Establishment Of Chemical-Inducible Activation-Tagging Lines In Arabidopsis And Biochemical, Expression Analysis Of AtCRK3

This project is composed of two major parts. One experiment is generation of a lot of chemical-inducible activation-tagging Arabidopsis mutant lines and screen of two salt signaling mutants. Another experiment is biochemical and expression analysis of an Arabidopsis CDPK-related protein kinase.In plants functional genomic research, mutants play a very important role in studying gene functions. Arabidopsis is recognized as an important model plant because of its relative small genome size, mature transformation technique, and completion of the genomic sequencing of Col-0. So it is an important to establish an efficient gene tagging system in Arabidopsis. Chemical-inducible promoter/enhancer system PER16 is a powerful approach for identifying novel mutant, which is generated by randomly integrated PER16 into the genome of Arabidopsis. In the PER16 system, the transcription of target genes is strictly regulated by estrogen. Upon induction by estrogen, target genes expression levels can be eight fold higher than that transcribed from 35S promoter. In the absence of an estrogen, the transcription of target genes is undetected. And no apparent toxicity to Arabidopsis transgenic plants is detected with this system. So the advantage of this PER16 system will allow us to recover mutants that display severely abnormal plant growth and development or lethality under the control of constitutive enhancer. The main experiment results are indicated as follows:In Arabidopsis transformation experiment, we used Arabidopsis of Col-0 ecotype, Agrobacterium of GV3101, transformation method of floral dip. The efficiency of transformation was about l%-2%. Such high efficiency of transformation maked it available to obtain a large amount of T-DNA inserted Arabidopsis lines. Up to now, a total of 22,000 T-DNA insertional lines of Arabidopsis plants transformed with PER16 system have been generated. Approximately 97% of the independent transformants were PCR-positive. Each transformant contained on the average 1.5 copies of the T-DNA tagging using kanamycin identification, suggesting that a total 33,000 sites in Arabidopsis genome were targeted. Fromthese T-DNA insertional lines, we have found several mutants with various phenotypes. The phenotype of one mutant among these was generated by chemical-inducible activation of a certain gene, in which loss of function mutations caused no apparent phenotype.We have carried out a large-scale screening of 200,000 seeds of Arabidopsis that originated from these parental mutant lines, with the aim of isolating mutants capable of germinating on agar medium supplemented with 250 mM NaCl. One high salt tolerant 1 (hstl) mutant was isolated. Further analysis indicated that hstl was more tolerant to NaCl stress than wild-type plant at seed germination and seedling development stages. The hstl mutant also had higher ratio of germination under high LiCl, whereas the effects of elevated levels of Mannitol and ABA were similar between the mutant and wild type. Therefore the mutants were primarily ionic tolerance for sodium. There were no significant phenotype differences between hstl and wild-type plant that grow on the medium without any stress. Further genetic analysis indicated that the hstl was a monogenic recessive mutant, and the phenotype of salt resistance was not linked to the KanR marker. So we concluded that hstl was an untagged mutant.In addition, we have screened a mutant with aberrant phenotype. Under the normal conditions, the phenotype of mutant exhibited shorter root, little rosette leaves, dwarfism, shorter siliques. In addition, the flower time of this mutant was late 10 days than that of wild type, but the rosette leaf numbers is same to that of wild type. Further analysis indicated that the mutant was more sensitive to 150 mM NaCl stress than wild-type plant at seed germination stages. We designed it as high salt sensitive 1 mutant (hssl). The hssl mutant was also more sensitive to high LiCl and ABA, whereas the effects of elevated levels of Mannitol were similar between the mutant and wild type. Further genetic analysis indicated that the hstl was a monogenic recessive mutant, and the phenotype of salt resistance was linked to the KanR marker. So we concluded that