Identification And Function Analyses Of GhMAPKKK49 And GhMAPKKK65 From Cotton

Plants have evolved complex signal regulatory mechanisms against from external stress. Mitogen-activated protein kinase pathway(MAPK) is one of the most vital intricate regulatory mechanisms. MAPK signaling cascades pathway play very important roles in involving in regulating cell division and differentiation, responsing to biotic and abiotic stress and program cell death and participating in other life activities. MAPK signal cascade pathway includes three gene family: MAPK, MAPKK and MAPKKK. In all the studies of MAPK signaling cascades, the gene research of MAPK family and MAPKK family has been very thorough, but the genetic research in MAPKKK family, especially in Raf gene subfamily, is rarer. Currently, the studies of MAPK signaling cascades are mostly concentrated in Arabidopsis, rice, mice and other model plants, while the research for cotton, an important cash crop, is limited respectively.In this study, we used cotton(Gossypium hirsutum L.)(Lumian 22) as the experimental material, and cloned two cotton MAPKKK genes by using the homologous cloning way, which named GhMAPKKK49 and GhMAPKKK65. By amino acid sequence analysis and phylogenetic analysis, we found that GhMAPKKK49 and GhMAPKKK65 belong to the Raf gene subfamily of MAPKKKs. Then, we analysed these two cotton MAPKKK genes more for finding the identification of biological function study. The results are as follows:(1) The transcription level of GhMAPKKK49 gene under a variety of environmental stresses were studied by using the realtime fluorescence quantitative PCR. The results show that the transcription level of GhMAPKKK49 is induced by drought(15% PEG6000), high salt(NaCl), Wounding and a variety of signaling molecules(ABA, SA, MeJA, ET, GA3,6-BA, NAA, H2O2) and other factors. Meanwhile, the expression of GhMAPKKK49 is suppressed by some biological stress, such as Ralstonia solanacearum and Rhizoctonia solani. Thus, GhMAPKKK49 may mediate a variety of biological and abiotic stress process and participate in a variety of complex pathways of signaling molecules.We have also confirmed that GhMAPKKK49 interacted with each of GhMKK4 and GhMKK9, rather than with GhMKK5 or GhMKK6 by yeast two-hybrid experiments, which means GhMAPKKK49 is the upstream activating genes of GhMKK4 and GhMKK9.(2) By analysis of the promoter of GhMAPKKK65, we found that there are a series of stress, growth, light response and hormone response and other related cis elements in the promoter of GhMAPKKK65. The expression of GhMAPKKK65 were induced by R. Solanacearum, R. solani, drought, cold, wounding and a variety of signaling molecules, such as 6-BA, GA3, ET, SA, MeJA and H2O2. Therefore, GhMAPKKK65 may mediate a variety of biotic and abiotic stresses and participate in a variety of complex signaling molecular pathways.We constructed GhMAPKKK65 overexpression vector and obtained the transgenic benthamiana plants by Agrobacterium infection. As the analysis of the overexpression plants showed, GhMAPKKK65 gene overexpression plants were sensitived to the R. Solanacearum and R. Solani, and exhibited a relatively high accumulation of ROS and a low activities of antioxidant enzymes. Therefore, GhMAPKKK65 gene may respond to plants against pathogens signaling pathways.We also described GhMAPKKK65 did not interact with GhMKK4 or GhMKK5 or GhMKK6 or GhMKK9 by yeast two-hybrid experiments.These results suggest that, GhMAPKKK65 may be involved in mediating plant MAPK signaling pathways against other pathogens signal process.