Cloning And Functional Characterization Of Dehydroascorbate Reductase Gene From Chimonanthus Praecox (L.) Link

Dehydroascorbate reductase (DHAR) is one class of glutathione S-transferase superfamily,DHAR gene from Arabidosis,rice,spinach and other plants have been cloned,but it has no report about the DHAR gene have been cloned in Chimonanthus praecox (L.) Link. DHAR plays a critical role in regeneration of ascorbic acid,it catalyzes the re-reduction of Dehydroascorbate to ascorbate by glutathione,and increases the accumulation of ascorbic acid in the most higher plant.it is critical for protection of cellular components against exogenous stress.DHAR has an important role in plant growth,too.This study reported the cloning and characterictics of a DHAR from Chimonanthus praecox (L.) Link,and named CpDHAR. The structure characteristics of CpDHAR protein were analyzed with bioinformatic method;We have constructed prokaryotic expression vector, expressing CpDHAR in E. coli.;and the content of CpDHAR from flowers of Chimonanthus praecox (L.) Link was analysised by the real time quantitative PCR.The main findings are as follows:1.Cloning and Structural prediction of CpDHARWe have found the cDNA sequence with the size of the 890bp,by rough randomly selection from cDNA library sequencing. Sequence analysis showed that the CpDHAR gene has 651bp in opening reading frame,and it encodes a protion of 216 amino acid residues with a calculated molecular mass of 24.02KD.it has a PloyA in 3'-untranslated region.The anayle of bioinformatic method shows that CpDHAR protein contained Alpha helix(44.44%), Radom coil extend (45.37%) and Extend strand(10.19%),and it has contained the abundant hydrophobicity Region and the 13 phosphorylation site.2.the construction of recombinant PET-CpDHAR and the analysis of prokaryotic expressionAt first,the characteristics of the vector and the frgment of CpDHAR was analysised, and CpDHAR is amplified by PCR using the primers with SacⅠand Hindlll digestion sites at the end.PMD19-T was used the intermediate,while the CpDHAR was inserted into SacⅠ/HindⅢ-digested PET-32(a) vector.Verified by sequencing and double digested properly,the resulting plamid PET-CpDHAR was transformed in the expression strain BL21(DE3).The result showed that a fairly good expression can be gained under the condition of 28℃,1Mm IPTG and overnight induced by optimizing the induced condtions,and the result was verified by SDS-PAGE. The result indicated that the recombinant PET-CpDHAR was efficient expressionde in BL21(DE3).3.The different organizations and different periods of CpDHAR from flowers of Chimonanthus praecox (L.) Link was analysised by the real time quantitative PCRWhen the flowers of Chimonanthus praecox (L.) will be bloom,we picked the flowers which come from the different flowering time and the organ of the flowers.The real time quantitative RT-PCR was used to analysis the expression of CpDHAR gene.The results reavled that the content of CpDHAR is the highest in the flower budding dates.In the flowering dates the content of CpDHAR is increasing,but when the flowers was withered,the content of CpDHAR was decreased.In the different organ,the result shows that it is the highest of the content of CpDHAR in the stamen,and the lowest is outside flap of the flower.