Isolation And Function Analysis Of A Cotton Mitogen-activated Protein Kinase Kinase Gene,GhMKK1

During their development, plants often suffer from environmental influences.Mitogen-activated protein kinase kinases (MAPKKs) play crucial roles in MAPK cascadesthat mediate a variety of stress responses in plants. A large number of researches showed thatMAPKKs were not only involved in biotic stresses, but also regulated resistance to abioticstresses. However, studies about MAPKKs in response to environmental stress mainly focuson model plants, such as Arabidopsis thaliana, the functions of MAPKKs in cotton(Gossypium hirsutum) have not been well studied. Cotton (Gossypium hirsutum) is one of themost important crops. Therefore, in this study, we selected the economically importantspecies cotton (Gossypium hirsutum L.) as the experiment material, and we isolated a novelgroup A MAPKK gene, GhMKK1, from cotton. A series of studies have been conducted onthe isolation, sequence and expression analysis, function identification of cotton GhMKK1,the main results are as follows:(1) The analysis of a full sequence indicated that the full-length cDNA of GhMKK1is1234bp long, including a21bp untranslated region (UTR), a127bp3′UTR and a1086bpopen reading frame (ORF). This ORF encodes a polypeptide of361amino acid protein with acalculated molecular mass of39.945kDa and an isoelectric point of5.08. Amino acidsequence alignment and a phylogenetic analysis derivated by GhMKK1showed thatGhMKK1shared high homology with PcMKK2, LeMKK1, AtMKK1and AtMKK2, whichall belong to group A MAPKK. All above results indicated that the gene we isolated is groupA MAPKK from cotton, denominated by GhMKK1.(2) RT-PCR analysis showed that the transcript levels of GhMKK1in response toenvironmental stress. The data showed that the transcript levels of GhMKK1could beaccumulated when cotton seedlings were treated with different abiotic stresses, such as highNaCl, wouding, H2O2, PEG, low temperature (4C), and biotic stress such as Ralstoniasolanacearum. Exogenous signaling molecules for plant defense response, such as salicylic acid (SA) and abscissic acid (ABA) can also induced the expression of GhMKK1. Thoseresults indicated that GhMKK1may played an important role in defense response.(3) A fusion protein35S-GhMKK1::GFP was constructed and then transformed intoonion inner epidermal cells using particle bombardment method. The obtained fluorescencepattern was observed using a confocal laser scanning microscope, and the analysis suggestedthat the GhMKK1was localized in both the nucleus and the cytoplasm, which providedstructural basis for functional analysis of GhMKK1.(4) A sense expression vector pBI121-GhMKK1was constructed and transformed intoplant (Nicotiana benthamiana) with Agrobacterium tumefaciens-mediated leaf disc method.RT-PCR was used to analyze the expression level of GhMKK1in transgenic plants. Theresults indicated that GhMKK1had been expressed successfully in the transgenic plants. Onthe basis of the molecular identification, the expression level of GhMKK1in T1transgenicplants was determined by RT-PCR. T3transgenic plants were used for functionalidentification, and wild-type plants were used as the controls. The data showed thatoverexpression of GhMKK1enhanced the resistance to abiotic stress, such as salt and drought.These results indicated that GhMKK1may positively regulated the response to abiotic stress.(5) Overexpression of GhMKK1resulted in the elevated sensitivity to Ralstoniasolanacearum and Rhizoctonia solani, and the expression levels of several disease-responsivegenes in N. benthamiana were decreased, which suggested overexpression of GhMKK1increased the pathogen susceptibility of the transgenic plants. These results indicated thatGhMKK1may be also involved in various biotic responses and GhMKK1may positivelyregulated the response to biotic stress.Taken together, these results showed that overexpression of GhMKK1increased theresistance to abiotic stress and the sensitivity to biotic stress in transgenic plants. The findingsnot only extend the knowledge of the biological function of group A MAPKKs, but alsoprovide evidence for further exploration of GhMKK1in the regulation of plant stressresponses.