Study On Molecular Strategies For Salt-Tolerant Rice Breeding

Rice is an important and salt sensitive crop plant. High soil salinity limits rice growth, development and productivity. Therefore, developing new cultivars with enhanced salt stress tolerance would undoubtedly have an enormous impact on global food production. We decided to improve salt stress tolerance by transforming rice with several salt-related genes such as SsNHX1, CAT1, GST, AVP1, and SOD2 coming from Suaeda salsa, Arabidopsis and yeast, respectively. The primary subjects of this study are as follows: First, heterogenous expression of the single GST and co-expression of the Suaeda salsa GST and CAT1 in transgenic rice in order to comparative analysis of those stress resistance level. Second, introduce the individual Suaeda salsa SsNHX1 and both SsHNX1 and Arabidopsis AVP1 into transgenic rice for purpose of comprehensive understanding of those salt tolerance mechanisms. Third, expression of the yeast SOD2 in transgenic rice for characterizing those salt tolerance levels. The main results of this study summarized below: 1 Transgenic rice plant carrying the SsNHX1 and SsNHX1/AVP1 enhanced salt tolerance 1) Heterogenous expression of the SsNHX1 in transgenic rice to examine salt tolerance by the transformants exposure to different concentrations of NaCl. It had been observed that there were no visible differences in growth and development between the transformants and the non-transformants under normal growth conditions. However, transgenic rice plants exhibited improved salt tolerance in comparison with non-transformed controls in the present of salt stress. 2) Comparative analysis of the transformants expressing the single SsNHX1 and co-expressing SsNHX1/AVP1 for salt tolerance, the results indicated that co-expression of these two genes conferred higher level of salt tolerance to transformed rice lines than that of the individual one. 3) There were more Na+, K+, Ca2+ and Mg2+ contents in SsNHX1 and SsNHX1/AVP1 transgenic rice than in non-transgenic rice controls upon salt stress imposition. The increase in cation concentration demonstrated a positive correlation between increased root proton export capacity and salt tolerance in transgenic rice plants. 4) There were no obvious differences in proton activity among all experimental lines in the absence of salt stress. However, the V-ATPase hydrolytic activity was much higher in SsNHX1 and SsNHX1/AVP1 transgenic rice plants than in non-transformed controls under salinity conditions. The results suggested that V-ATPase provided the driving force for Na+ compartmentation. Furthermore, the V-PPase activity remarkably increased in SsNHX1/AVP1 transgenic rice plants, and the increasing extent was related to the development period of rice seedlings, which was much higher during early development stage in comparison with that of late stage. 5) The MDA as well as the H2O2 content was obviously lower whereas the photosynthetic rate was higher in transgenic rice plants in comparison with that of controls under salt stress conditions. The results implied that the less oxidative stress might result from the Na+ sequestration in these transformants. 2 Expression of yeast SOD2 in transgenic rice resulted in increased salt tolerance 1)Over-expression of the yeast SOD2 in rice to characterize the salt tolerance level of the transformants with different concentrations of salt treatment, the data showed that expression of the SOD2 in transgenic rice plants resulted in increased salt tolerance. 2)The SOD2 transformants had elevated K+, Ca2+ and Mg2+ levels with higher K+/Na+ ratio and less Na + than those of non-transformed controls. These data indicated that the SOD2 may play important roles in Na + extrusion from the cytoplasm in transgenic rice plants.3)The P-ATPase hydrolytic activity was more than two times in SOD2 transgenic rice plants in comparison with those of controls. The data implied that P-ATPase might provide driving force for Na+ extrusion from the cytosol. Furthermore, the root proton export capacity was closely related to P-ATPase activity in the transformants. 4)The photosynthetic rate was higher while the MDA and H2O2 contents were lower in SOD2 transgenic rice plants than in those controls, which suggested that less ROS generated owing to the Na+ extrusion from the cytosol. 3 GST and GST/CAT1 transformants improved multiple stress resistance 1)Introduced the GST into rice to determine the stress tolerance capacity of the transformed rice plants with multiple stress treatments. The results provided evidence that GST could contribute to resistance to multiple stresses such as salt, lower temperature and so on, which maight result from the increased GST activity thereby reduced oxidative stress in the transformants. 2)Comparative analysis of the GST and GST/CAT1 transformants for stress resistance, and it had been observed that both the CAT1 and GST activity were increased. Interestingly, the SOD activity was also increased along with the activity of CAT in GST/CAT1 transgenic rice plants compared to those of controls. The elevated levels of CAT and GST as well as SOD clearly lead to increasing oxidative stress protection in GST/CAT1 transformants. 4 Different genes made different contributions to salt tolerance in transgenic rice plants Although as a result of foreign gene inserted position and posttranscriptional regulation and other problems, it was difficult to compare the salt tolerance efficiency of transformants with different genes. However, taken all of the results account, we may find that transgenic rice plants carrying both of the SsNHX1/AVP1 were superior to the single SsNHX1 in salt tolerance. The Na+/H+ antiporter gene (such as SsNHX1) may take an advantage over the antioxidant enzyme gene (such as GST) in transgenic rice plants for salt tolerance. Co-expression of both GST/CAT1 in rice conferred higher stress resistance to transformed rice lines thanthat of the single GST. The SOD2 seemed not as good as the SsNHX1 for salt stress resistance in transgenic rice lines. 5 Salt tolerance mechanisms in transgenic rice plants Through general analysis of the salt tolerance mechanisms in transgenic rice plants we also found that although only one or two foreign genes was inserted in the rice, the transformants were distinctive from the non-transformants in many aspects such as proton activity, photosynthetic rate, antioxidant level and so forth. The results may imply that up-regulation of one or two genes in rice could cause pleiotropic up–regulation of other salt-tolerant related genes (or of the activity of the gene products) to confer higher level of salt tolerance to transgenic rice plants. 6 Posttranscriptional regulation of gene expression In the present study, we also provided evidence that although the foreign gene such as CAT1 driven by the strong constitutive CaMV 35S promoter, their mRNA level was lower under non-stress conditions in comparison with those of stress conditions. The results suggested that the mRNA accumulation maybe posttranscriptionally controlled. The main innovation points of this study generalized as follows: 1 Developed the efficiency genetic transformation system of rice in our laboratory, for the first time. 2 For the first time, heterogenously expressed the Suaeda salsa vacuolar Na+/H+ antiporter gene-SsNHX1 in rice, and made a general analysis on the salt tolerant mechanisms in the transformants. 3 Co-expressed the SsNHX1 and AVP1 in rice, and compared the salt stress resistance capacity in SsNHX1 and SsNHX1/AVP1 transgenic rice plants, for the first time. 4 For the first time, over-expressed the SOD2 in rice, and characterized the salt tolerance mechanisms in the transformed rice lines. 5 For the first time, introduced the GST coming from Suaeda salsa into rice, and offered a general analysis of the multiple stress resistance in the transgenic rice plants.6 Co-expressed the GST and CAT1 in rice, and presented a comparative analysis of the stress tolerance capacity of GST and GST /CAT1 transformants, for the first time. 7 For the first time, to provide a comparative analysis of different gene contributions to salt tolerance in transgenic rice plants.