Identification Of Mitogen Activated Protein Kinases Kinases Genes And Screening Of Drought-related Functional Genes In Potato

MAPKK(Mitogen-activated protein kinases kinases)is mainly found in animal and plant cells,which is a signal transduction,and playing an important role in the cell.MAPKK is a key component of MAPK cascade reactions.It can be different extracellular implused such as hormones,cytokines and other things,which can regulate cell growth,differentiation,and other important physiological processes.It generally present in all eukaryotes.So far,the MAPK cascades have been investigated a series of research in only a limited number of plants,such as Arabidopsis,rice and poplar.However,the study of MAPKK family gene have not been reported in potato(Solanum tuberosum L.),particularly the drought-related MAPKK gene have remained elusive.In this research,we carried out bioinformatics analyses of MAPKK genes in potato,and identified 5 MAPKK family genes.We passed on some biotic and abiotic stress in potato,selecting drought-related MAPKK gene.In addition,we constructed the plant expression vector of MAPKK1 gene and Gateway-MAPKK1 interference and through the Agrobacterium mediate genetic transformation to obtain the transgenic potato plants.We use qRT-PCR analysis the expression of MAPKK1 gene in transgenic plants.The research results are as follows:1.Through the bioinformatics analysis,we identified 5 StMAPKK genes in potato.They can be divided into four groups: A,B,C and D group.The StMAPKK gene in potato,average length of cDNA is 1001 bp,ranging from 1547 bp to 1574 bp.StMAPKKs in potato encoded amino acid sequences from 515 to 333 amino acids amino acids,the molecular weight of predicted protein in StMAPKKs changes from 37.26 kDa to 57.45 kDa.The isoelectric point(PI)by derived peptides from 5.47 to 8.87.However,the location of MAPKK gene in chromosome was mainly concentrated in chromosome 12 and 3.2.Analysis the relative expression levels of StMAPKK family gene in potato under biotic and abiotic stress(4? and 45?,20% PEG,200 mM,NaCl,10 mM,H2O2,100 ?M,MeJA,100 ?M SA and 100 ?M ABA).The relative expression levels of stMAPKK gene under drought stress,most of which were upregulated.Especially,StMAPKK1 and StMAPKK5 raised the highest degree.3.The MAPKK1 gene which has the most obvious upregulated in the relative expression levels of 5 StMAPKK genes under drought stress,was used to construct expression vectors.And the expression vector of drought-related MAPKK1 gene used the Agrobacterium mediate genetic transformation to obtain transgenic plants by PCR identification.By the q RT-PCR,the relative expression in levels of St MAPKK1 in transgenic ‘Atlantic' is 3.2 times,comparing with the control group potato ‘Atlantic'.And the transgenic potato ‘Zihuabai' of StMAPKK1 gene,the relative expression levels of StMAPKK1 up to the control group ‘Zihuabai' varieties of 7.2 times.4.The interference expression vector of MAPKK1 gene was constructed using gateway technology.After genetic transformation by Agrobacterium,we obtained transgenic plants by PCR identification.Analysis by the qRT-PCR,the relative expression levels of StMAPKK1 in transgenic potato ‘Atlantic',is 0.09 times of notransgenic ‘Atlantic'.And the transgenic ‘Zihuabai',compared with no-transgenic ‘Zihuabai' is 0.4 times.Therefore,the relative expression levels of StMAPKK1 genes were significantly inhibited.