Bioinformatics Analysis Of Pear MAPKs Family And Funnctional Study Of PbrMAPK15

The response to the different external stimuli in plants is a complex process involved in genes,signal transduction pathways,and gene expression products.This process can be divided into four phases:plant perception of stress signals,transferring of stimuli signals,identification of stimuli signals by membrane receptors,and expression of the corresponding gene.Among these,the protein kinases are an important class of plants regulatory enzyme,they perceived the external environmental signal by membrane receptors,to regulate the changes of some ions and molecular from intracellular concentrations,and then to activate protein phosphorylation pathway.Mitogen-activated protein kinase(MAPK)cascades play an important role in regulating plant growth,development and signal transmission.Pear is a perennial woody plant,which is economically important and can adapt to many different environments.The function of MAPK has been identified mostly concentrated in model plants.Therefore there is still very little pear MAPK information in the public databases.With the completion of the pear genome analysis,it became possible to analyze the pear MAPK genes in whole genome level,which may help to reveal MAPK signal transduction regulation mechanism in pear.MAPK family genes were identified based on the genome database of Rosaceae,the classification and phylogenetic analysis of the predicted and known MAPK family of Rosaceae by their amino acid sequences and conserved domains.The evolutionary process was then illustrated with bioinformatics analysis.The The expression of MAPK gene in pear pollen tubes was analyzed by qRT-PCR in different treatment.PbrMAPK15,screened out based on bioinformatics and homologous with AtMPK6,was cloned and subcellularly localized.The biological function of PbrMAPK15 gene was also studied with transgenic technique.The main results of this study were as follows:1.87 MAPKs family genes were identified in the Rosaceae genome,including 23 in pear(PbrMAPK),26 in apple(MdoMAPK),15 in peach(PpeMAPK),11 in strawberry(FveMAPK),and 12 in plum(PmuMAPK).Their protein domains,amino acid conserved sequences,phylogenetic trees,replication patterns,linear relationships and evolutionary times were analyzed and discussed.The expression of MAPK gene in pear roots,stems,leaves,fruits,pollen and styles of was detected by RT-PCR.The results indicated that the PbrMAPK gene family may be involved in the all life of pear.Induced by temperature stress and plant hormones(ABA,BR,SPD,SPM,IAA),by analyzed the expression of MAPKs gene in pear we can see that,except at low temperature stress,only two of PbrMAPK13 and PbrMAPK14 gene expression levels were significantly up-regulated,most genes were responsive to various treatments,and different pear MAPK gene had different response patterns to different treatments.These results suggest that the MAPK gene family may play an important role in plant responses to external stimuli.2.The AtMPK6 homologous gene PbrMAPK15 was screened based on the results of phylogenetic analysis and protein domain analysis of MAPK gene family in Pear and Arabidopsis thaliana.The gene was cloned and sequenced,and it was found to locate in cell nucleus,just same as AtMPK6.A stable expression vector specifically expressed in pollen was constructed with LAT52 promoter,transformation of PbrMAPK15 into atmpk3mpk6/+mutant plants was conducted mediated by agrobacterium.The progeny positive transgenic plants were selected to obtain T3 stable generation with the genotypy of atmpk3mpk6/PbrMAPK15-GFP.3.Vector driven by 35S promoter was constructed and PbrMAPK15 was transformed into atmapk6 mutant plants mediated by agrobacterium to obtain stable and genetic positive transgenic plants.The results indicated that PbrMAPK15 can recover the phenotype of mapk6 mutant of Arabidopsis thaliana seed and pod.