| Sugarcane is not only the first major sugar crop, but also an important energy providing plant in China and in the world. Sugarcane is a native to the warm temperate and widely cultivated in tropical and subtropical regions, and the need for appropriate temperature and humidity for normal growth. During recent years, the world’s climate changes dramatically as extreme low temperature increased frequently. Sugarcane production has been occurred huge losses due to frost damage and chilling injury. Therefore, it is of great significance to carry out research on the mechanism of cold resistance in sugarcane. At present, researches on effects of low temperature stress on morphology and anatomy of root system, physiological and biochemical metabolism, molecular levels and other relevant researches in sugarcane have been seldomly reported. In this study, roots of sugarcane seedling were treated with low temperature stress. In order to reveal the relationship between root growth, physiological characteristics, relavent gene and cold resistance, we conducted comparative analysis of root morphology, cell ultrastructure, physiological and biochemical characteristics. We cloned a-tubulin gene and analyzed the gene expression in sugarcane. Moreover, the microtubule a-tubulin gene was over expressed in prokaryote(E.coli) and a-tubulin protein was purified and prepared for monoclonal antibody. The transformation of a-tubulin gene in tobacco and sugarcane was carried on by Agrobacterium mediated, and the a-tubulin transgenic tobacco and sugarcane were confirmed with PCR, report gene GFP and Bar gene respectively. The function of a-tubulin gene from sugarcane in cold stress was analyzed in transgenic tobacco. The main research contents and results are as follows:1. Low temperature stress significantly inhibited the root growth. Compared with the normal growth condition, roots under cold stress appeared yellow, brown, and necrotic near the apical area; and the length and volume were significantly lower than those of the control. The root biomass in two varieties (ROC22 and GT28) under low temperature was obviously lower than that in control, while there was no significant different in the ratio of root and shoot. Compared with variety GT28, the upper part and lower part of variety ROC22 were influenced significantly by cold stress since the cells and organelles of ROC22 were injured more than those of GT28. The results of ultrastructure showed that low temperature stress resulted in circular-shaped root tip cells (normally square-shaped), structural disorder, organelles decreases, cell membrane disruption, and nuclear disintegration; also, the vague mitochondrial cristae and gradually vacuolated, partial dissolute.2. The contents of soluble protein and sugar and the activities of protease were affected under low temperature stress. The soluble protein of ROC22 initially increased slightly but then decreased, while the soluble protein of GT28 first increased, then decreased, but then increased again and maintained at a high level. The amino acid contents of both varieties first increased and then decreased, in which that of ROC22 was significantly lower than that of the controls on the 5th and 9th day, however, that of GT28 was always higher than that of the control group. The contents of soluble and insoluble sugar of ROC22 decreased gradually, which were significantly different from the control, while the soluble sugar content of GT28 increased gradually which was higher than the control group, but the insoluble sugar content in GT28 first increased and then decreased. The endopeptidase activity in roots first increased and then decreased, and it was higher under weak acid condition than that in alkaline condition activity. The activity of GT28 was higher than that of ROC22. The activities of carboxypeptidase and aminopeptidase under short periods of time in low temperature did not change significantly. However, on the 9 days of cold stress, the carboxypeptidase activity surged and aminopeptidase activity decelerated in ROC22, whereas in GT28 the two enzyme activity change was not significant.3. The prokaryotic expression vector of pET30a(+)-α-tubulin was constructed successfully, and the fusion protein was induced with IPTG. The results of prokaryotic expression demonstrated that the sizes of combined proteins were 49.6 kDa of pET-α-tubulin. The purified and concentrated proteins of pET-α-tubulin was obtained using Ni2+-NTA column affinity chromatography, and dialysis, that was prepared for the monoclonal antibody. The titer of a-tubulin antibody was finally evaluated by ELISA. The results showed that the titer of α-tubulin monoclonal antibody was greater than 1:512000. Using the western blot a-tubulin in sugarcane was detected that indicated that α-tubulin monoclonal antibody has been successfully made in the present study.4. QRT-PCR. technique was used to analyze the expression of α-tubulin gene in sugarcane. The results showed that the expression of α-tubulin gene was at its highest in stalk, the root was the second and the leaf was the lowest. Under low temperature stress, the expressions of α-tubulin gene in root, stalk, and leaf first increased and then decreased, but eventually the differences mitigated. Western blot method was used to detect the protein expression of α-tubulin gene in sugarcane and the results showed that the expression of a-tubulin in ROC22 decreased gradually, whereas that in GT28 increased first and then decreased but was higher compared to that in control group.5. The expression vectors of pBI 121-a-tubulin for tobacco and pCAMBIA3300-α-tabulin for sugarcane were constructed successfully. The transformation with pBI121-α-tubulin under control of CaMV35S promoter and with pCAMBIA3300-α-tobulin under control of Ubi promoter was transferred to WT tabacco, and sugarcane variety ROC22, respectively using the Agrobacterium mediated method. The transgenic tobacco was detected by PCR and GFP report gene, while transgenic sugarcane was also confirmed by PCR and Bar gene (Ubi).6. Functional verification of low temperature stress on transgenic α-tubulin gene in tobacco. The results showed that there was no different in the chlorophyll contents bwteem wild and transgenic tobacco. At the room temperature (28℃), photosynthesis including Pn, Gs and Tr was higher in transgenic tobacco than those in WT tobacco, while Ci was lower in transgenic tobacco as compared with WT. Under low temperature (0-4℃), photosynthesis was inhibited in wild and transgenic tobacco and also the respiration was shifted from strong to weak respiration as time passed. Under low temperature, the osmotic substances including soluble protein, soluble sugar, and proline contents in transgenic tobacco were more stable compared to those in wild tobacco. The protective enzyme system, the activities of SOD and POD first increased and then decreased, but the changes were significantly higher in the wild tobacco in comparison to that of transgenic tobacco; the CAT activity in wild tobacco decreased gradually, while the CAT activity in transgenic tobacco decreased siginificantly. The results suggested that physiological and biochemical characteristics in transgenic tobacco under cold stress was excessive expression of α-tubulin dependent.7. Using qRT-PCR technology, transgenic α-tubulin gene from sugarcane was analyzed the expression in tobacco. The results showed that the expression of α-tubulin gene in the two genotypes of tobacco was the highest in vein, stem and the lowest in leaf. Compared with WT tobacco, gene expression of α-tubulin in transgenic tobacco was higher in leaves and stem, but lower in vein. Under low temperature stress, the expression of α-tubulin gene in wild tobacco leaves decreased gradually, while it in transgenic tobacco leaves decreased and then increased, but it was obviously higher than that of wild tobacco. Western blot method was used to detect the expression of α-tubulin protein and it was found that under low temperature stress, the wild tobacco α-tubulin protein expression first increased and then decreased, whereas the transgenic tobacco α-tubulin protein expression exhibited and inverse parabola shaped where it first decreased, then increased, again decreased, and lastly increased. The results indicated that excessive a-tubulin gene increased the low temperature resistance of transgenic tobacco. |
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