| Summary: | 碩士 === 國立臺灣大學 === 生化科技學系 === 99 === Bambusa oldhamii is characterized by its fast growth. This feature might relate to physiological role of cytokinin. Two putative cytokinin receptors genes (CRE, cytokinin response element) are cloned from bamboo cDNA library, designated as BoCRE1 and BoCRE2p. However, both genes lack the DNA fragment encoding the N-terminal sequences by comparing with CRE1 from other species, such as rice and Arabidopsis.
To obtain the full-length cDNA encoding BoCRE1 and BoCRE2p, TAIL-PCR, 5’-RACE and RT-PCR were used to amplify the fragment of the 5’-end. The deduced protein sequences of BoCRE1 is similar to the OsCRE1. The deduced protein sequences of BoCRE2p is more similar to the ZmHK, but lacks of histidine kinase (HK) and receiver domain (RD). In an attempt to analyze BoCRE1 biochemical properties, recombanant protein of HK, RD and the HKRD domains were heterologously expressed in BL21(DE3). Recombinant HK and HKRD were expressed in inclusion bodies, whereas recombinant RD was expressed in soluble fraction. Polyclonal antibody against purified recombinant RD was prepared. To confirm the identity of BoCRE2p in Bambusa oldhamii, 3’-RACE were employed to identify the stop codon. Probes designed based on the conserved sequences of BoCRE1 and BoCRE2p were prepared for Northern blot analysis to determine RNA expression level of these genes. The expression profiles of these two genes in the different bamboo tissues and the different growth stages were analyzed by quantitative real-time PCR. The levels of BoCRE1 mRNA showed significant changes in the base and the top regions of bamboo at different growth stages, whereas the levels of BoCRE2p mRNA showed significant changes in the middle regions at different growth stages.
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