Evaluation Of Cold Tolerance Of Different Varieties Of Papaver Nudicaule And Cloning Of Cold Inducible Gene PnDREB1

In this study, we have compared cold tolerance of different varieties of Papaver nudicaule by investigating the influence of low temperature on leaf morphology and physiological and biochemical indices. Additionally, based on the known sequences, full coding sequence of a cold-induced transcription factor DREB1 was obtained from P. nudicaule, designated as PnDREB1. The cold-induced expression levels were evaluated and the in silica sequences analyses and structural prediction were also performed. The main results obtained are as following:1. The cold-tolerance evaluation of different P. nudicaule varietiesFour P. nudicaule varieties,’Champagne Bubbles", domestic variety,’Wonderland’and ’Garden Gnome’, in mature period are analyzed. The effects of low temperature on total of 11 morphological, physiological and biochemical traits were investigated, including leaf morphology, relative electrical conductivity (REC), malondialdehyde (MDA), etc. The results showed that:(1)After 24-hours treatment under different low temperatures (from 3℃ to-9℃), REC in four varieties rose following a "S"-like " trands along with the temperature decline. The Semi-lethal temperatures (LT50) of ’Champagne Bubbles’ domestic variety.’Wonderland’, and’Garden Gnome’were-7.7℃,-5.2℃,"-1.9℃, and-4.5 ℃, respectively; (2)After 0h,24h,48h and 72h of the-3 ℃ treatment,4 varieties were damaged with varying degrees. Their freezing indexes were 0.30,1.46,2.34,1.67, respectively, after 72h low temperature treatment. When low temperature stress continued, the MDA content of all varieties were initially decreased and then increased; The soluble protein (SP) content of’Champagne Bubbles’rose after the initial decrease, the others were increased firstly and then decreased; The proline content of’Garden Gnome’ continued to rise, the others were firstly increased and then decreased; The SOD activity of ’Champagne Bubbles’ continued to rise, the other three varieties were increased firstly and then decreased; In all of varieties, the activity of POD were increased initially and then decreased; the activity of CAT showed the "Up-down-up" trend; the changes of APX activity were same as POD; GR activity and AsA content were both decreased first and then increased. These showed that different varieties exhibited diverse adaptability to the cold stress; (3)We have analyzed 9 physiological indicators by using principal component analysis and subordinate function. The results showed that the order of cold tolerance in 4 varieties of Papaver nudicaule was’Champagne Bubbles’> domestic variety>’Garden Gnome’>’Wonderland’. This result was similar to the order of Semi-lethal temperatures and freezing indexes. These results provide basis for identifying cold indicators and screening new varieties in Papaver nudicaule.2. Isolation and characterization of PnDREBlA new DREB1 transcription factor, PnDREB1, was cloned by RT-PCR and RACE from’Champagne Bubbles’. It is 1035bp in length, containing an continuous open reading frame(ORF) of 699bp and no intron was found. It encodes a putative protein of 232 amino acids with a predicted molecular mass of 26.3kDa. The predicted PnDREBl protein contains a conserved AP2/ERF domain with 58 amino acids. In the N-terminus of the domain, there was an alkaline-rich region which was predicted as nuclear localization signal (NLS):PKR(K)K (P)AGRxKFxETRHP. The domain also contains DSV(A)WR sequence in the downstream. These two polypeptides are characteristic of DREB1/CBF gene family. The predicted PnDREB1 protein shares highly homologous with DREBs of Nelumbo nucifera, Prunus mume, Chrysanthemum, Pyrus pyrifolia, Arabidopsis thaliana, etc. Phylogenetic analysis showed that PnDREBl belongs to A-l subgroup of DREB/CBF transcription factor subfamily, and is closest with DREB1 of Chrysanthemum.Bioinformatics analysis showed that PnDREB1 protein is hydrophilic without transmembrane structure and signal peptide. It may be located in the chloroplast or the nucleus. Secondary structure and tertiary structure prediction showed that the protein contains an a-helix, three β-pleated sheets and several β-corners.Semi-quantitative RT-PCR analysis showed that the PnDREB1 gene expression level in leaf was very low before cold treatment. After-3 ℃ treatment, the transcriptional level of PnDREB1 was increased first and then decreased when the low temperature treatment continued. The relative expression maximum level at 8h of teatment.