Isolation And Functional Analysis Of Protein Kinase Genes Relevant To Osmotic Stress In Glycine Soja

High salinity, drought and low temperature are the major environmental factors that limit plant growth, development, geographical distribution and adversely affect crop production in the world. With the rapid development of molecular biology and bioengineering, using the bioengineering strategies to improve stress tolerance in crops has been an important aspect of modern agricultural research. However, stress tolerance is a complex physiological process in which many genes are involved. Using the bioengineering strategies to transfer a key controlling gene (eg: kinase gene, responding and transducting the stress signal) is a more efficient way to slove the problem. If the key kinase gene regulating signal transduction pathways in the plant resistance to stress could be gained, it would provide important gene resource for molecular breeding and theoretic support to the study of plant molecular mechanism of osmotic stress resistance.Wild soybean (Glycine soja) is characteristic of better stress-resistance and adaptive capacity. It is an important gene resource in molecular breeding by means of transgenic technology. In this study, the stress-responsive kinase genes of wild soybean were selected according to gene expression profiles under salinity, drought and cold stresses, which had been established in our laboratory previously. Full-length sequences of three kinase genes were obtained by in silico cloning and SMART-RACE technique. We analyzed the expression patterns of target genes under different stresses. Subcellular localization, kinase activities and physiological function were also detected. This research will provide key gene for plant genetic engineering of osmotic stress and important information for stress tolerance researches. The main results were summarized as follows:1. Selection of kinase ESTs early responding to osmotic stresses from G.sojaESTs of Glycine soja downloaded from dbEST database were aligned with more than 60 000 probe-sets of the Affymetrix soybean chip, and the expression patterns of ESTs were infered using gene expression profiles of wild soybean under salinity, drought and cold stress. The initial result showed that there were 18 kinase ESTs up-regulated under the 4oC treatment, 20 kinase ESTs up-regulated under the NaCl treatment and 27 kinase ESTs up-regulated under the PEG treatment. According to the annotations of UniProt and Go database, we choose the ESTs of GsAPK, GsCBRLK and GsLRPK for the full-length genes isolation.2. Isolation of protein kinase genes early responding to osmotic stresses from G. soja The full-length cDNA of the three kinase genes were isolated by in silico cloning method and the modified SMART-RACE technique. The full-length cDNA of GsAPK is 1020bp, encoding a 399 amino acid protein. The full-length cDNA of GsCBRLK gene is 1362bp, encoding a 453 amino acid protein. The full-length cDNA of GsLRPK gene is 2145bp, encoding a 714 amino acid protein. The bioinformatical analysis revealed as follows:GsAPK protein contains the catalytic domain of protein kinase family in its C terminal; myristyl-sitesbut without signal peptide in its N terminal, which suggests this protein might be regulated or activated by post-translation modifications. In addition, it has a transmembrane region from residue 184 to 201GsCBRLK protein has a CaM binding domain, a myristyl-sites, no signal peptide in its N terminal and the catalytic domain of protein kinase family in its C terminal.GsLRPK protein contains the feature of LRR-RLK family, LRRs motifs, transmembrane region, protein kinase catalytic domain and a signal peptide which can introduce the protein to the secretory vacuole.3. Expression patterns analysis of kinase genes under various stress treatmentsSemi-quantitative RT-PCR was used to analyze the expression patterns of kinase genes under different stresses. GsAPK, GsCBRLK and GsLRPK genes were induced by low-temperature, drought, high salinity and ABA stress treatments which demonstrate that they are likely involved in ABA-dependent signaling pathways and might serve as a master regulator in plant abiotic stress response. The tissue specific expression pattern in leaves and roots likely indicate the existence of different regulatory mechanisms in these two tissue types.4. Subcellular localization of kinase proteinsThree subcelluar localization vectors were constructed and the GFP-fusion kinase proteins were transiently expressed in tobacco leaf cells via the Agro-bacterial infiltration. GFP fusion kinase proteins were observed solely to be localized to the plasma membrane using a confocal laser scanning microscope.5. Protein GsCBRLK binding assayThe full-length GsCBRLK, three truncated forms and GsCaM were cloned into the downstream of HIS6 tag into the pET-32b expression vector. The constructs were then transformed into E. coli strain BL21(DE3) pLysS and fusion proteins were induced by IPTG. Using the CaM binding assay, we found that the CaM-binding property of GsCBRLK is Ca2+-dependent and the CaM-binding site is located at amino acids 147-169 in GsCBRLK.The full-length GsCBRLK, three truncated forms and GsCaM were cloned into the Y2H vectors and introduced into the yeast strain Y187. The activity ofβ-galactosidase indicated that GsCaM and GsCBRLK could interact in vivo. 6. Enzymatic activity assayIn vitro phosphorylation assays were performed to detect the enzymatic activity of target kinase proteins. The results showed that GsAPK is an ABA-activated Ca2+-independent protein kinase. GsCBRLK kinase activity is Ca2+ dependent and is possibly up-regulated through the direct interaction between CaMBD and CaM. In addition, Mn2+ did not activate GsCBRLK autophosphorylation while Mg2+ only had a weak effect. Interestingly, strong activity was observed in the presence of Ca2+. However, autophosphorylation of GsCBRLK is Mn2+-activated, not Mg2+ or Ca2+. GsLRPK is a cold and drought activated protein kinase.7. Overexpression of target kinase genes and the phenotypic analysis of transgenic plantsTwo binary vectors harboring the HIS6-tag-fused-kinase proteins were constructed and introduced into Arabidopsis plants by Agrobacterium tumefaciens-mediated transformation. DNA gel blot and the semi-quantitative RT-PCR showed the presence of kinase genes in the two independent T4 generation transgenic lines.Root growth assays were performed to detect the seedlings response to various stresses. The results showed that the growth was less significantly inhibited in the wild-type plants compared to that of GsAPK over-expressor lines under NaCl stress. Conversely, in ABA treatment, growth of transgenic Arabidopsis plants is better than wild-type plants. Statistical analysis confirmed that over-expressing GsAPK can significantly alter plant tolerance to salinity and ABA stress. GsCBRLK gene can significantly improve plant tolerance to salinity and ABA stress. GsLRPK gene functions in cold stress response of yeast cells.