转桑树MAPK5基因拟南芥在不同逆境胁迫下的抗性分析

摘要

[Objective] Mitogen-activated protein kinases ( MAPK ) play key roles in response to biotic and abiotic stresses in plants. To investigate the function of mulberry ( Morus notabilis) MnMAPK5 in response to stress conditions, the activities of the promoter of MnMAPK5 in transgenic Arabidopsis thaliana under stress conditions, subcellular localization of MnMAPK5 and its overexpression in A. thaliana were analyzed.[Method] The promoter of MnMAPK5 was inserted into the plant expression vector pBI121 and transformed into A. thaliana by the floral dip method . The activity of GUS was dectected in the transgenic plants treated respectively with high temperature,low temperature,NaCl and PEG. The overexpression vector was constructed and transformed into A. thaliana and the T3 transgenic plants were used for resistance analysis.[Result] MnMAPK5 gene was found encoding a cytoplasm-and nucleus-localized protein. The results of GUS staining showed that the activity of MnMAPK5 promoter was induced by high temperature,low temperature,salt and drought. The MnMAPK5-overexpressed transgenic A. thaliana showed lower germination rates,shorter length,and worse growth than wild A. thaliana plants under salt,drought,low temperature and H2 O2 . Under salt stress,MDA content and POD activity were higher in the MnMAPK5-overexpressed transgenic plants while CAT activity,soluble sugar content and H2 O2 content were lower. Under drought,the MDA content and H2 O2 content in the MnMAPK5-overexpressed transgenic plants were higher than in the wild plants while CAT activity and POD activity were lower. Under low temperature stress,CAT activity,POD activity,proline content and soluble sugar content in the transgenic plants were lower than in the wild plants. Under H2 O2 stress,MDA content was higher in the transgenic plants than in the wild plants while proline content and soluble sugar content were lower. Besides,the expression levels of AtPOD,AtCAT,AtRD22 and other stress related genes were lower in the transgenic plants than in the wild plants under all the above stresses.[Conclusion] The activities of MnMAPK5 promoter in the transgenic A. thaliana showed responses to different abiotic stresses. The MnMAPK5-overexpressed transgenic A. thaliana showed declined resistance against abiotic stresses,and indicated that MnMAPK5 may be a negative factor in response to stress conditions.%[目的]植物促分裂原活化蛋白激酶(MAPK)在响应生物与非生物胁迫中发挥重要作用.本研究通过分析桑树(川桑)B家族MAPK基因MnMAPK5的亚细胞定位和启动子活性,并将其转入拟南芥中进行过表达研究,探讨MnMAPK5在逆境胁迫下的作用机制.[方法]将MnMAPK5的上游启动子连接到pBI121载体上,通过花粉介导法转入拟南芥中,经高温、低温、NaCl和PEG处理后进行GUS染色分析.通过构建过表达载体,将MnMAPK5转化到拟南芥中,用T3转基因植株进行抗逆境胁迫分析.[结果]MnMAPK5基因的编码蛋白定位于细胞质和细胞核中.在拟南芥中检测MnMAPK5启动子驱动的GUS活性的结果显示,MnMAPK5启动子活性受到了高温、低温、高盐和干旱胁迫的诱导.在高盐、干旱、低温和H2 O2处理条件下,过表达MnMAPK5抑制了转基因拟南芥的萌发和生长.在盐胁迫下,过表达MnMAPK5提高了转基因植株中的MDA含量和POD活性,降低了CAT活性、可溶性糖含量和H2 O2含量;在干旱胁迫下,过表达MnMAPK5提高了转基因拟南芥中的MDA和H2 O2含量,降低了CAT和POD活性;在低温下,转基因拟南芥相比野生型植株表现出更低的CAT活性、POD活性、脯氨酸含量和可溶性糖含量;在氧化环境胁迫下,转基因拟南芥的MDA含量显著高于野生型拟南芥,而脯氨酸含量和可溶性糖含量要低.此外,在各个胁迫环境条件下,AtPOD、AtCAT和AtRD22等胁迫相关基因的表达量在转基因植株低于野生型拟南芥.[结论]MnMAPK5启动子在转基因拟南芥中的活性受到了不同非生物胁迫的诱导.过量表达MnMAPK5降低了拟南芥对非生物胁迫的抵抗能力,表明MnMAPK5在逆境胁迫下起着负调控作用.