Molecular Cloning And Characterization Of Cold-regulated Genes From Brassica Pekinensis

It has been found that changes in gene expression occur during cold acclimation in a wide range of plants. Using homology gene clone method, with the technique of RT-PCR, two cDNA clones from Brassica pekinensis,KT680 and RT750, were obtained. The 5'-UTR and 3' -UTR of RT680 and the 3'-UTR of RT750 were further acquired using RACE technique. The full-length of RT680 with 5'-UTR and 3-UTR is 955bp containing a 663bp ORF,which encodes 220 amino acids. The molecular weight of its deduced polypetid is 25KDa, so we name it cor25. The full-length of RT750 with 3'-UTR is 979bp containing an 810bp ORF,which encodes 269 amino acids. The molecular weight of its deduced polypetid is 30KDa, so we name it cor30. Analysis of the deduced full-length protein sequences suggests that both cor25 and cor30 are cold-regulated genes. The deduced amino acid sequence alignment shows that COR25 and COR30 are similar to COR47 and ERD10 from Arabidopsis thaliana. COR25 has 53.8% identity with COR47 and 59.4% identity with ERD10, while COR30 has 59.4% identity with COR47 and 60.2% identity with ERD10.The number of copies of cor25 in the genome and the expression pattern of cor25 were investigated. Southern hybridization indicates that there may be more than one copy of cor25 in the genome of Chinese cabbage. Northern hybridization shows that the expression of cor25 can be induced by low-temperature, dehydration and ABA, respectively. The transcript leval of cor25 increases after cold treatment(5℃) for 2h and reaches the a maximum within 1d. The maximum level remains unchangeable under the cold condition up to 10d. Moving the cold-acclimatized plants back to the control temperature(26℃) can result in rapid decrease of the transcript leval of cor25 within 2h. Drought stress can cause the increase of the transcript of cor25 within 2h to a leval much higher than that induced by 8h cold treatment. The expression cor25 can also be inducedby the treatment of 100uM ABA for 4h. According to the expression pattern, we presume that cor25 may be involved in the cellular potection against low temperature induced by dehydration.Transgenic research was conducted. The coding sequence of the mature COR25 profein was inserted into pET30a vector. The recombinant vector was transferred into the expression host bacteria BL21. The target protein was produced by the induction of IPTG The cor25 was inserted into the multiple cloning site of pCAMBIA2300 and the recombinant vector was transferred into Agrobacterium tumefaciens EHA105 by free-thaw method and transformation of tobacco cv.Bairihong and rice cv. 1826 were performed.