BdMKK6.2Gene Cloning From Brachypodium Distachyon And Its Genetic Transformation In Tobacco

Plants are sessile, they do not move about like animals while avoiding unfavorableconditions, which made plant gradually formed a stress response network system differentfrom animal, to adapt to changes in the environment in the evolutionary process.Mitgogen-activated protein kinase (MAPK) cascades play essential roles in plant signaltransduction pathways in response to bio-and abiotic stimulation. MAPK cascades havebeen implicated in diverse cellular processes including cell growth, differentitation,proliferation, program cell death and in responses to diversity of hormones and stress.MAPK cascades are constituted by three kinases (MAPKKKâ†'MKKâ†'MAPK), whichcould transfer the phosphate group of ATP to serine, threonine, or tyrosine of downstreamproteins to regulate their activity. Phosphorylating of target protein could realize thetransmission and amplification of the signal. According to the reports, the MAPKKs havethe minimum number in MAPK cascades in plant. MAPKK may be activated by morethan one MAPKKK and could activate multiple MAPK at the same time, so multipleMAPK cascade may share one MAPKK and MAPK cascades may have interaction. Now,there are evidences indicate that the MAPKK6may be involved in a variety of signaltransmission of bio-and abiotic stresses. It has been found that, AtMKK6in Arabidopsisand NQK1in tobacco have the function of regulatory role in the process of cell division.Brachypodium distachyon is a new model plant species for grass and its genome’ssequecing has been completed, but there are still few reports about BdMKK. First wecloned the cDNA sequence of BdMKK6.2genes from Brachypodium distachyon byRT-PCR, and then we analyzed it with bioinformatics methods. The cDNA is1056bplong which encodes a protein with351amino acid residues. The analysis of structuraldomain indicates that BdMKK6.2contains protein kinase domain and Serine/Threonineprotein kinases active-signature. Amino acid sequence alignment shows that BdMKK6.2contains11conserved subdomains of protein kinases and the consenses sequenceS/T-X3-5-S/T of plant MAPKKs. Phylogenetic analysis reveals that BdMKK6.2is highlyhomologous to TuMPKK1, AetMAPKK1and OsMKK6, all of which are classified asgroup A MAPKKs. In order to further study the functions of BdMKK6.2in plants, its overexpressionvector was constructed by inserting it into pCAMBIAl304under the control of CMV35Spromotor. Then the overexpression vector was transformed into tobacco byAgrobacterium-mediated transformation method. Five positive transgenic tobacco lineswere confirmed by PCR and RT-PCR. These overexpression transgenic tobacco lines canbe used in subsequent study.