Cloning And Functional Analysis Of BdCKX Gene In Brachypodium Distachyon

Cytokinin is a major type of important plant hormones that participate in the regulation of many physiological functions and development processes in plants, including inhibitory effect on primary root elongation, seed dormancy breaking, organ development regulation,responses to environmental stimuli, etc. The irreversible degradation of endogenous cytokinin is dependent on the activity of the cytokinin oxidases. Therefore, research on cytokinin oxidase will allow insightful understanding of plant development, and consequently facilitate the genetic improvement of crops. In this research, two cytokinin oxidase genes in Brachypodium distachyon, BdCKX1 and BdCKX2, were selected to investigate their differential expression profiles at different developmental stages in various tissues using qPCR approach. In addition, an overexpression construct was made by ligating the BdCKX1 gene with the pCXSN vector, which was subsequently transformed into both Arabidopsis and Brachypodium. The resulting morphological phenotypes of the transgenic plants were further analyzed. The significant findings from this research can be summarized as the follows:( 1) Expression spectra showed that expressions of BdCKX1 and BdCKX2 both reached their peak levels in leaf of 40 d old plants, whereas they remained low levels in the root. In addition, within 50 days after germination, both genes had lower expressions in root than in leaf, suggesting that 40 days post germination might be the crucial stage for the functionality of BdCKX genes in Brachypodium distachyon. This also implied that expressions of both BdCKX1 and BdCKX2 exhibited tissue specific patterns, which might not be affected by the long distance transport of cytokinin.( 2) Through the construct for overexpressing BdCKX1, the BdCKX1 gene was ectopically expressed in Arabidopsis. It was found that germination of the transgenic seeds(T3 generation) was delayed for two days from the control, showing a phenotype in cytokinin deficiency. This suggested that monocot plants probably utilized a similar conserved mode on cytokinin regulation to dicot plants.( 3) In Brachypodium transgenic plants overexpressing the BdCKX1 gene, the above-ground tissues of T1 generation transgenic plants suffered from developmental defects, exhibiting weak growth, a clear indication for deficient cytokinin. This implied that BdCKX1 participated in cytokinin degradation and provided preliminary evidence for itsfunction.