Responses Of Arabidopsis Thaliana Mutant To Abiotic Stresses As Well As Cloning And Expression Of CBF1

Plants are usually facing the abiotic stresses during their growth and development. They can mitigate the stresses by activating special stress signal pathways to produce proteins with protective function. 155 mutant, which is obtained by T-DNA insertion, exhibits notable differences in phenotype comparing with wild type C24. Previously sequencing result indicates that the gene traped by T-DNA insertion may encode the protein related with the HSP101 (heat shock protein), which is a subunit of ATPase with the activity of molecular chaperon. Moreover, experimental result proved that the gene traped by T-DNA play a role in plant's heat-resistance.Since there are the extensive crosstalks between different stress signal pathways, this thesis is mainly focused on the difference between mutant and wild type on the sensitivity and endurance of stresses, such as low temperature, drought, high salinity and extreme light intensity. We try to analyze the correlation between different stress signals, then measure the concentration and accumulating rate of osmoprotestants such as proline and deoxidize sugar under the normal condition and several stress pressure. The accumulating rate of mutant 155 is different from wild type C24. The results are as below:1. Although the difference in freezing tolerantce is not remarkable between mutant and wild type, wild type is more freezing-tolerant than mutant. Curiously, this difference only occurs in plants grow in soil, In contrast, plants grow in culture medium has no singnificance difference in freezing tolerantce between wild type and mutant. 5-weeks-old plants are more freezing-tolerant than 7-weeks-old plants both in wild type and mutant material, which may reflect the stress tolerance of plant, or at least the freezing tolerance is dynamic along the whole lifecycle.2. The expression of CBF1 in mutant after cold treatment (4°C, 2h) can not be inspected by RT-PCR, while the expression in wild type is clear and strong. From this result, it seems that the freezing tolerance of mutant should be much weaker than the wild type, but the experimental result is not. We suggested that the controversy may because of the compensation of other cold responsive pathways. Furthermore, it is also illustrate the complexity and diversity of the stress signal transductions.3. The gene clone of CBF1, the main transmission activator of the cold acclimation, and preparatory work of vector construction was done.4. The increase of the salinity of culture medium exhibits the tendancy of "promoting firstly and then suppressive"way. It means under the relatively low concentration, the increase of salinity promote the growth of both the mutant and wild type. When the salinity reaching a critical point, the growth of both wild type and mutant plants were suppressed. Obviously, there is an optimum salinity for plants, or at least, for Arabidopsis. We have not seen any observed different reaction between wild type and mutant on medium.5. The concentration of proline and deoxidize sugar is higher in mutant than in wild type under normal conditions. When plants are facing the cold acclimation and drought stress, the total amount of accumulation of proline and deoxidize is much higher in mutant than in wild type, higher when cold acclimated for 7 days than drought stressed 7 days, higher when cold acclimated for the first 7 days than the second 7 days. These results indicate that the accumulating rate of proline is decreased as time goes on, moreover, cold acclimation is more effective than drought stress to increase the content of proline. Mutant resists drought treatment longer than wild type. This observation is coincident with the higher concentration of osmoprotentant (for example, proline and deoxidize sugar) in mutant.6. Extreme light conditions such as high or dark affect the growth, plants exhibit physiological reactions such as prolonged petiole, etiolated and coiled leaves, withered stem and so on. Extreme light conditions hurt wild type more severely than mutant. The reason may be the differential expression of signal transduction between wild type and mutant, or the smaller leaves and transpiration of mutant. Stresses such as low temperature, drought, high and dark light condition delayed the flowering time in both wild type and mutant. Moreover, both mutant and wild type plants treated under dark conditions blossom later than high light treatment.All the results above illustrate that the notable difference on stress sensitivity and endurance between mutant and wild type are because of the loss-of function of mutated gene by T-DNA insertion. Therefore, we conclude that there is some kind of crosstalk between the signal pathways of heat shock reaction and other stresses. When the HSP101 gene is blocked in mutant, the expression of CBF1 is severely affected, the thermotolerance of mutant is weaker than wild type. Whereas the drought tolerance of mutant is stronger than wild type, for mutant possess higher content and accumulating rate of proline and deoxidize sugar. Moreover, the endurance of extreme light condition in mutant is better than wild type as well. These observations may prove the presumption that the crosstalk between different pathways is very complex and multi-linear.