GhMAPKKK42 Responds To Environmental Stress And Is Invovled In Growth And Development In Cotton

Mitogen-activated protein kinase(MAPK) cascades are a class of serine/threonine protein kinases exist in a variety of eukaryotic. They can convert extracellular stimuli into a wide range of cellular responses through conserved signal pathway MAPKKK-MAPKK-MAPK. MAPK cascade pathways are commonly found in animals, plants and yeast systems. When plants are subjected to external biotic and abiotic stresses, MAPK cascades will be started. These stresses include: drought, high salt, low temperature, mechanical damage, pathogen infection and reactive oxygen species(ROS). So far, reports on MAPK cascades are focused on members of the MAPK and MAPKK family, while rarely on MAPKKK, especially in the important crops of cotton. Cotton is an important fiber-producing and oil-bearing crops, so we choose cotton(Gossypium hirsutum) as our experiment material. In this study, we isolated a novel MAPKKK gene named Gh MAPKKK42 through homologous cloning, and has carried on the sequence analysis, expression pattern analysis and biological function analysis, the specific research results are as follows:(1) We obtained a cDNA fragment of 1558 bp by homologous cloning, including a 5’UTR(untranslated region) of 215 bp, a 3’ UTR of 218 bp and an open reading frame(open reading frame, ORF) of 1125 bp. ORF encoded a protein of 374 amino acids with the isoelectric point is 8.89 and molecular weight of the polypeptide is 42.573 kDa. Through the amino acid sequence analysis and cluster analysis, we found that the amino acid sequence contains a C terminal kinase domain and had a high homology with that of RAF subfamily. Therefore, we conclude that this gene belongs to RAF subfamily of MAPKKK family. The subcellular localization analysis showed that the protein is localized in the nucleus, suggesting that it may play a specific biologica role in the nucleus.(2) We analyzed the expression patterns of cotton from outside environmental stresses through real-time fluorescence quantitative PCR(qPCR) method. And results showed that, the transcriptional level of GhMAPKKK42 will be up-regulated under the treatment of low temperature(4 ℃), mechanical damage and signaling molecules GA3 and NAA, but down-regulated under the treatment of drought, high salt, R. solanacearum, R. Solani and signaling molecules MeJA and ABA, indicating that GhMAPKKK42 responds to various biotic and abiotic stresses and the regulation of signal molecules.(3) By using the method of hi-TAIL PCR, we got the promote sequence of GhMAPKKK42 of 1104 bp. Analysis carried on Plantcare showed that the sequence contain the cis-acting elements HSE and LTR, which related to temperature stress, GARE-motif and P-box, which responds to signaling molecules GA, and GCN4_motif and as-2-box, which related to tissue-specific and development, indicating that gene GhMAPKKK42 may participate in the temperature stress and invovled in the process of growth and development in Cotton.(4) Functional analysis of the transgenic tobacco plants are as follows: Firstly, seeds germination of transgenic plants are lower to that of wild-type tobacco under drought and salt treatment; By detecting the expression of antioxidant enzyme genes, we found that the expression of genes are down-regulated in transgenic plants, so the ability of transgenic plants to remove reactive oxygen species is decreased, indicating that transgenic plants are sensitive to drought and salt treatment. Secondly, there is a large area of the macula and more H2O2 accumulation in transgenic tobacco leaves after pathogen infection. Besides, the expression of resistant genes PR1 c, PR2 and PR4 are down-regulated, indicating that transgenic plants are sensitive to R. Solanacearum, and the sensitivity are associated with the expression of pathogen resistant genes. Thirdly, transgenic plants displayed slight injury under low temperature, and accumulated fewer malondialdehyde(MDA) but more soluble sugar, the activity of antioxidant enzymes also higher than that of wild-type tobacco, indicating that transgenic plants are resistant to low temperature.(5) Using the yeast two hybrid system, we identified that GhMAPKKK42 can interact with GhMAPKK9, GhMAPK4 and GhMAPK13. How the specific signaling pathway form need further verification.