In Silico Bioinformatic Analysis Of MAPKs Gene Family And Cloning, Expression Analysis Of TaMPK1in Wheat (Triticum Aestivum L.)

Plants are frequently challenged by various harsh environmental factors that limitplant growth, development and biomass productivity throughout their life cycle. Tocombat these stresses, plants have developed a variety of complex and sophisticatedcellular signal transduction network. As a molecular switch, the reversible phosphory-lation of proteins plays a vital role in fast and accurate cellular signal transfer. Amongthem, the protein phosphorylation is catalyzed by protein kinase. Mitogen-activatedprotein kinase (MAPK) cascades are universal and evolutionarily conserved signaltransduction modules in eukaryotes. They play a key role in plant environmentaladaptation through regulating cytoplasmic proteins’ function and/or gene expression.MAPK cascades are minimally composed of three kinase modules, MAPKKK, MAPKKand MAPK, which are linked in various ways to amplify and transmit exogenous signalfrom upstream receptors to downstream targets. Because wheat is one of the importantgramineae food crops in the world, studies on the molecular mechanisms of its stresstolerance has been received special attention. However, there is few report on wheatMAPKs genes because of its genome complexity and its unavailability genome sequence.Based on the wheat EST database information, in silicon cloning approach was usedto predict and analyze putative MAPK gene family in wheat genome. Besides, full-lengthcDNAs of three wheat MPAK genes, designated TaMPK1,TaMPK4and TaMPK8, weresuccesfully cloned by RT-PCR technique. TaMPK1, which was at the same evolutionbranch with OsMPK2and AtMPK1, was selected for further analysis of its nucleic acidcharacteristics, protein information and expression pattern.The main results are as follows：1) In silico bioinformatic analysis of wheat MAPKsBased on OsMPKs (Oryza sativa MAPKs) and AtMPKs (Arabidopsis thalianaMAPKs) genes and wheat EST database, a total of11putative TaMPKs were predictedthrough in silico analysis of ESTs in wheat genome. characteristics of their sequences,ORFs, subcellular localization information and expression patterns were analyzed usingvarious softwares. According to the evolutionary relationship with OsMPKs, AtMPKs, NtMPKs and ZmMPKs, the cladogram of52MAPKs from the five species were drawnand the11putative TaMPKs were renamed in a systematic way.2) Cloning and characterization of a group C MAPK gene TaMPK1in wheatAccording to the in silico analysis results, one contig having high homology withAtMPK1was selected for cloning with specific primer pairs by RT-PCR using total RNAextracted from wheat seedlings as template. The amplified cDNA fragment of AtMPK1gene contains a1110bp ORF, which encoded a protein,including369amino acid residueswith a calculated relative molecular weight of42.2kD, and designated TaMPK1. Analignment of the amino acid sequences of TaMPK1with other plants MAPKs showed thatthe TaMPK1contained all11complete subdomains of serine/threonine specificity proteinkinases, specially, it contained the TEY motif. A phylogenetic tree analysis indicated thatTaMPK1was highly homologous to OsMSRMK2, ATMPKl, ATMPK2, ATMPK7,NtNTF3and PsMPK2, all of which were classified as Group C MAPKs．3) TaMPK1subcellular localization researchBioinformatics prediction and subcellular localization experiment of maizehomologous gene showed that TaMPK1had a higher probability in the nucleus. While ourtransient gene expression result in onion epidermal cell transformed with TaMPK1::GFPshowed that TaMPK1::GFP fusion protein was distributed in whole cell.4) TaMPK1expression analysis1) Expression of TaMPK1gene in different tissues of wheat was examined byReal-time quantitative PCR. The results showed that TaMPK1expressed in all wheatorgans checked including roots, stems, leaves, stamens, pistils as well as lemma,while its expression level was the highest in stem.2) The Real-time quantitative PCR was used to examine expression of TaMPK1gene after various abiotic stress treatments on wheat seedlings such as lowtemperature, PEG and high-salt. It was found that expression of TaMPK1wasup-regulated under40C cold,20%PEG6000and200mmol/L NaCl treatmentsalthough their expression patterns were different. Therefore, TaMPK1was consideredto be related with the response of wheat to these aboitic stresses.3) Signal molecules including ABA and hydrogen peroxide were used to treat wheat seedlings to investigate whether there are involved in regulation of TaMPK1expression. The result of Real-time quantitative PCR showed that TaMPK1geneexpression was increased under ABA and hydrogen peroxide treatments, indicatingthat ABA and hydrogen peroxide signaling pathways are involves in TaMPK1generegulation in response to these aboitic stresses.The information of MAPK genes in wheat genome in this research providedimportant reference for their cloning and functional study.