Isolation And Functional Analysis Of A MAPKK Gene ZmMKK1in Zea Mays

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling model in plants, animals and yeast. The MAPK cascade is composed of three protein kinases: mitogen-activated protein kinase kinase kinase (MAPKKKMKKK7MEKK/MAP3K), mitogen-activated protein kinase kinase (MAPKX(MKKMEK/MAP2K), mitogen-activated protein kinase (MAPK/MPK). MAPK cascades form the complex signaling network through the "crosstalk". MAPK cascades link different cell membrane receptors with cell responses to regulate various biotic and abiotic stresses, and play an important role in plant hormone signals, cell division, growth and development. Plant MAPK cascade gene models show that the number of MAPKKs is less than that of MAPKs, suggesting that the same MAPKK may participate in several MAPK modules. MAPKKs play an important role in integrating signals from MAPKKKs and transducing to multiple MAPKs. At present, the structure and function of multiple MAPKKs have been researched in Arabidopsis, tobacco, alfalfa and so on.Various biotic and abiotic stresses seriously inhibit the growth and yield of maize which is as a worldwide food crops. Recent researches also suggest that many protein kinases in maize play important roles in resistance to various stresses. So far, three MAPKKs have been separated from maize. ZmMKK3could regulate the osmotic stress. ZmMKK4regulates osmotic stress by reactive oxygen species scavenging system and enhances the resistance to salt and cold tolerance. ZmMEK1functions in the proliferation of root tip in maize. In this study, a group A MAPKK gene, named ZmMKK1, was isolated from maize root (Zea may L. Zhengdan958). Sequence characteristic, expression pattern analysis, functional studies in overexpressing-ZwMKK1Arabidopsis thaliana and tobacco were analysis and ZmMKK1homeotic transforming system in maize was established. The main results were as follows:(1) A mitogen-activated protein kinase kinase gene ZmMKK1was isolated from maize root. The length of ZmMKK1gene is1641bp, contains1053bp ORF, and encodes350amino acids. It is predicted the molecular weight of ZmMKK1is38.8kDa and pI is6.3. Protein sequence analysis shows that ZmMKKl has11conserved domains and contains a conservative S/T-X3.5-S/T motif in Ⅶ-Ⅷ the domain, and its N-terminal has a conserved MAPK docking site. The phylogenetic tree analysis showed that ZmMKK1belongs to A subgroup.(2) The transient expression of ZmMKK1-GFP and its activation form ZmMKKlDD-GFP in onion epidermal indicated that ZmMKK1was located in the cytoplasm, and ZmMKK1DD was located in cytoplasm and nucleus.(3) qRT-PCR analysis found that ZmMKK1specifically expressed in the maize root, stem and leaf, and the highest expression in the stem. NaCl, PEG,12℃, SA, H2O2and CaCl2could induce ZmMKKl expression and ETH, ABA and JA inhibited ZmMKK1expression. H2O2and Ca2+mediated12℃-induced up-regulation expression of ZmMKK1.(4)PROKⅡ-ZmMKK1expression vector was constructed, and successfully transformed into tobacco and Arabidopsis thaliana. qRT-PCR analysis showed that ZmMKK1could stably express in transgenic plants.(5) Under low temperature stress, the seed germination rate in overexpressing ZmMKK1tobacco was high than that the Vec tobacco, and the transgenic tobacco seedlings grew better than the Vec seedlings. The transgenic tobacco accumulated more compatible substances such as proline, soluble sugar, had higher activity of antioxidant enzymes, and improved the expression of stress-related genes compared with the Vec tobacco.(6) Overexpressing ZmMKK1tobacco enhanced the tolerance to bacterial pathogen, and enhanced the sensitivity to necrotrophic pathogen.(7) Under salt and drought conditions, the overexpressing ZmMKK1Arabidopsis thaliana enhanced the tolerance to salt and drought stresses by ROS-and ABA-dependent ways.(8) pYES2-ZmMKK1expression vector was constructed and successfully transformed into yeast, Saccharomyces cerevisiae. Under salt and drought stresses, ZmMKK1enhanced salt and drought tolerance to Saccharomyces cerevisiae.(9) ZmMKK1interacted with ZmMEKK1by yeast two-hybrid assay.(10) PZP211-ZmMKK1sense and antisense expression vector were constructed and homeotic transgenic maize was gained. qRT-PCR analysis showed that sense ZmMKKl stably expressed in transgenic maize.