Cloning And Characterization Of RHsp90 Gene In Rice (Oryza Sativa L.) Under Stress

Plants frequently encounter stresses such as salinity, drought or extreme temperature;they adversely affect plant growth, development or productivity. One of the methods to solve such problem is to research on the molecular mechanisms of stress tolerance, finding stress related genes and breeding for stress tolerance in crops. Hsp90 fulfills a housekeeping function in contributing to the folding, maintenance of structural integrity and proper regulation of a subset of proteins under normal and stressful conditions. It co-chaperones with a subset of substrate proteins involved in cell cycle control and signal transduction. In this study, the aim gene-rice heat shock protein 90 kDa gene (rHsp90) was cloned under the carbonate stress, and basing on the recent advance of Hsp90, reaserch mainly focus on its molecular mechanism of stress tolerance and transgenic engneering. The main results are as follows:The rice (Oryza sativa L.) heat shock protein 90 kDa gene (rHsp90, GenBank accession no. AB037681) was cloned from the rice grown under carbonate (NaHCO₃) stress by using differential display method. The full length of rHsp90 gene was 2 731 bp, which contained an ORF (2 433 bp) with a predicted molecular mass of 92.8 kDa and pI 4.89. The BLAST algorithm identified two proteins with high similiarity to rHsp90 protein, and they were from Barley (GenBank accession no. X67960, 91% amino acid identity) and the Madagascar periwinkle (GenBank accession no. L14594, 80% amino acid identity), respectively. The amino acid sequence had a feature containing a eukaryotic secretary signal C-terminal tetrapeptide lys-asp-glu-leu (KDEL) motif that typically located and retained in ER shared with H. vulgare and C. roseus. Southern blot analysis showed the result that rHsp90 was a single-copy gene in rice genome. To understand the responsive relationship between rHsp90 and environmental stress, experiment studied the profile of rHsp90's expression by Northern blot analysis using young rice seedlings under several stresses. The results showed that the accumulation of rHsp90 mRNA was observed in salts (NaCl, NaHCO₃ and Na₂CO₃), PEG, high pH conditions (pH 8.0 and pH 11.0) and heat shock (42 ℃ and 50 ℃) conditions, and the intensity of salt and heat shock stresses influenced the expression of rHsp90. These results suggested that the expression of rHsp90 responded to several abiotic stresses.To determine whether rHsp90 protected cells against environmental stress, experiment used pYES2 as expression vector, and pYES2-rHsp90 was transformed into yeast (Saccharomyces cerevisiae). The results showed that the growth of cells suitable expressing rHsp90 was better than control under NaCl, KC1, LiCl, heat shock (50 ℃) stress, especially Na₂CO₃ and NaHCO₃. These results suggested the expression of rHsp90 in yeast increased the cells' tolerance to various stresses, especially the carbonate stresses. To further investigate a role of the rHsp90 gene in salinity tolerance, the gene was cloned into pBI121, and transformed into tobacco by using Agrobacterium-mediated transformation method. The six independent and stable expression lines of over-expressing rHsp90 were confirmed by PCR, Southern and Northern blot analysis. Salt tolerance of the six independent transgenic lines was examined, and the transgenic plants grew well under NaCl and NaHCCb concentrations as high as 200 mM and 2 mM respectively. At such stress treatments, the wild-type plants grew little and showed some chlorosis and decreased vigor. These results indicated that rHsp90 played an important role in plant response to salt.The rHsp90 gene was expressed as a GST fusion protein by transformation of E. coli BL21 cells with plasmid pGEX-6p-3-r/ft/?90. The GST-rHsp90 was expressed and purificated by using Glutathione-Sepharose 4B under different temperature and the concentrantion of IPTG, and obtained high purity and yield protein. The fusion protein was digested by PreScission Protease, and rHsp90 was obtained after cleavage of the GST tag. It promotes us to identify the proteins that interact with rHsp90 chaperon, and to further study on the real molecular mechanism of rHsp90 tolerance for stress conditions.