Isolation And Identification Of Small Molecule Calcium-binding Protein Genes In Rice

Calcium signaling protein families, including CDPK, CaM/CML and CBL-CIPK, play a vital role in plant calcium signal tansduction pathways. The biochemical characteristics and gene functions of several members in these families have been extensively studied. But there is little research about the function of the small molecule calcium binding protein (smCaBP) with only one EF-hand motif in the response to stress in plants. Until now, a smCaBP gene EFA27 has been first showed to be induced by abscisic acid and osmotic stress and named for smCaBP1. In this study, we screened out two new rice smCaBP genes from the rice low temperature expression microarray and the stress-induced expression profiling, which were named as smCaBP2 and smCaBP3. Therefore, we compared the protein structure and calcium binding properties of these three smCaBPs, and identified their expression patterns and function in response to abiotic stress, these results are as follows:1. Portein structural analysis showed that, plant smCaBPs contain one EF-hand motif, and can be classified into two types, smCaBP2 of 12.33 kDa and smCaBP3 of 13.9 kDa belong to the first type. smCaBP1 of 27.1 kDa in the second type was 58% and 40% similar to smCaBP2 and smCaBP3 respectively. smCaBP2 and smCaBP3 have one glutamic acid (E) or aspartic acid (D) in the EF-hand loop more than smCaBP1.2. In the in vitro calcium binding assay, after 5 ?g GST-smCaBPs were incubated with 0.5 ?mol Ca2+, Mg2+ or both at 4? overnight in the 50 ?L reaction buffer followed by Native-PAGE, except for GST-smCaBP1, Ca2+binding decreased the electrophoretic mobility of both GST-smCaBP2 and GST-smCaBP3, but Mg2+ have little effect, suggesting the effects of Ca2+binding on the conformation change of GST-smCaBP2 and GST-smCaBP3 in vitro.3. RT-PCR showed that, smCaBP1 transcription was induced by 200 mmol/L NaCl and 20% PEG-6000, smCaBP2 were induced to express by 4?,200 mmol/L NaCl and 20%PEG-6000, transcript levels of smCaBP3 was enhanced by 20%PEG-6000. Interestingly, smCaBPl,2,3 expression were significantly induced by 100 ?mmol/L ABA treatment, indicating their involvements in the ABA signaling pathway.4. Hygromycin and PCR detected that, all smCaBP1,2,3 T-DNA insertion mutants are heterozygous. Compared with wild-type, the endogenous gene expressions were decreased to some extent. The mutants were little lower and but normal in the growth and development. The smCaBP1,2,3 overexpressing transgenic plants had higher expressions and normal growth and development.5. ABA sensitivity analysis showed that, compared with the wild type, the growth of smCaBP1 and smCaBP2 overexpression transgenic plants shoot were suppressed more significantly by ABA. Moreover, the root was more sensitive to ABA than the shoot.6. The salt tolerance and drought tolerance showed that, under 20% PEG-6000 treatment for 7 days and recovery after 7 days, the survival rates of overexpressing smCaBP1,2 plants were 36.65% and 25.15% higher than the wild type plants, respectively, the survival rates of overexpressing smCaBP1,2 plants were 36.2% and 2.93% higher than wild type plants under 200 mmol/L NaCl treatment for 4 days followed by the recovery of 7 days, respectively. Therefore, smCaBP1 enhanced the rice tolerance to both salt and drought. However, smCaBP2 enhanced the rice tolerance to drought only. In the study, the discovery, calcium binding properties and gene functionidentifications of these smCaBPs in rice have been discussed, and provide the newdirections for the future research in this field.