| Carbonic anhydrase (CA, EC:4.2.1.1), a zinc-containing metalloenzyme, which catalyzes the reversible transformation between CO2and HCO3-and regulates CO2supply level in the photosynthesis of C3and C4plants. It was found that there was an association between CA activity and photosynthetic rate. In recent years, several studies indicated that CA was also involved in plant response to abiotic stresses. Among different rice varieties, there were variations in CA activity and photosynthetic characteristics, however, a clear association between CA activity and photosynthetic rate has not been reported. In this study, a preliminary analysis concerning the relationship among CA activity, OsCA gene expression level and photosynthesis of functional leaves at rice grain filling stage was conducted. This study also determined the regulative effect of CA gene and its protein by foliar spraying of zinc ion aiming to improve the physiological function of leaves in late growth stage of rice.In this study, four rice varieties (R49, N22, TXZ-25and ITA312) were chosen as experimental materials. The OsCA gene transcription level, the expression level of OSCA protein and its enzyme activity and photosynthetic characteristics of the flag leaf of these four varirties in the different grain filling stages (5,15and25d after flowering) were analyzed. Moreover, OsCA gene/the OsCA protein sequence were analyzed by bio-information to design and synthesize polypeptides, which were used to immunize animals to get polyclonal antibodies and further detect the regulatory effect of CA protein level by exogenous zinc ion.The main results are as follows:1. The CA activity of flag leaf were significantly different among different varieties; for the same variety at different grain filling stag (5,15and25d after flowering), the CA activity of flag leaf showed a firstly increased and then decreased trend, and its enzyme activity reached a maximum15days after flowering and subsequently decreased with the progress of leaf senescence. The net photosynthetic rate of flag leaf of four varieties all showed a gradual downward trend, and there was a positive correlation between leaf CA enzyme activity and its net photosynthetic rate. The trends of the OsCA relative expression and the CA enzyme activity were consistent, but varied among different varieties. In view of the significant association between OsCA gene expression/CA enzyme activity and rice leaf photosynthetic rate, the OsCA gene/the OSCA protein level could be used as an indirect indicator to identify leaf functional difference in terms of variety photosynthetic rate as in rice varieties.2. Rice variety N22was treated with foliar spraying of zinc (0,0.1%,0.2%,0.5%,1.0%). The results showed a first increase and then decrease trend of CA activity with the increase of zinc concentration. Within certain zinc concentration (0.1-0.2%), foliar applications of exogenous zinc ion could significantly improve the activity of CA enzyme and the content of leaf chlorophyll (SPAD). Once exceeding resistant zinc range (0.5%) of rice leaf, excess exogenous zinc ions inhibited the activity of leaf CA enzyme and reduced SPAD, and caused poison to the blades with yellow spots. When the zinc ion concentration was0.2%, leaves CA activity reached the maximum, and at the same time, the OsCA gene expression level by RT-qPCR was corresponding with the trend of CA enzyme activity, which suggested that CA enzyme activity could be regulated by promoting transcription level through adding exogenous zinc ions.3. Polypeptide was designed by the predicted antigenic determinant concerning the gene sequence information. The polyclonal antibody against CA has been successfully prepared and then the results of Western blot showed that the peptide antibody had a high specificity.4. Western blot results showed that zinc treated and control leaves all appeared two approximately20KDa molecular weight of OsCA protein bands. Banscan5.0software was used to analyze the value of the control and treated leaves gradation of the OSCA protein bands. Quantitative analysis results showed that rice leaf OsCA protein content in treated group(0.1%0.2%,0.5%and1%) respectively increased by11%,17%,23%and24%in comparison with those in control group. Especially in the zinc concentration of0.1%, the increase range of content of OsCA protein reached to the maximum, indicating that foliar spraying of zinc treatment could induce the accumulation of OsCA protein in rice tillering stage. |
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