Functional Analysis Of Mitogen-activated Protein Kinase Kinase Kinase 16 Gene From Arabidopsis Thaliana

The natural environment of plants consists of a complex series of abiotic stresses and biotic stresses.Plants respond equally to these stresses.In the transcriptome analysis,under the condition of low temperature stress,7870 genes of the heat-resistant 93-114 and the cold-sensitive cultivar reken 501 of Hevea brasiliensis Muell.Arg were differentially expressed,among which MAPK and MAPKKK(two groups of MAPK cascade)were up-regulated expression.The mitogen-activated protein kinase(MAPK)cascade is a conserved and important signaling component in response to various environmental stresses.In plants,the MAPK signaling network plays a Vital role in many biological processes,such as cell division,hormonal response,ROS homeostasis,development,aging,and biotic and abiotic stress.At present,the MEKK subfamily and MAPKKK related to stress resistance need further study.In this study,the Arabidopsis thaliana MAPKKK15 and MAPKKK16 genes were found to be homologous to this gene by comparing the gene in the rubber tree with the Arabidopsis MAPKKK family,and the Arabidopsis thaliana T-DNA insertion mutant was used as experimental material.The genetic function of the MAPKKK15 and MAPKKK16 genes in the MAPK family of Arabidopsis thaliana was studied by genetics and biochemical indicators,RT-PCR and genetic transformation.The main results were as follows:1.The bioinformatics method was used to analyze the gene in the rubber tree and the homology alignment with the Arabidopsis MAPKKK15 and MAPKKK16 genes.The results showed that the gene in the rubber tree had higher homology with Arabidopsis MAPKKK15 and MAPKKK16.2.Six T-DNA homozygous mutants of Arabidopsis thaliana were successfully identified by "three primer method",and the expression of two genes in Arabidopsis mutants was detected by real-time fluorescence quantitative technique.Three T-DNA insertion mutants of MAPKKK15 were MK3-15-1,MK3-15-2,MK3-15-3,except that MK3-15-2 is wild type,the other two can be screened for homozygous mutants.Homozygous mutants were obtained in three T-DNA insertion mutants MK3-16-1,MK3-16-2,and MK3-16-3 of MAPKKK16.None of the two genes in the selected homozygous mutants were expressed,indicating that the "three-primer method" screening results were effective.3.The selected Arabidopsis homozygous mutants were subjected to drought,freezing damage and pathogen stress treatment.The phenotype was observed.After the freeze damage treatment and inoculation of the pathogens,the survival rate of the mutant plants was greater than that of the wild type plants,and The expression of MAPKKK15 and MAPKKK16 gene was down-regulated,which was opposite to the up-regulation of MAPKKK gene expression in rubber trees,but the difference was not obvious under drought conditions.Further dehydration rate measurements showed no significant difference,and the relative conductivity results showed MAPKKK15,MAPKKK16 gene mutants.These results indicate that the MAPKKK15 and MAPKKK16 genes may be involved in the regulation of Arabidopsis stress response.4.The complementary expression vector and overexpression vector of MAPKKK16 gene were constructed,and the Arabidopsis thaliana mutant plants and Arabidopsis thaliana wild type plants were transformed respectively.The positive transformed plants were obtained by resistance screening and PCR molecular identification.5.These findings revealed an important role of MAPKKK16 in Arabidopsis stress response,providing a basis for further study of the role of MAPKKK15 and MAPKKK16 genes in the regulation of stress response in Arabidopsis thaliana.