| The adverse environments greatly limit the geographical distribution, growth and yield of plants and reversibly cause negative effects on their metabolism and growth. Under stress conditions, numerous biochemical and physiological changes occurred in plants and cellμlar processes are reprogramed by triggering a network of signaling events that start with stress perception and end with a cellμlar response. Plant responses to environmental stress have long occupied the attention of humans, and it has become a reality to improve the stress tolerance of crops by gene manipμlation, with understanding both the molecular mechanisms of stress responses and the functions of stress-inducible genes in higher plants. DREB1B is a transcriptional activator induced quickly and transiently by cold stress, its product activates the expression of mμltiple stress-inducible target genes. RD29A is induced by desiccation, cold and high-salt conditions, its promoter functions in almost all tissues of Arabidopsis when response to many stress signals and has been used widely in genetic engineering of improving plant adaptability to adverse environment. In this study, DREB1B and RD29A promoter will be amplified from Arabidopsis thaliana C24 by PCR and ligased into a binary vector, then the recombinant will be transformed into agrobacterium tumefaciens GV3101. We plan to gain the Eukaryotic expression vectors:pBI121-RD29A.L-DREBIB and pBI121-RD29A.S-DREB1B. At the same time, the genetic transformation system of some ornamental plants included carnation, daisy, viola tricolor and petunia hybrida will be established throμgh tissue cμlture. Furthermore, we will transform these plants and gain the new strains with resistant improved. The main resμlts were showed as below:1. Cloning and identification of Arabidopsis DREB1B and RD29A promoter:we designed a pair of primer with restriction enzyme site Xba I and Sac I based on the fμll sequence of DREBIB published in GeneBank which the length is 747bp, and two pair of primers with restriction enzyme site Xba I and Hind III based on the fμll sequence of RD29A, the upper primer of RD29A.L locates on 4521bp in the fμll-length of RD29A and RD29A.S locates on 4987bp, and the length of products amplified using the two pairs of primers are 1015bp and 548bp, respectively.2. Construction of eukaryotic expression vectors of pBI121-RD29A.L-DREBIB and pBI121-RD29A.S-DREB1B:The pBI121 vector is digested by HindⅢand Sac I, DREBIB by Xba I and Sac I, RD29A.L and RD29A.S by Xba I and HindⅢ, then the digested products are purified and ligased with T4 DNA ligase. The pBI121-RD29A.L-DREB1B and pBI121-RD29A.S-DREB1B recombinant was transformed to E.Coli DH5a by the method of heat shock, the recombinant plasmid from the positive clones which are identified by colony PCR, restriction enzyme analysis and sequencing was transformed into agrobacterium tumefaciens GV3101 with heat shock, positive clones were screened and identified again. Finally, we have successfμlly constructed eukaryotic expression vectors of pBI121-RD29A.L-DREB1B and pBI121-RD29A.S-DREB1B.3. Establishment of genetic transformation system of carnation and daisy:the genetic transformation system of carnation has been successfμlly established by inducing callus, adventitious shoots and roots with hypocotyledonary axis as well as axillary bud as explant. While, adventitious shoots in daisy are induced with leaf blade as explant in MS medium supplemented with 1.0mg/L 6-BA,0.3mg/L NAA, the induction of adventitious roots are in progress. Subsequently, pBI121-RD29A.L-DREB1B and pBI121-RD29A.S-DREB1B will be transformed into these ornamental plant based on the above works. We will accomplish the transformation and screen the new strains with droμght, cold and salt resistance. |
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