Cloning, Expression And Function Analysis Of The BADH And LEA Gene From Chorispora Bungeana

Chorispora bungeana Fisch. & C.A. Mey is a rare and typical alpine subnival plant species that is highly capable of resisting freezing environment. However, it does not possess special morphological characteristics that helped it surviving under freezing environment, physiological and molecular mechanisms were assumed to account for its adaptation to the freezing environment. Therefore, it is a valuable species for stress-related genes cloning and research on the mechanism of cold resistance. In our work, BADH gene (CbBADH) and LEA gene (CbLEA) were cloned from C. bungeana. The pattern of CbBADH expression and accumulation of betaine under various treatments were investigated; the function of CbLEA protein in stress protection was investigated by transgenic approach. The summary is as follows:1. CbBADH gene is 1729 bp in length with an open reading frame (ORF) of 502 amino acids, corresponding to a protein of predicted molecular mass 54.5 KD and an isoelectric point of 5.30. The deduced amino acid sequence possesses a decapeptide (VSMELGGKSP), which is involved in the enzyme active site and NAD⁺ binding. The putative amino acid sequence of the CbBADH showed 73.3-95.4% similarity to the BADH of other plants. All of these indicate that CbBADH gene is the homologus gene of BADH.2. CbBADH was expressed in and purified from Escherichia coli. Through investigation the function of the enzyme coded by CbBADH, we found that recombinant CbBADH protein showed high activity for the oxidation of betaine aldehyde, and the optimal pH of CbBADH was 8.0. These results indicated that CbBADH gene encoded a functional BADH enzyme, which could catalyze betaine aldehyde to betaine, and the enzyme activity was not affected by the substitution of M for L in the decapeptide.3. The expression patterns of CbBADH gene were investigated under cold stress. The CbBADH gene transcript progressively increased under 4°C and increased much more distinctly and quickly under 0°C and -4℃. This result suggests there is a close relationship between the expression of CbBADH and cold stress, CbBADH expression is sensitive to low temperature. CbBADH plays an important protective mechanism in signal transduction to resist cold stress in C bungeana. Lower temperature could trigger the expression more quickly exactly, this phenomenon reflects fitly that C. bungeana has the ability to adapt to acute temperature change in the growth environment.4. Like other higher plants, C. bungeana accumulated betaine in response to low temperature stress. Lower temperature could trigger the accumulation more quickly exactly. Moreover, the increase in the endogenous betaine levels in C. bungeana seemed to have a maximum level, which increased 3-fold, under different low temperature stresses. These results implied that the accumulation of betaine was one but not the main protective mechanisms in C. bungeana to resist cold stress, other special cold-hardiness mechanism still existed in C. bungeana5. CbLEA gene is 842 bp in length with an open reading frame (ORF) of 169 amino acids, corresponding to a protein of predicted molecular mass 17.9 KD and an isoelectric point of 6.45. Hydrophilicity prediction shows CbLEA is a highly hydrophilic small protein. The secondary structure analysis shows CbLEA protein contains 83.43% alpha helix and 16.57% random coil. CbLEA protein shares lower similarity with LEAs from other plants (35.84%⁵2.78%) except A.thalian, indicating that CbLEA is a novel LEA gene.6. CbLEA gene was over-expressed in tobacco by Agrobacterium-mediated leaf disc transformation with a construct containing the CbLEA ORF under control of CaMV 35S promoter. The transgenic tobacco plants were confirmed by polymerase chain reaction, real time RT-PCR and Western bolt analysis. To investigate the function CbLEA protein, the three transgenic lines were chosen to test whether over-expression of the CbLEA protein increased their ability for tolerance against stress, based on accumulated amounts of CbLEA protein.7. Under stress condition, the transgenic T₁ plants show better performance than non-transgenic plants, and maintaine higher RWC and chlorophyll content, less MDA and EL, when compared with non-transgenic plants. Moreover, the LT50 (the killing temperature for 50% of the cells) values was significantly altered in transgenic plant, which was declined to -3°C, the LT50 value is -2°C in non-transgenic plants, and the transgenic plants survived longer time than non-transgenic plants at the constant freezing treatment of -2°C. These results indicated that CbLEA gene was an excellent stress-tolerance gene, and the transgenic tobacco plants exhibited significantly increased tolerance to salinity and cold.8. Furthermore, the extent of increased stress tolerance shows a positive correlation to accumulation level of the CbLEA protein, indicating that the CbLEA gene product function in cell protection under stress condition.In conclusion, the accumulation of betaine was also found in C. bungeana, indicating that there is a conservative mechanism in plant cold-resisting process. However, the increase in the endogenous betaine levels in C. bungeana seemed to have a maximum level under cold stress, and the accumulation of betaine was one but not the main protective mechanisms in C. bungeana to resist cold stress, other special cold-hardiness mechanism still existed in C. bungeana. Tthe analysis of stress tolerance in transgenic plant show that the CbLEA protein may protein cell through binding water, maintaining membrane structure, the stress-tolerance gene from C. bungeana seem to be more efficient than the stress-tolerance gene from other plant in cold tolerance.