Analysis Of Expression And Function Of GhMAPK6 Gene In Cotton (Gossypiym Hirsutum)

The mitogen-activated protein kinase (MAPK) cascade is involved in various biotic and abiotic stress responses, hormone responses, cell proliferation, differentiation, and developmental processes in plants. A typical MAPK cascade consists of three steps:a mitogen-activated protein kinase kinase kinase (MAPKKK) activates a particular mitogen-activated protein kinase kinase (MAPKK) through phosphorylation on two serine/threonine residues in a conserved S/T-X3-5-S/T motif, and then the activated MAPKK can in turn phosphorylate a MAPK on threonine and tyrosine residues in the invariant sequence TXY.Abscisic acid (ABA) is an important cellular signal and stress hormone. It plays crucial roles in reporting environmental stresses, initiating stress responses and preparing plants to cope with the stresses. Evidence for ABA in triggering the production of H2O2 has been provided, and H2O2 has been shown to be a signal mediating various stress tolerance responses. The response chains following abiotic stresses that alter MAPK signaling may, in fact, be mediated at least in part through oxidative processes, because H2O2 has been shown to be able to activate these MAPKs. In this thesis, one gene encoding MAPK protein was isolated from cotton roots cDNA library, designated GhMAPK6.Our studies mainly focused on the cloning, expression, subcellular localization and the functional analysis. The main results are as follows:1. Isolation and characterization of GhMAPK6By screening the cotton cDNA library, one cDNA encoding MAPK protein was isolated. The putative MAPK clone from cotton exhibited high levels of sequence similarity to Arabidopsis AtMAPK6, so the novel cotton MAPK gene was designated as GhMAPK6. A 3,907-bp genomic fragment of GhMAPK6 was isolated from cotton genomic DNA by PCR, using gene-specific primers. Comparison of the sequences between the GhMAPK6 genomic clone and its cDNA revealed that five introns were present in the gene. Phylogenetic analysis revealed that GhMAPK6 shared high similarities to AtMAPK6, NtSIPK and NbSIPK, which were all group A members.2. GhMAPK6 expression was induced by ABA, salt and drought stressesTo evaluate the role of the isolated GhMAPK6 gene in plants, we first analyzed its expression pattern. Quantitative PCR analysis showed widespread expression of GhMAPK6 gene in cotton tissues, with relatively high levels in roots and hypocotyls. To investigate whether GhMAPK6 expression is regulated by abiotic stresses, cotton seedlings were subjected to NaCl. PEG (polyethylene glycol 4000) and ABA treatments.The expression levels of GhMAPK6 were significantly up-regulated by drought and NaCl. In particular, the transcriptional level of GhMAPK6 was utmostly increased after ABA treatment. After ABA treatment for 1 hour, GhMAPK6 transcripts began to be accumulated in roots, and reached its highest levels within 5 hours. These results suggested that GhMAPK6 may be involved in plant response to ABA signaling.3. GhMAPK6 protein localizes in cytoplasmTo investigate the subcelluar localization of GhMAPK6 protein, green fluorescent protein (GFP) fused to C-terminal of the GhMAPK6 was expressed in cotton callus cells under the control of a cauliflower mosaic virus 35S promoter. The experimental results revealed that GhMAPK6:eGFP fluorescence signals were detected on cytoplasm of the transformed cells. The results suggested that the GhMAPK6 protein may localize in cytoplasm.4. Overexpression of GhMAPK6 recovers the wild-type phenotype of Atmkk1 mutantTo investigate its role in plants, GhMAPK6 under the control of cauliflower mosaic virus (CaMV) 35S promoter was expressed in homozygous Atmkk1 mutant plants. Transgenic Arabidopsis plants were obtained on a selection MS medium with 250mg/L hygromycin B. Through hygromycin B-resistance assay and PCR analysis, the homozygous transgenic progeny lines were selected for further study. The results indicated that overexpression of GhMAPK6 in Atmkkl T-DNA insertion mutant could recover the defects caused by Atmkk1 mutation, suggesting that GhMAPK6 has a similar function to AtMAPK6 in ABA-induced CAT1 expression and H2O2 production.