Function Analysis Of PHK4 Gene From Chinese Cabbage

Two-component signaling system is an important way to sense and transmit the environmental signals in prokaryotic and eukaryotic cells. In recent years, important progresses have been made in studies on the compositions and functions of the plant two-component signaling system. Study results show that two-component signaling systems are involved in transduction and transmission of signals of plant hormone (ethylene, cytokinin), osmosis and light.Cytokinins are very important for the development and growth of plants. They take part in the regulation of various physiological processes in plants, such as promoting cell division and bud differentiation; inhibiting root elongation; regulating apical dominance; promoting the formation of vascular bundles, the development of flowers and fruits, and the production of anthocyanin; participating in gamete cell and embryo development, biosynthesis of chloroplast, stress response and pathogen resistance; delaying blossom time and leaf senescence, and so on.The cytokinin receptors belong to the family of histidine protein kinases, they are elements of two-component signaling system. Studies on the Arabidopsis showed that AHK2, AHK3 and AHK4/CRE1/WOL are the cytokinin receptors, which are involved in combining with cytokinins and transmitting the signals into the cells. AHK2 and AHK3 express in the roots, leaves, stems and flowers. AHK4 expresses mainly in the roots. The cre1-1mutants showed a lack of cytokinin responses, such as cell proliferation, greening and shoot formation. The wol mutants display defects in the root vascular system. In yeast expression systems, the conserved His and Asp mutants abolish a cytokinin-dependent growth, while the CRE1/AHK4 complement the yeast mutants that lack the endogenous His protein kinase and restor the ability to grow in a cytokinin-dependent manner. In Arabidopsis, expression of AHK4/CRE1/WOL enhanced binding of t-Zeatin.Both Chinese cabbage and Arabidopsis are of cruciferous plants, but the genome of Chinese cabbage has not been completly sequenced. Our lab isolated the PHK4 gene which is homologous with AHK4. The isolated cDNA sequence of PHK4 is 3156bp.In this paper, the PHK4 gene was transferred into the yeast mutant TM182 and the transformation of arabidopsis AHK4 mutant was conducted in order to confirm the function of PHK4 by the function complement experiments. The main results are as follows:1. The yeast expression vector pCUY315-PHK4 was constructed and transferred into TM182. Under the stimulation of cytokinin, the loss-of-funtion mutant TM182 which is lethal on glucose medium could restore growth. This preliminary showed that PHK4 funtions as a receptor for cytokinin.2. The plant expression vector p130035SI-PHK4 was constructed and transferred into Agrobacterium tumefaciens strain GV3101. Studies on the transformation of ahk4-1 mutant are under the way.