Isolation And Functional Identification Of A Novel Mitogen Activated Protein Kinase Gene, GhMAPK, In Cotton (Gossypium Hirsutum L.)

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays pivotal role in the regulation of stress and developmental signals in plants. The MAPK cascades transfer signals through phosphorylations of MAPKKK- MAPKK- MAPK in turn.Numerous MAPK have been isolated and characterized from different species, and many of them had close relations with plant disease resistance. Cotton (Gossypium hirsutum) is an economically important species, its MAPKs, however, have not been reported before. In this thesis, a series of studies have been conducted on the isolation, sequence and expression analysis, function identification of cotton MAPK (GhMAPK). The main results are as follows:1. A novel gene, termed Gossypium hirsutum MAPK (GhMAPK), was isolated from cotton. The full-length cDNA of GhMAPK is 1832bp long, and encodes for a 372 amino acid protein that contains all 11 of the MAPK conserved subdomains and the phosphorylation-activation motif, TEY. Amino acid sequence alignment revealed that GhMAPK shared high identity with group-C MAPK in plants and showed 83%~89% similarities with MAPKs from Arabidopsis, apricot, pea, Petunia, and tobacco.2. Southern blot analysis indicated that the GhMAPK belonged to a multygene family in cotton. Two introns were found within the region of genomic sequence. Northern blot analysis revealed that the transcripts of GhMAPK accumulated markedly when the cotton seedlings were subjected to various abiotic stimuli such as wounding, cold (4℃), or salinity stress; Furthermore, GhMAPK was upregulated by the exogenous signaling molecules, such as salicylic acid (SA) and hydrogen peroxide (H2O2), as well as pathogen attacks. These results indicate that the GhMAPK, which has a high degree of identity with group-C plant MAPKs, may also play an important role in response to environmental stresses.3. Construct a sense expression vector pBI-GhMAPK, and transformed it in to the tobacco plant (NC89). We also transformed the empty vector into tobacco at the same time as control. We carried out Northern blot and Western blot on some transgenic plants, and found that GhMAPK has been expressed successfully in the tobacco plants.4. On the basis of the molecular identification, we analyzed the biological function of progeny T1 generation plants. The transgenic plants exhibited much milder symptoms than the nontransgenic plants upon challenge with Phytophtora parasitica var. nicotianae Tucker, but only 10% transgenenic plants showed resistance to TMV virions.5. Constructed an E.coli expression vector pET-GhMAPK and induce it to express in E.coli strain BL21(DE3). The strong induced fusion protein bands were collected into PBS solution and immuned little mouse to obtain antiserum. The value of antibody reaches 1:1000.