In Silico Cloning And Bioinformatic Analysis Of Maize Mitogen-Activated Protein Kinases

Based on EST (expressed sequence tag) database and maize genome database, wepredicted maize mitogen-activated protein kinase (MAPK) gene family, using an in silicocloning method. The putative MAPK genes were identified with the aid of various types ofbioinformatic software and designated to be ZmMPKs (Zea mays MAPK). Subsequently, threeZmMPKs were cloned, i.e. ZmMPK5, ZmMPK6, and ZmMPK9. The main results of this studywere:(1) Based on OsMPK (Oryza sativa MAPK) genes and Maize Genetics and GenomicsDatabase (Maize GDB), 11 putative ZmMPKs were predicted through in silico analysis ofESTs; 3 putative ZmMPKs were found through searching in cDNA database and 6 putativeZmMPKs were found in maize protein database using HMMer 2.3.2. According to theevolutionary relationship in OsMPKs, AtMPKs (Arabidopsis thaliana MAPK) and ZmMPKs,the cladogram of 58 MAPK genes from the three species were drawn classifying 21 putativeZmMPKs which were renamed to be ZmMPK1 through ZmMPK21.(2) The 21 putative ZmMPKs were analysed by various types of bioinformatic software:ProtParam for analyzing protein physico-chemical property; ProtScale for analyzinghydrophilicity and hydrophobicity; TMpred (Prediction of Transmembrane Regions andOrientation) for analyzing transmembrane regions; COILS for analyzing coiled-coils; TargetPfor the subcellular location; Pfam and PlantsP for analyzing the protein functional domains;and the SWISS-MODEL for making homology modeling. The results showed that theproteins of ZmMPK7, ZmMPK8, ZmMPK9, ZmMPK10, ZmMPK11, ZmMPK12,ZmMPK21 protein were stable, while the rest of other putative ZmMPKs were unstable. Allputative ZmMPKs were found to be hydrophilic with no transmembrane region and fewcoiled-coils; ZmMPK14, ZmMPK17, ZmMPK18 and ZmMPK20 are likely localized insideof mitochondrion, while the others ZmMPKs were in the cytoplasm. This characteristics wascoincided with MAPKs from other species. Furthermore, the 3D structure of 19 putativeMAPKs was illustrated.(3) According to the sequences of the putative ZmMPKs, the PCR primers weredesigned and 3 ZmMPK genes, i.e. ZmMPK5, ZmMPK6, and ZmMPK9 were clonedsuccessfully,the special fragment of ZmMPK2,ZmMPK3,ZmMPK4,ZmMPK5,ZmMPK17,ZmMPK18 and ZmMPK20 were also cloned, and RNAi vectors were contructed.