Cloning Of DREB Gene From Island Cotton (Gossypium Barbadense L.) And Its Functional Analysis In Transgenic Tobacco

DREB(dehydration responsive element binding protein)transcription factors are a family of functionally diverse proteins.They are unique to plants and play an important role in regulation of plant growth and development,homone reguation and responses to various stresses.The DREB transcription factor can specifically combine with the DRE(dehydration responsion element)and activate expression of many stress-induced genes under the stress and increasing the capacity of resistance and endurance to adversity of plant,is the best adversity resistance gene in plant by far.In this study,homology clonging methods in the new cloned from island cotton,a DREB transcription factor genes into tobacco by its,its basic characteristics and biological functions have been studied and proved than genes DREBs of transcription factors basic characteristics,which in transgenic tobacco plants over-expression can improve the capacity of resistance to abiotic stress.The major findings are as follows:1.According to published gene sequence of land cotton GhDREB1 transcription factor,specific primers P1 and P2 were designed and a gene with 651bp in length were amplified by RT-PCR.The gene contained a complete sequence encoding 216 amino acids,named XHDREB.Homological analysis showed that 85.19% amino acids in the sequences were similar to cotton GhDREB1 gene,47.32% amino acids in the sequences were similar to AtCBF1/DREB1B and 46.64% amino.acids in the sequences were similar to AtDREB1A/CBF3.2.Analysis indicated that the deduced relative molecular weight of the amino acid was 24.6 kDa for XHDREB.It contains the typical characteristics to DREB1 transcription factor:58 amino acids constitute a DNA binding region AP2 and two typical characteristics amino acids sequence to DREB1/CBF:PKKRAGRKKFRETR and DSAWR.3.The sensed specific primer DR-1 and DR-2,containing Kpnâ… and Pstâ… restriction sites,were designed.Plasmid containing XHDREB gene and pCAMBIA2300-35SOCS vector was digested with Kpnâ… and Pstâ… ,respectively.The T4DNA ligase was used to be ligated,sensed plant expression vector pC-XHDREB was built.4.Transforming tobacco by using agrobacterium-mediated.Screening and identification of positive tobacco by Kan resistance,PCR and RT-PCR.The drought,high salt resistance and other resistance of transgenic tobacco were analysised.And the biochemical and physiology quota were measured,the results indicate that the transgenic tobacco had some of resistance ability to these abiotic stress.5.The expression of XHDREB in transgenic tobacco were analyzed by using real time quantitative RT-PCR.There is difference on expression levels of XHDREB under drought stress between upland GhDREB1 and island cotton,But the expression levels of XHDREB in island cotton is similar to the DREB1/CBF genes of arabidopsis and other plant.The expression levels of XHDREB under high salt stress in island cotton is similar to upland GhDREB1,arabidopsis and other plants.But the expression levels of XHDREB under cold stress in island cotton is different from upland GhDREB1,arabidopsis and other plant.