Analysis Of PHK4 Gene Of Chinese Cabbage

Cytokinin as a kind of common hormones in higher plants participates in regulating a variety of physiological processes during the individual growth of plants.Two-component signaling network exists in prokaryotic and eukaryotic cells.The organisms sense and transmit the environmental signals by this system.The cytokinin receptors are elements of the two-component signaling net.They belong to the family of histidine protein kinases.AHK2,AHK3 and AHK4/CRE1/WOL are the cytokinin receptors of Arabidopsis,which are involved in combining with cytokinins and transducing the signals into the cells.AHK2 and AHK3 are expressed in the roots,leaves,stems and flowers;AHK4 is expressed mainly in the roots.Both Chinese cabbage and Arabidopsis are of cruciferous plants.Our lab has amplified the genomic DNA,CDS and 3' UTR sequence of a gene homologous with AHK4 from Chinese cabbage self-line AB-81 which we named PHK4.In this work,the 5' UTR of PHK4 was amplificated by 5' RACE.The study results of PHK4 gene in this paper are as follows:(1)The prokaryotic expression vector pET-30a-PHK4 was expressed successfully in E.coli.By changing the IPTG concentration and induction time,the expression condition of PHK4 gene was studied.The target protein was in the soluble form and the relative molecular mass was about 116 KD identified by the SDS-PAGE.(2)The in vitro adventitious bud regeneration system for wild-type Arabidopsis and the callus induction system for both wild-type Arabidopsis and the ahk4-1 mutant were established.On the media of MS+KT 0.1-0.5 mg/L+2,4-D 0.1-0.5 mg/L,the induction frequencies all reached 100%.The calli inducted on the medium of MS+2,4-D 0.1 mg/L+KT 0.5 mg/L showed the highest differention rate after being transferred to differention media.MS+6-BA 0.5 mg/L +IAA 0.1 mg/L was the effective medium for bud differentiation,the regeneration efficiency of leaf and stem was respectively 82.30%and 69.83%on this medium.The plant over-expression vector p130035SI-PHK4 was transformed into the Arabidopsis thaliana ahk4-1 mutant by leaf disc transformation and the transformed calli were obtained.The root elongation of the transgenic ahk4-1 mutant calli was inhibited in the presence of the cytokinin.This functional complementation test showed that PHK4 has cytokinin receptor function.(3)The 1607 bp sequence upstream of the PHK4 gene was cloned and was analyzed by PLACE and Plant CARE softs.It was predicted that the promoter region has not only the basic transcription elements but also the regulatory elements,such as the anaerobic induction element,the fungal elicitor-responsive element,the regulatory element of endosperm expression,the response element of heat stess,and the response element of low temperature.The PHK4 promoter-driven GUS gene expression vector was constructed,which has laid the foundation for the future study of the expression of PHK4 gene.