Cloning And Functional Analysis Of DREB Homologous Genes In Chrysanthemum

Chrysanthemum（Dendranthema×morifolium Ramat.） is one type of herbage plants in chrysanthemum genus of composite.With important ornamental value,chrysanthemum grows in temperate zone and subtropics in Asia.Chrysanthemum is the fist one in the four dominating cut flowers in the world and it is very important in the people’s life and the production of flowers.Moreover,with strong vitality and adaptability,chrysanthemum has good tolerance to abiotic stresses.Therefore,the study on molecular mechanism about stresses in chrysanthemum is valuable.Plants are affected by many abiotic stresses,such as drought,high salinity,low/high temperature and hormone and so on,in the process of growth and development.DREB transcription factors play the key roles in response to abiotic stresses.DREB transcription factors specifically bind the DRE/CRT cis-acting element and trigger the downstream target genes.The coding products of target genes were functional proteins and enzymes such as proline,sucrose,LEA,aquaporin（AQP） and praline synthetase et al.In transgenic Arabidopsis plants,overexpression of DREB1A can introduce at least 40 downstream genes and increase the tolerance to abiotic stresses.One transcription factor introduces many downstream genes and improves the resistance activity to stresses.It had very large potential value and widest foreground to study transcription factors in plant breeding.The thesis aims included:firstly,two DREB-like genes were isolated from chrysanthemum and the relate function were studied;secondly,some information was obtained by the SNP analysis in chrysanthemum using DmDREBb gene.To analysis the molecular mechanism of DREB in chrysanthemum,two DREB-like genes were isolated from chrysanthemum cv.’Hong Feng’ using degenerate PCR and RACE methods in this study.These cDNAs were 872bp and 675bp in length containing a single open reading frame encoding 191,185 amino acids with predicted molecular masses of 21.66 and 20.99 kDa,and the pI were 6.72 and 6.80,respectively.Both sequences had been deposited to the GenBank database under accession numbers EF490996 and EF487535 for DmDREBa and DmDREBb,respectively.The genome sequences were obtained by the same primers with cDNAs and the lengths of both genes were also same with cDNA sequences.The results indicated that there was no intron in the two chrysanthemum DREB-like genes.The sequence alignment analysis showed that DmDREBs genes had high similarity with other DREB genes and had higher similarity with DREB1 genes.Blast analysis suggested high similarity between DmDREBa and DmDREBb with the overall identities 88%at amino acid level and 90%at nucleotide acids except for one obvious gap of MPGVIDS（152—159） in amino acids level.The DmDREBs proteins contained a conserved DNA-binding domain of 57 amino acids that presented in a large family of plants.DmDREBs genes revealed a typical DREB1-type nuclear localization signal（NLS） consensus PKKRAGGKKFMETRHP before the EREBP/AP2 domain and a DSAWR sequence after the domain,and that domain existed in all DREB1-type proteins from various species.Threeβ-sheets and an amphipathic a-helix were also found in the EREBP/AP2 domain of DmDREBa and DmDREBb,which may be responsible for DNA-binding activity.Thus,sequence analysis indicated that DmDREBa and DmDREBb were putative DREB1 homologs.Phylogenic analysis suggested that DmDREBs were attributable to the DREB1 subgroups comparing to DREB2 transcription factors. Interestingly,DmDREBs shared the highest similar to Capsicum annuum CaCBF1A （AY368482） and Vitis vinifera VvCBF1（AY390372）at amino acid level,two species that are chilling sensitive.Furthermore,Vitis vinifera is one of woody plants.Southern blot analysis was used to determine the approximate number of the DREB-like genes in chrysanthemum.Probing with the DmDREBa sequence indicated that chrysanthemum contains multiple DREB-like genes/alleles.To investigate the expression patterns of DmDREBs in chrysanthemum,the expression of the two genes at different organs was analyzed.Both genes were highly expressed in leaves and stems,but with low expression in roots or flowers at 15℃.The expression levels of DmDREBa and DmDREBb were different in several stresses.Under cold stress（4℃）,DmDREBb was expressed highly at 0.5h,but DmDREBa was highly expressed in 24h under salt condition.The expression level of DmDREBa was also reduced during the whole process but a little expression was observed after 24h with 100μM ABA treatment.However,the expression level of DmDREBb was variable under 100μM ABA condition.The mRNA level of DmDREBa was inhibited significantly under PEG6000 and hot condition（40℃）.The expression of DmDREBb was continued only 0.5h,and then was also inhibited strongly.These two proteins were found to have transcriptional activity and had the DRE-binding capacity as shown by yeast one-hybrid system and were localized to the nucleus of cells.These studies indicated that the two proteins were DREB transcription factors.The DmDREBa gent was overexpressed in tobacco and 43 transgenic plants were obtained by PCR method.And southern blot analysis indicated that 1-3 copies of DmDREBa were randomly inserted.The tolerance of transgenic tobacco plants was increased comparing to the wild tobacco plants under low temperature,drought and high salinity conditions.The content of MDA in wild tobacco plants was 0.619×10^-3μmol/g which was more than 0.481×10^-3μmol/g in transgenic plants,which indicated the tolerance ability to low temperature of transgenic plants was increased.The lost water of transgenic tobacco was less than wild plants after natural transpiration.Further,the density of the stoma in the epidermis was observed and found that the numbers of stoma in upper or lower epidermis of transgenic plants were reduced comparing to wild tobacco.The result of t test suggested that the differences between the upper and lower epidermis of transgenic plants and wild type were significant.The transgenic tobaccos had better character than the wild plants under low temperature,drought and high salinity conditions.At last the SNP research was carried out in chrysanthemum cultivars and wild plants using DmDREBb gene.The main mutations were transversions in chrysanthemum wild plants with more mutations of transitions in chrysanthemum cultivars.The results showed that the diversity of SNP in wild plants（π=0.02368） was higher than that in chrysanthemum cultivars（π=0.00963）.The results indicated that artificial selection affected the evolution of chrysanthemum cultivars.The value of Ka/Ks was less than 1 suggested that the nucleotide acids sequence was very conservative and purifying selection affected the evolution of chrysanthemum cultivars.The analysis of LD was suggested that the strength of LD was decreased with the extrending of distance.The decreased trend of chrysanthemum wild plants was very evidence,but the trend was gentle in chrysanthemum cultivars.The structure of LD was changed during the domestication of chrysanthemum cultivars that affected diversity drop.