Functional Analysis Of Cold Responsive Genes OrCr1 And OrCr2 Down-regulated By RNAi In Chaling Wild Rice

Chaling wild rice is one of the two wild rice polulations which exist northernmost in the world with strong cold tolerance. In order to clone its cold resistance-related gene(s), a global genome expression profiling of it under cold stress was performed using the 60 K Gene Chip Rice Genome Array(Affymetrix). Some up- or down-regulated genes were identified. The data obtained by Gene Chip analysis was confirmed by the real-time quantitative PCR analysis. Among the upor down-regulated genes, the Or Cr1(Oryza rufipogon cold responsive gene 1) and Or Cr2 were two candidates of the cold resistance-related genes selected for further studies.To investigate whether two genes are cold tolerant or others, two RNA interference vectors named p HB-Or Cr1 and p HB-Or Cr2 drived by the promoter 2×Ca MV35 S, were constructed. Then, the vectors were transformed to japonica rice Nipponbare with Agrobacterium-mediated method. Eleven and thirteen independent RNAi events were obtained for Or Cr1 and Or Cr2, respectively. The expression quantities of both genes in RNAi plants were analyzed by RT-q PCR method, and the results showed that the expression quantities of two genes of many RNAi plants decreased remarkably, indicating that the effect of the interference was positive. Furthermore, the T0 generational RNAi plants produced adequate T1 generational seeds. The majority of them were able to germinate and become seedlings on the culture medium containing hygromycin. Finally, the cold tolerance and other phenotype of the T0 and T1 generational plants were analyzed. The results were showed as follows:1) After natural low temperature(average temperature: 4.7°C) for 3 days, the increase of the relative electrolyte leakage of the T0 generational Or Cr1-RNAi plants was lower than that of Nipponbare( as wild type control), however, the increase of the relative electrolyte leakage of the T0 generational Or Cr2-RNAi plants was higher than that of Nipponbare.2) After natural low temperature(average temperature: 4.7°C) for 3 days, the decrease of the total chlorophyll of the T0 generational Or Cr1-RNAi plants was not significantly different from that of Nipponbare, while the decrease of the total chlorophyll of the T0 generational Or Cr2-RNAi was higher than that of Nipponbare.3) After natural low temperature(average temperature: 4.7°C) for 3 days, the decrease of the net photosynthetic rate of the T0 generational Or Cr1-RNAi plants was lower than that of Nipponbare, while the decrease of the net photosynthetic rate of the T0 generational Or Cr2-RNAi plants was higher than that of Nipponbare. Under the low temperature, the Fv/Fm of both kinds of wild type and RNAi plants decrease. However, the decrease of the T0 generational Or Cr2-RNAi plants was more obvious.4) After treated with artificial low temperature(constant temperature of 4.0 °C) for 5 days, the survival rates of Or Cr1-RNAi seedlings and Or Cr2 RNAi seedlings in T1 generation are 36.5% and 10.3%, respectively, and that of wild-typical Nipponbare seedlings is 12.5%. Additionally, the dehydration rate of the Or Cr1-RNAi seedlings was slower compared with those of wild type and Or Cr2-RNAi seedlings.5) Under the non-stress condition, the panicle length, spikelets per panicle, 1000-grains weight, seed set percentage and plant height of T0 generational Or Cr1-RNAi plants were lower than those of wild type Nipponbare. Besides, 1000-grains weight and seed set percentage of T0 generational Or Cr2-RNAi plants were lower than those of wild type Nipponbare.The above results indicated that the cold tolerance of the rice plant enhanced by inhibiting the expression level of Or Cr1 gene. On the other hand, the cold tolerance of the plant decreased by inhibiting the expression level of Or Cr2 gene. They may be the cold resistant gene, and may be relative to the development of rice plant.