Cloning And Initial Function Analysis Of Cytokinin Oxidase/Dehydrogenase Genes From Wheat

As important plant hormones, cytokinins (CK) are considered to play a key role in plant growth and development, including cell division and differentiation. The homeostasis of endogenous CK levels in plants is regulated by biosynthesis and metabolism. The cytokinin oxidase/dehydrogenase (CKX) was thought by far to be the only enzyme in plants to catalyze the catabolism of the active CK into inactive products by cleaving the N6-unsaturated side chain of the specific CK. CKX is considered to be correlated to crop productivity and anti-stress ability. Cloning and characteristic analysis of TaCKX genes are carried out for studying the function of these genes in wheat growth and development. These researches can provide a clue for wheat breeding by using TaCKX gene family.Two novel genes of TaCKX were cloned from wheat and their expression characteristics were analyzed in the work. The function of TaCKX1 and TaCKX3 was studied initially. The main results are as follows:1. Two novel genes, TaCKX3 and TaCKX8, were cloned from wheat and their sequences were analysed. The open reading frames (ORF) of TaCKX3 and TaCKX8 encode a 516-polypeptide and a 522-polypeptide respectively. The deduced sequence of TaCKX3 has no signal peptide, and therefore, TaCKX3 may carry out functions in cytoplasm, whereas TaCKX8 has signal peptide. As shown in the alignment, both of the predicted sequences of TaCKX3 and TaCKX8 have two conserved domains for FAD binding and CK binding. The phylogenetic tree revealed that the putative polypeptide of TaCKX3 has significant homology with ZmCKX10, and TaCKX8 has high homology with OsCKX3. TaCKX3 and TaCKX8 have low homology with reported TaCKX1,TaCKX2-1 and TaCKX2-2.2. Prokaryotic expression vector pET-TaCKX1 was constructed and the pET-TaCKX1 plasmid was transformed into BL21 (DE3) plysS for expression. The expression of TaCKX1 gene was increased with the time induced by IPTG. TaCKX1 fusion protein had been expressed successfully in the form of inclusion bodies. The fusion protein which is 58.9kD was purified for TaCKX1 polyclone antibody purified.3. The real-time quantitative reverse transcription PCR showed that TaCKX3 and TaCKX8 can be significantly induced by 6-BA. The expression of TaCKX8 was down-regulated by PEG in leaves,root and shoot tip of wheat seedling. Under NaCl treatment, the expression of TaCKX8 decreased in leaves and shoot tip, but increased in root. This result suggests that TaCKX8 gene may be involved in the regulation of root growth under salt stress. The expression analysis showed that TaCKX3 and TaCKX8 could regulate plant growth under abiotic stress, but the mechanism of their function may be different. The diverse expression pattern of TaCKX8 in different parts of wheat revealed that TaCKX8 may perform distinct regulating functions in those parts.4. Plant expression vectors and RNA interference vectors for TaCKX were constructed successfully. Transgenic TaCKX3 and TaCKX8 Arabidopsis were obtained by Agribacterium tumefaciens-mediated transformation. Under drought stress, TaCKX1 can promote root elongation of Arabidopsis, but cause the decrease of germination rate. The result suggested that TaCKX1 may be involved in the regulation of root and shoot parts growth under drought stress to adapt to the stress environment. TaCKX3 was proved that it can promote root elongation in TaCKX3 transgenic Arabidopsis.In conclusion, we cloned two novel TaCKX genes from wheat, and analyzed their sequence characteristics and expression patterns. Plant expression vectors of TaCKX were constructed. The functions of TaCKX1 and TaCKX3 were analyzed by studying Transgenic TaCKX1 and TaCKX3 Arabidopsis. The above works established a foundation for using these novel genes in wheat transformation and genetic improvement.