZnPPIX could inhibit CoCl2’s effect and the addition of CO was able to reverse ZnPPⅨ’s inhitition.As a conclusion, HO/CO system was involved in CoCl2-induced lateral root development process in tomato seedlings by regulating the expression of cell cycle genes.更多还原