Isolation Of LcMKK And LchERF Genes From Lycium Chinense And Functional Analysis Of The Genes For Abiotic Stress Tolerance

Abiotic stresses such as drought and salinity are major reasons that affect the growth and development of plants. However, during the long process of evolution, plants have developed a variety of elaborate mechanisms to perceive stress signals and regulate expression of stress-responsive genes to protect themselves. The MAPK cascade typically consist of three functionally interlinked protein kinases: MAPK, MAPKK and MAPKKK. They are functionally linked and act as an important network for transducing signals from upstream receptors to the downstream cellular effectors. The numbers of genes show that MAPKK is the crucial component of the MAPK cascade. Although many MAPKK genes have been isolated and the roles of them in response to environmental stresses have been studied in model and crop plants, the functional role of MAPKKs in MAPK cascades needs to be further explored.In this study, a novel MAPKK gene, LcMKK, was isolated from Lycium chinense. NaCl, PEG and 4℃ treatment lead to a significant increase of LcMKK transcript. The results show that the LcMKK gene is involved in response to multiple stresses. To investigate the function of the LcMKK gene, the coding region of LcMKK was introduced into the vector pCAMBIA2300 under the control of the CaMV 35 S promoter and then transformed into wild-type tobacco. Under dehydration conditions, compared to WT plants, LcMKK-overexpressing lines exhibited remarkably less water loss and electrolyte leakage. Under drought conditions, LcMKK-overexpressing lines exhibited higher levels of germination rate, chlorophyll content, plant survival rate, antioxidant enzyme activity, lower electrolyte leakage and less accumulation of ROS. Additionally, overexpression of LcMKK enhanced the expression of stress-responsive genes under drought conditions. The transcript level of NtERF2 was nearly increased 2.6 times in transgenic tobaccos. All these results indicate that LcMKK is probably a promising candidate gene for drought improvement in crops.The transcript level of NtERF2 was increased in LcMKK-overexpressing tobaccos, showing that LcMKK can regulate the transcription of ethylene-responsive transcription factors(ERFs) in transgenic tobaccos. Therefore, a novel ERF gene, LchERF, was cloned from L. chinense and the function of LchERF was investigated in this study. PEG, NaCl, and exogenous ethylene treatment lead to a significant increase of LchERF transcript. The results show that the LchERF gene is involved in response to multiple stresses. Then the coding region of LchERF was introduced into the vector pCAMBIA2300 under the control of the CaMV 35 S promoter and transformed into wild-type tobacco. Under salt stress, compared to Vec plants, LchERF-overexpressing tobaccos showed significantly higher levels of germination rate, chlorophyll content, plant survival rate, proline content and less accumulation of H2O2. Overall, our results demonstrate that LchERF play an important role in the regulation of plant responses to abiotic stresses and mediate various physiological pathways that enhance salt stress tolerance in plants. The gene has important application value in the production of adverse-resistant plants.