Identification And Molecular Biological Analysis Of Interaction Proteins With Wheat WK Protein Kinase

Abiotic stress such as drought, extreme temperature and high salt seriously affect plant growth and production, plants were evolved to adapt to these environment stresses. In the process, the signal was transmitted both in intracellular and extracellular. Phosphorylation and dephosphorylation of proteins are important biochemical reactions involved in the energy metabolism and signal transmission. In addition, the phosphorylation of proteins is complished by protein kinases. What’s more, research on protein kinases of wheat have great meaning for the mechanism of physiological metabolism and stress resistance. This study focuses on the identification of the relationship that WK interacts with WD40and MAPK3respectively, which were screened by yeast two-hybrid with WK. In addition, both WD40and MAPK3were analyzed about the characteristic of expression, tissue specificity and the subcellular localization.(1) The full length of WD40and MAPK3were structured to the vector of pGADT7respectively, meanwhile WK were structured to pGBKT7vector. pGADT7-WD40and pGADT7-MAPK3were recpectively co-transformed in yeast cells with pGBKT7-WK. As results, yeast can grow on the SD/-Ade/-His/-Leu/-Trp/X-a-gal and the spots are blue, what’ more, the sequence of those genes were absolutely right, which indicated that WK interacts with WD40and MAPK3in yeast respectively.(2) The wheat seedings were treated with low-temperature, drought, ABA, NaCl respectively, and the characteristic of expression of WD40were analyzed. The wheat seedings were treated with ABA, drought, NaCl, MeJA respectively, and the characteristic of expression of MAPK3were analyzed.It is showed that WD40was highly up-regulated in drought treatment. Meanwhile, MAPK3has a great response to ABA and salt stress.(3) The WD40and MAPK3fragments were cloned into the green fluorescent protein (GFP), Subcellular localization of GFP expression in onion epidermal cells was monitored by confocal microscopy after particle bombardment fusion protein coated microprojectiles, GFP control vectors were transiently expressed in all onion epidermal cells,WD40fusion protein just was monitored in cell nucleus, MAPK3fusion protein was monitored in cell nucleus and cytomembrane. The results indicate that WD40protein located in the cell nucleus and MAPK3located in the cytomembrane and cell nucleus.(4) The WD40and MAPK3fragments were cloned into the pBI121, the eukaryotic expression vector. And then transformed into C58C1, Agrobacterium strain. Selectived transgenic Arabidopsis lines by Agrobacterium-mediated dip flora using50mg/L kanamycin. Four transgenic Arabidopsis lines of WD40and ten transgenic Arabidopsis lines of MAPK3were obtained, which set a confirm foundation for the further research of stress resistance.