| Ascorbic acid is a molecule against oxidative stress,and sustains dynamic equilibrium of redox in apoplast.Ascorbate oxidase(AO)is responsible for the oxidation of ascorbic acid to dehydroascorbic acid,redox state regulation of apoplast.AO was involved in various abiotic stress responses and physiological processes.Our previous study found that the expression level of tobacco AO was affected by low temperature,but the exact mechanism is unclear.In order to understand the molecular response mechanism of AO to cold stress,AO promoter of tobacco(Nicotiana tabaccum cv.K326)was cloned and analyzed AO promoter activation after part deletion.The cis-acting elements in AO promoter response to low temperature were defined to understand the molecular mechanism of of AO expression induced by cold.The main findings are as follows:The genome walking primers were designed according to the 5’ regionof the first AO exon sequence,and after two rounds of nested PCR,a length of 1669 bp sequence of the promoter was obtained.The AO promoter region was analyzed by online promoter analysis software such as PLACE The results showed,in addition to the basic core promoter elements,there were a lot of adversity related cis-acting elements in AO promoter,including hormone response element,light response element,dehydration response element,sugar response element,etc..Cis-acting elements assiociated with low temperature were MYB1 AT,MYBCORE,MYCCONSENSUSAT and so on.We speculated that the expression of AO may be regulated by hormone,sugar signal and adversity related signals.According to the types and distribution of cis-acting elements in the result of AO promoter analysis,seven 5’end deleted fragment were then constructed by ligating the cloned fragment to the vector p Cambia1391,used for transformation of Arabidopsis by floral dip method.PCR and Hygromycin were used to screening T2 generation,each promoter fragments were obtained three or more transgenic homozygous lines.Transgenic lines were low-temperature treated for various durations and afterwards GUS expression level in the leaf blades was measured.It is found that one-day low temperature treatment resulted in a downregulation of GUS expression in all the transgenic lines.However,as the promoter prolongs,the expression level in P7 becomes higher.On the other hand,the expression in P6 and P5 are significantly reduced and in P4 the expression is rescued to 87% of that in the control group.These results indicates that there may be a cis-acting,low-temperature responsive element between P6 and P7 and between P4 and P5 which exerts opposite regulatory effects.Histochemical staining against GUS in low temperature-treated transgenic Arabidopsis lines was performed.The results show that the staining intensities in the leaves exhibited indiscernible change following low temperature treatment,which is in consistency with q PCR results;whereas in the root and petiole,the low temperature induced effect is profound.It is speculated here that tobacco AO promoter may contain some tissue specific expression related element.Additionally,there is the possibility that exogenous gene promoter may perform differently compared with the species where it origins. |
