Bioinformatic Analysis And Function Investigation Of Arabidopsis Lectin Receptor Like Kinase LecRK-b2

Lectin protein kinase (LecRK) belongs to receptor-like protein kinase which is widespread in plants. It is generally believed that they participate in varieties of signal transduction and the identification of sugar signals. But to date, little is known about their gene function. In this study, Arabidopsis lectin receptor-like kinase LecRK-b2 was analyzed with bioinformatics methods and its gene functions were investigated. The results of detailed research are as follows:(1) Using the bioinformatics methods, the amino acid sequence and functional domains of LecRK-b2 were analyzed. Applying the homology modeling, the three-dimensional structure model of lectin receptor domain of LecRK-b2 protein was constructed. The results showed that LecRK-b2 contained three functional domains: N-terminal was the soybean-agglutinin-similar ligand recognition domain, the middle part was a transmembrane domain, C-terminal contained a serine/threonine kinase domain. The 24 amino acids nearby the N-terminal were predicted by the software for the signal peptide sequence. Hydrophobicity analysis results showed that, except for an obviously hydrophobic areas which formed a transmembrane structure, there were no other significantly hydrophobic domains. The total number of hydrophilic amino acid was slightly more than hydrophobic amino acids, so LecRK-b2 was a hydrophilic protein. Three-dimensional structure model showed that the N-terminal domain of LecRK-b2 protein was very similar to the plant lectins whose crystal structure was determined inβ-sheet and carbohydrate binding site. plantCARE analysis showed that there existed a large number of hormone-related boxes, as well as stress-induced associated boxes in the regulatory region of LecRK-b2 gene. The predicted results of PROSCAN program showed that LecRK-b2 protein might contain 19 phosphorylation site, a serine/threonine protein phosphorylation active site, two ATP-binding site. It seemed that LecRK-b2 was a serine/threonine protein kinase.(2) RT-PCR analysis revealed that LecRK-b2 gene had very low expression levels in various tissues and organs in wild-type Arabidopsis, but its expression level significantly increased in the first 24 hours during germination process, and its declined to a very low level after the completion of germination. ABA, NaCl, mannitol, salicylic acid, wounding, and senescence could induce the transcription of LecRK-b2 gene. GFP fusion protein analysis revealed that the LecRK-b2 localized in the plasmid membrane. Promoter::GUS test results showed that the 904bp sequence in the upstream of LecRK-b2 gene had a completed promoter activity, temporal and spatial specificity expression patterns, and controled LecRK-b2 gene kept in a high level of expression during germination. While the transcription of LecRK-b2 gene was almost unable to be detectedin adult plants..The seeds germination of T-DNA insertion mutants (lecrk-b2) was insensitive to ABA. Analysis results indicated that there was no significant difference in ABA content between lecrk-b2 and wild-type seeds. It illustrated that the ABA-insensitive germination of lecrk-b2 seed was not caused by the different endogenous ABA content, but had relationship with the ABA-related signal transduction. In addition, germination and seedling root elongation of lecrk-b2 was insensitive to salt stress and osmotic stress.Transgenic plants were obtained by constructing 35S::LecRK-b2 plasmid and transforming lecrk-b2. The results showed that there was no significant difference in germination between overexpression transgenic lines and wild-type on different concentrations of ABA containing medium. This revealed that the T-DNA insertion mutant SALK₀20262 could be restored to the wild-type phenotype by 35S::LecRK-b2. It indicated that LecRK-b2 gene deletion led to ABA-insensitive phenotype of lecrk-b2 seeds.(3) Quantitative PCR results showed that, during the process of seed germination, transcription levels of LecRK-b2 gene were significantly declined in abi3 mutant. It indicated that LecRK-b2 gene transcription was positively regulated by ABI3. Dual-LUC analysis showed that the transiently over-expressed ABI3 protein could active the expression of the LUC, which was controlled by LecRK-b2 gene promoter in vivo in Arabidopsis thaliana. LecRK-b2 gene promoter sequence analysis displayed that there was a binding site of ABI3 named RY/G motif. Combining these results, we concluded that LecRK-b2 gene transcription was regulated by ABI3 protein in Arabidopsis in vivo.(4) By applying pColdTF vector, we expressed LecRK-b2 protein in Escherichia coli and obtained purified soluble recombinant protein. By using 32P-isotope labeled adenosine triphosphate ([γ-32P] ATP) to provide phosphate, we confirmed that the recombinant protein was autophosphorylated and the autophosphorylation activity of recombinant LecRK-b2 was divalent manganese ion dependent. After the recombinant LecRK-b2 protein labeled with 32P hydrolyzed completely in 6N of HCl, the thin-layer chromatography (TLC) was used for the analysis. It was found that the serine and threonine of recombinant protein was phosphorylated in the self-phosphorylation process. It indicated that the LecRK-b2 belonged to serine/threonine kinase.(5) The in vitro pull-down assay showed that recombinant LecRK-b2 protein and the lectin domain could interacted with itself respectively, but the C-terminal kinase domain could not self-interact. Yeast two-hybrid results showed that the lectin domain had self-interaction, and the C-terminal kinase domain did not interact with itself. This result was consistent with that of pull-down assay. Finally, split luciferase complementary assays displayed that the N-terminal and full-length LecRK-b2 protein had self-interaction in vivo. It indicated that LecRK-b2 protein could form dimer in living cells of Arabidopsis thaliana, and the lectin domain of N-terminal played a major role for the formation of protein dimers; and a separate C-terminal kinase domain could not interact with itself, it did not contribute to the formation of dimer structure of the entire receptor protein.