Identification Of Cold-resistance Of Descurainia Sophia And The Clone, Sequence Analysis, Prokaryotic Expression And Purification Of DsCBF

Temperature is the key factor that restricts the growth and distribution of plants. The low temperature will hurt pants and it is the major factor of reductions of output of many crops in the north.C-repeat/dehydration responsive binding factor (CBF) is positively cold-regulated gene found in the last century. When the higher plants are under cold stress, they will cause the cold-hurt and make plants dead. The CBF will be induced quickly when the cold-hurt happened. It activates all kinds of cold-resistance mechanism and the transcription and translation of the cold-resisted protein to clear up this harmful effect.Descurainia sophia can endure cold stress and hold much adaptability. It can live in lots of place and is a good material for the study of cold-resistanee. Medicago sativa is excellent forage with good nutrition but can't endure cold. So it is important to improve its ability of cold-resistance.We have studied the cold-resisted characters of D. sophia and M. sativa and cloned the CBF gene of D. sophia. It is important to the alteration of alfalfa with methods of genic engineering. It will enlarge the growth range of alfalfa which play important role in improving environment, making use of the wastelands in the high and cold region.We confirmed the cold-resisted characters with measuring indexes about superoxide dismutase(SOD), proline (Pro) and electric conductivity. The result showed that with 4℃ treatment, the activity of SOD increased early, then decreased and at last it kept stabilization; Pro content increased during the whole chilling treatment; The electric conductivity of D. sophia increased slowly with chilling treatment above -5℃, but increased significantly below -6℃ due to injury ofplasma membrane. It showed that D. sophia could endure the temperature of -5°C. The same as it, the electric conductivity of M. sativa increased significantly below -6 °C to prove that it could endure -3°C. All above appeared that D. sophia owned the stronger ability of cold-resistance.We aligned CBF genes of some species, and designed the degenerate primers according their structural conservative region. The 250bp EST sequence of D. sophia CBF (DsCBF) gene have been gained using homologous clone. Then new primers were designed according EST and the 844bp DNA sequence of DsCBF have been gained at last with 3'RACE and 5'RACE.During we clone the 5' end of DsCBF with 5'RACE, we have compared and optimized the three method of 5'RACE, which concluded Circular or concatemeric first-strand cDNA-mediated .RACE (cRACE), Terminal deoxynucleotidyl transferase RACE (TDT-RACE) and CLONTECH reverse transcriptase RACE (CLONTECH- -RACE). cRACE couldn't got visible segment whether optimized or not. TDT-RACE have got dispering segments, and could see one blurry segment about 450bp after optimization. The result of CLONTECH-RACE is good and after optimization it have gained the only clear segment about 450bp. Here we not only cloned the 5' end of DsCBF but also set up 5'RACE experimental flat of clone technology.We used bioinformatics tools analyzing this clone of DsCBF and got the whole open reading frame (ORF) and-3' untranslated regions (3'UTR). The results proved DsCBF belong to CBF family of AP2 DNA binding protein superfamily. It have AP2 binding region with two special sects of short polypeptides of PKK/RPAGRxK FxETRHP and DSAWR and a putative active acidic region composed with acidic amino acids. DsCBF has no nuclear localization signal, no signal peptide and no transmembrane structure. There is no obvious possibility of helical coiled-coil structure existence. It is different in the acidic amino acid proportion and the second structure of active acidic region between DsCBF and Arabidopsis thaliana CBF, which maybe is the reason thatD. Sophia has stronger ability of cold-resistance.At last we expressed DsCBF through pET-32a prokaryotic expression system and purified it.