hstl was a tagged mutant.The roles of Ca2+ are mediated by a group of Ca2+-binding proteins including Ca2+-dependent protein kinases (CDPKs) and CaM via its binding proteins including CaM-binding protein kinases. Drought, salt, injury, cold, light and hormones can influence the expression or activity of different CDPKs in plants. Arabidopsis contains eight putative CDPK-related protein kinases (CRKs). In the sequence tree, they are located on a branch that is most closely related to a subgroup containing three CDPKs, indicating that there is a high degree of similarity between their catalytic domains. In the previous studies, the activity of AtCRKl for both autophosphorylation and substrate phosphorylation was also Ca2+-independent as indicated in other CRKs, but this activity is stimulated by several Arabidopsis CaM isoforms in a Ca2+-dependent manner. It is suggested that CRKs in plants are perhaps responsive to calcium signaling. In this study, the expression and function ofAtCRK3 in Arabidopsis have been investigated during growth and development by using techniques of molecular biology, cytogenetics, histochemistry, cell biology and so on. The results provide valuable information for understanding the molecular mechanisms of signal transduction pathway controlling plant growth and development. The main results are indicated as follows:A cDNA clone from Arabidopsis corresponding to a typical CDPK related protein kinase, AtCRK3, was isolated. AtCRK3 contained all conserved kinase domain, and had one N-terminal myristoylation sites and C-terminal domain containing apparently degenerate EF-hands that were predicted cannot bind to calcium. AtCRK3 was expressed in Bac to Bac system, and then purified by Ni-NTA resin column. Biochemical analysis showed that the kinase activity of AtCRK3 for both autophosphorylation and substrate phosphorylation were neither enhanced by Ca2+ nor inhibited by EGTA, which was similar to other CRKs. The Fmax and Km of AtCRK3 were 35.5 nmol min" mg" and 6.2 uM respectively when using histone Ills as substrate. Our experiments also showed that the presence of Ca2+/CaMs couldn't stimulate the kinase activity compared with those in the presence of Ca +. Capillary electrophoresis showed that autophosphorylation of AtCRK3 occurred on threonine residues. These data showed that AtCRK3 was a serine/threonine protein kinase. pH, MgC^ and NaCl had effects to the kinase activity of AtCRK3. When pH was 8.0, [MgCb] was 10-15 mM, and [NaCl] was 0 mM, the enzymatic activity of AtCRK3 was the highest.The results of Northern blot indicated that the transcript of AtCRK3 expressed in all tested organs and highest levels in roots and flowers, less in leaves, stems and siliques, which were similar to the results of AtCRK3 promoter-GUS stain. And there was a significant induction of AtCRK3 in the roots of Arabidopsis under the treatments of cold and ABA. The transcript ofAtCRK3 gradually accumulated by cold stress and reached its maximal level at 12 h, and this increased response slightly decreased at 24 h after cold stress. In the ABA treatment, it was found that AtCRK3 transcript obviously increased at 2 h and attained its maximal level at 4 h after exposure to ABA treatment and then gradually reduced until the end of the ABA treatment (10 h) in this study. The results of RNA in situ hybridization showed that: during Arabidopsis vegetable growth, AtCRK3 mRNAs were expressed mainly in actively dividing and vigorously metabolizing cells and tissues, whereas in quiescent cells, AtCRK3 mRNAs were expressed at very low levels or not expressed. In addition, AtCRK3 mRNAs distributed in vascular tissues in all tested tissues and organs, implying that AtCRK3 might play an important role in nutrition transportation. And during pollen development, tapetum cell development and degeneration, the transcription of AtCRK3 also expressed obviously in a temporal and spatial difference manners. Using the onion cells transient expression assay and confocal microscopy, the results showed that the distribution patterns ofAtCRK3::GFP similar to that of free GFP, including nuclear and cytosolic fluorescence in onion cells, indicating that the location of AtCRK3 was different from predication according to the sequences. In sum, our studies showed that AtCRK3 might be an important component in plant signalling transduction pathway.