The Cloning Of Arabidopsis CBF1 And RD29A Promoter,Constructing Of Expression Vectors And Their Transformation Into Daisy

Low temperature is one of the adverse environmental factors that most affects plant growth and development.Currently,the best documented genetic pathway leading to gene induction under low temperature conditions is CBF/DREB(C-repeat binding factors/dehydrate responsive element binding factor).CBFs are induced by ICE(Inducer of CBF Expression).CBFs act as transcription factors binding to the CRT cis-acting elements in the promoter regions of COR(cold regulated)genes and induce the expression of about 500 COR genes.These genes encode proteins related to the cold resistance and will increase the freezing tolerance of plants.However,in addition to their role in cold acclimation increasing freezing tolerance,CBFs also promote growth retardation and reduce GA levels.As a result,the accurate regulation of CBFs expression is very important.On the other hand,the RD29A promoter,with both DRE and ABRE elements,is an optimal stress-inducible promoter activated by dehydration,high salinity and low temperature.In this experiment,constructs containing Arabidopsis RD29A::CBF1 in the eukaryotic expression vector pBI121 have been transformed into Daisy using the Agrobacterium tumefaciens-mediated method.The main experiment and results are as follow:1.Two primers are designed according to the CBF1 sequences provided in the NCBI website.The XbaI and Sac I restrict enzyme site are placed at the two terminal of the primer,respectively.The length of CBF1 is 728bp.Two pairs of primers are designed according to the RD29A sequences provided in the NCBI website,one pair of which the upstream primer begins at the 4521 bp site,and the other one at 4987bp site,while the downstream primers are at the same 5516bp site.The Xbal and Hind III restrict enzyme sites are placed at the two terminal of each pair of primer,respectively.The cloning lengths of this two pairs of primers are long RD29A(RD29A-LP):995bp,short RD29A(RD29A-SP):529bp,respectively.After compared the sequences of CBF1,RD29A-LP and RD29A-SP to NCBI,the similarities are 99.87%,98.70%,99.87%,respectively.2.Three expression vectors,pBI121-RD29A-SP-CBF1,pBI 121-RD29A-LP-CBF1,pBI121-35S-CBF1 were constructed using the binary vector digested by according enzymes.These vectors were successfully transferred into Agrobacterium tumefacien LBA4404 and GV3101 by direct DNA transfer.LBA4404 was chose as a final medium to translate the vectors into Daisy.3.The leaves of Daisy were chosen as explants to construct its regeneration systems.To Daisy,the best shoot germination culture was MS+6-BA 1.0mg/L+NAA 0.3mg/L;the regeneration rate was 79.20%;the best root culture was MS+6-BA 1.0mg/L,and the regeneration rate was 88.9%.4.The optimum selective pressures of Kan in Daisy and Cef in the suppression of LBA4404 were determined.The optimum selective pressure of Kan in shoot germination was 50mg/L;and that of root was 5mg/L.The optimum concentration of Cef in the suppression of LBA4404 was determined as 200mg/L,which would minimize the negative affection.5.After the double selection of Kan in shoot germination and root regeneration,30 pBI121-RD29A-SP-CBF1 transplants and 20 pBI121-RD29A-LP-CBF1 transplants survived.The DNA of each transplants are extracted using CTAB.Two pBI121-RD29A-LP-CBFl transplants are considered to be positive after PCR confirmation.Phenotypes of these two transplants and wild type plants are observed,and found that the flowering of transplants are apparently late than that of control.Even the two transplants have also some differences in phenotypes.The leaves of transplants L3 were more and greener than those of L5,and the living status was also better in L3 than that in L5.After 2 days of cold treatments,the transplants L3 died and wild type plants still survived,and further work is undergoing.