Functional Analysis Of Calcineurin B-Like Gene OsCBL6 From Rice (Oryza Sativa L.)

Calcium is a ubiquitous secondary messenger in plants and play crucial roles in plant growth and development and responses to internal and external stimulation. The production and transduction of calcium signals are realized through the change of calcium concentration in cytoplasm. These Ca2+ signals is deciphered by various intracellular Ca2+ sensors which relays and enlarges Ca2+ signals into a wide variety of biological responses. In plant cells, CBLs proteins have been implicated as one type of the most important Ca2+ sensors in plant-specific calcium signaling. This thesis aimed to clone and functional analysis of OsCBL6, a member of OsCBLs gene families, related in plant environmental stresses.Based on the similarity of sequence, OsCBL6 gene was identified by searching the rice japonica genome database, which was located on chromosomes 12. To investigate the cellular function of rice OsCBL6 gene, two plant expression vectors carrying sense or antisense OsCBL6 gene under the regulation of cauliflower mosaic virus 35s promoter were constructed. The mature embryo derived calli were infected by Agrobacterium tumefaciens EHA105 with sense-pCAMBIA1304-OsCBL6 or antisense-pCAMBIA1304-OsCBL6, from which hygromycin resistant plants were obtained. PCR identification proved that the sense-OsCBL6 or antisense-OsCBL6 was integrated into the genomes of the T1 rice lines. The result of southern blot assay showed that WT had no band, but all of the transgenic strains had 1 or 2 bands. From the result that we could get the conclusion that OsCBL6 was steadily incorporated into genome of the WT and the copy numbers of OsCBL6 in transgenic lines were 1 or 2. So they could be used in the next experiments. Semi-quantitative RT-PCR assay showed that the expression of OsCBL6 extremely increased in sense transgenic rice lines, and decreased to some extent in antisense transgenic rice lines. There were no significant variation in plant height, tiller number, effective tiller number, panicle length,100-seeds weight and seed setting rate among non-transgenic line and 4 sense-OsCBL6 transgenic lines. Comparing the agronomic traits of 4 antisense-OsCBL6 transgenic lines with those of the non-transgenic rice plants, it was showed that the seed setting rate of A5-1 was significantly lower than that of non-transgenic plants while there was no significant difference in the agronomic traits among other antisense-CsCBL6 transgenic lines and non-transgenic plants. After the seedlings of T2 generation were treated by abiotic stresses, the contents of proline and soluble sugars in sense-OsCBL6 transgenic lines were much more increased than those in antisense-OsCBL6 transgenic lines and non-transgenic plants while the relative electrolyte leakages from antisense-OsCBL6 transgenic lines and non-transgenic plants were higher than that from sense-OsCBL6 transgenic lines. By evaluation of tolerance to 150 mmol NaCl, drought and low temperature (4℃) treatments, over-expression of OsCBL6 in rice significantly enhanced the tolerance of transgenic plants. It was discussed that the OsCBL6 protein might play an important role in the tolerance of rice to abiotic stresses.