Identification And Characterization Of NtKrp, A Novel Member Of Kinesin Related Protein Superfamily In Tobacco

Experiments were performed to investigate the role of NtKrp gene in development of Nicotiana tabacum L. cv Wisconsin 38. We analyzed the expression pattern, biochemical activity, in vivo localization and the transgenic plants' phenotypes of this gene. The main results were as fellows:1. Through screening the cDNA libraries of embryos at different stages, we succeed in cloning the full-length NtKrp gene, and efficiently performed a series of bioinformatic analyses on the putative protein sequence. A motor domain was found in this protein, which was highly conserved in kinesin superfamily. So it was supposed that this protein belongs to kinesin superfamily. Through an analysis of the motor domain, we classified this protein into the orphan subfamily with AtPAKRPl, which is a kinesin-related protein in Arabidopsis. This protein has no high homologues with other members in this superfamily. Up to now litter is known about this subfamily. And the putative spatial structure of this domain is similar with the other motor domains of kinesin superfamily. So this motor domain may have the ATPase activity and the binding capability with microtubules. All these results showed that NtKrp is one member of the kinesin superfamily.2. In order to study the function of this gene, the expression patterns of this gene was analyzed in tobacco. With RT-PCR technique, the expression in several tissues was firstly analyzed, such as leaf, root, stem and unfertilized flower. The expected signal was only detected in leaf. The expression of NtKrp gene during embryogenesiswas also analyzed with single-cell and single-embryo RT-PCR technique. The expected signals were detected in both zygote and embryos, but not in egg. Then in situ hybridization experiment was performed to analyze the localization of this gene in leaf and embryos. It was found that the expression of NtKrp in leaf was mainly limited to the palisade tissue of mesophyll. And there are distinct localization patterns of this gene in embryos. At the globular embryo stage, the expected singals distributed only in the embryo proper and a few of endosperm cells around the embryo. But at heart shaped embryo stage, the signal was only detected in the central area along the apical-basal axis and cotyledons. This distinct expression patterns provided an important cues about the function of this gene, and also showed the specificity of this gene. The difference in the expression pattern between NtKrp and AtPAKRPl suggests a difference in function although they are highly homologuous.3. In order to get more information about this gene, we analyzed the biochemical activity of motor domain with the enzyme coupled system and ITC method. This highly conserved domain of kinesin superfamily is a microtubule-dependent ATPase, which can hydrolyze ATP and move along the microtubules. The motor domain of NtKrp was expressed and purified through E. coli. The motor domain of NtKrp protein showed an obvious feature of microtubule-dependent ATPase. But compared to the other kinesins, the K_m mt of NtKrp is lower, so the binding of kinesin to microtubules is weak, which may also provided an important cues about the function of this protein. The affect of the tail region protein was also analyzed on the ATPase activity of motor domain. With enzyme coupled method, we tested the ATPase activity under different condition. The results showed that as the concentration of tail region protein was getting lower, the ATPase activity of motor domain was getting higher, indicating that the tail protein can inhibit the ATPase activity of the motor domain. This suggests this protein may regulate its activity status by self-folding.4. Through bioinformatic analysis, it was found that there were three parts in this protein: motor domain, central stalk region and tail region. All of them had their own contributions to the function of this protein. The in vivo localizations of full-length protein, motor domain and tail region were analyzed respectively. As the first step, wesucceeded in constructing three expression vectors, which expressed the fusion proteins with GFP. After transformed into the onion epidermal cells with particle bombardment, the in vivo localizations of these fusion proteins were observed. The in vivo localizations of full-length protein and the tail region showed a similar pattern. All of them located in the cytoplasm. But the motor domain showed a different pattern, which located in both cytoplasm and nucleus. This means that the tail region played an important role in the in vivo localization of full-length protein. Moreover, we found a highly conserved domain (coiled-coil) in the tail region through further bioinformatic analysis. It was well known that this domain in tail region always functioned in the interaction with the downstream cargo. We found a highly conserved motif (UG_FHA₁) in this domain, which is a threonine-phosphorylation dependent motif. After the phosphorylation of the threonine, this motif may introduce the interaction between the motif and a protein containing FHA domain. With site-mutant technique, we test the hypothesis. When threonine was deleted or replaced by alanine, the in vivo localization of full-length protein and the tail protein were also detected in nucleus. This result indicates that this motif plays an important role in their localization in vivo. It seems that the threonine-dependent phosphrolytion may be involved in the interaction between NtKrp and the motif.5. In order to get further information about the role of NtKrp in the development in tobacco, analysis was carried out with transgenic plants through the agrobacterium-mediated transformation with lead disk of tobacco. Several vectors for full-length protein, motor domain, tail region, anti-sense and RNAi were constructed. The primary analysis of the phenotype of the antisense transgenic plants was performed. Compared to the control seeds, the germination of this transgenic seeds was delayed and the metabolism was slower, which indicates that the transfer of the materials and the transform of the energy for seed germination were slower. The development of the transgenic plant showed much slower than that of the control plants. All these results suggest that NtKrp plays an important role in the development of tobacco. The experiments with other transgenic plants were still ongoing.