Isolation And Functional Identification Of A Mitogen-activated Protein Kinase Gene GhMPK16 In Cotton (gossypium Hirsutum)

Plants have developed sophisticated defense mechanisms to deal with diverse unfavorable environmental factors during their sessile life cycle. Reversible protein phosphorylation/dephosphorylation modifications play a key role in extracellular signaling pathways. MAPK cascades are composed of three interlinked protein kinases: MAPKK kinases, MAPK kinases and MAPKs. The mitogen-activated protein kinase (MAPK) cascade is a highly conserved pathway to phosphorylation a wide range of substrates that translates external stimuli into cellular responses in all eukaryotes.In plants, MAPK pathways are involved in the regulation of development, growth, programmed cell death and in responses to a diversity of environmental stimuli. So far only a few MAPK cascade components have been studied in detail. Cotton (Gossypium hirsutum) is one of the most important fiber and oil crops. Its growth and yield are severely inhibited by diverse unfavorable environmental factors. In this thesis, a series of studies have been conducted on the isolation, subcellular localization sequence and expression analysis, function identification of GhMPK16. The main results are as follows:1. A novel gene, termed GhMPK16, was isolated from cotton. GenBank accession number: FJ966889. The full-length cDNA of GhMPK16 is 2030 bp long, and encodes for a 554 amino acid protein that contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TDY. GhMPK16 exhibited high similarities to group D MAPK AtMPK16. GhMPK16 was the first group D MAPK isolated from cotton.2. A 2748 bp genomic fragment of GhMPK7 (GenBank accession number: FJ966896) was isolated from cotton genomic DNA. Comparison of the sequences between the GhMPK7 genomic clone and its cDNA revealed that ten introns are present in the gene. The comparative analysis of the homology genomic sequences indicated that the number of introns and the relative positions of exon-intron organization are conserved, and the sizes of their parallel introns are different. Southern blot analysis implies that there is a single GhMPK16 gene in the cotton genome.3. Transient transformation assay with onion epidermal cells revealed that GhMPK16 protein is localized in cell nucleus. GhMPK16 may act a set of target genes in the nucleus.4. Northern blot analysis revealed that the transcripts of GhMAPK accumulated markedly when the cotton seedlings were subjected to various abiotic stimuli such as high/low temperature, salinity stress or wounding; Furthermore, GhMAPK was upregulated by the exogenous signaling molecules, such as SA, MeJA and ABA, as well as some pathogen attacks. These results imply that GhMPK16 may play roles in plant defense responses and in the regulation of certain components of multiple stress-signaling pathways.5. To better understand GhMPK16 function under diverse stresses, we transformed a sense expression vector pBI-GhMPK16 into the Arabidopsis (Col). Transgenic plant confirmed with PCR and northern blot analyses.6. Bioassays in transgenic Arabidopsis demonstrate that overexpression of GhMPK16 significantly enhances the resistance of Arabidopsis against Colletotrichum than the wild-type lines. The overexpression-GhMPK16 transgenic plants showed a more sensitive to drought stress.