Isolation And Functional Analysis Of Extracellular Calcium-sensing Receptor (OsCAS) Gene From Rice (Oryza Sativa L.)

Calcium is a key nutrient for growth and development of animals and plants. Calcium signaling, one of the most important signal pathways, is involved in growth, development and stress response. It is well known that intracellular Ca²⁺ is an important second messenger that controls a varity of cellular functions. Yet in 1993, Brown et al. first cloned the G protein-coupled extracellular Ca²⁺ sensing receptor (CaR) from bovine parathyroid. This suggested that Ca²⁺might also function as an extracellular messenger . Recently, Han and Pei cloned a calcium sensing receptor (CAS) by expression cloning a cDNA library from Arabidopsis leaves in animal HEK293 cells and screening for external Ca²⁺-induced elevations of cytosolic Ca²⁺. The cloning of this receptor has demonstrated that Ca²⁺ also function as a 'first messenger' in plants. Blasting in GeneBank, we found a sequence homology to Arabidopsis CAS. We have cloned the rice OsCAS gene by homology cloning strategy and performed preliminary studies of its function. OsCAS contains 1164 nucleotides which encode 387 amino acids. Sequence analysis shows OsCAS has one transmembrane domain. Its carboxy terminus is possibly intracellular, which is likely to mediate protein-protein interactions, and it has a sequence similar to a rhodanese domain. This domain is also found in phosphatases and a variety of proteins such as sulfide hydrogenases and certain stress responsive proteins. Its N terminus is possibly extracellular. It has no significant homology to any known functional domains and to the extracellular amino terminus of Arabidopsis CAS. In this dissertation project, we investigate the function of OsCAS in the following aspects:1. Expression of OsCAS in HEK293 cells. HEK293 cells were transiently transfected with empty pcDNA₃ vector, OsCAS and Arabidopsis CAS. When [Ca²⁺]_o increased from 0.1 mM to 2.5mM, we observed that [Ca²⁺]_i significantly elevated in OsCAS transfectants. This suggests that OsCAS can sense an increase of [Ca²⁺]_o and result in an increase of [Ca²⁺]_i.2. Subcellular localization of OsCAS. UBQ p:: CAS :: eGFP and UBQ p:: eGFP were transiently expressed in onion epidermal cells by bombardment, we observed using Confocal Laser Scanning Microscope that CAS::GFP (green fluorescent protein) was localized to the cell surface.3. Expression pattern of OsCAS in Oryza sativa L. Total RNAs were prepared from leaves, root, and stem of Oryza sativa using Trizol Regeant and reversely transcribed into cDNAs. These cDNAs were used for quantitation with real time PCR. The result suggests that CAS is predominantly expressed in leaves, lesser extent in stem and bearly detectable in root.4. OsCAS expression and sensitivity to NaCl stress. We constructed OsCAS sense and anti-sense expression vector and transferred them into rice by Agrobacterium- mediated transformation. We obtained the OsCAS expression inhibited and overexpressed strains. The T₀ anti-sense plants showed a severe defect in development and growth. Studied its T₁ plants on the stress resistance, we found that after treatment with 200mM NaCl, the chlorophyll and proline content in the transgenic plants are much lower than the wild type. Adscititious Ca²⁺ could not mitigate the symptom of salt stress, but the symptom of wild type was alleviated under the same conditions.In summary, OsCAS is a calcium sensing receptor localized to the plasma membrane. OsCAS mediates [Ca²⁺]_o -induced [Ca²⁺]_i increases. OsCAS gene plays an important role in rice. The antisense transgenic plants show weak growth, late heading date, late flowering and salt sensitivity. We presumed there are two reasons leading to these phenomena: first, [Ca²⁺]_i oscillations are coupled to the [Ca²⁺]_o oscillations- CAS-IP₃ pathway. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP₃), which in turn directs release of Ca²⁺ from internal stores. When OsCAS was repressive, the IP₃ pathway and [Ca²⁺]_i oscillations was destroyed, so affected expression of the stress responsive gene. It has been reported that when the PLC pathway was destroyed, the accumulation of proline could be affected. Proline can enhance the stress resistance of plants, accumulation of proline is a kind of responsion to stress. Second, Ca²⁺ is necessary for enhancing K⁺ absorb and excluding Na⁺. Ca²⁺, as an important element in the plant signal transduction network, often participates in many signal pathways. Its molecular mechanisms are worthy being elucidated in the further research.