Expression And Functional Analyses Of Trehalose-6-phosphate Phosphatase Genes Promoter From Caragana Korshinskii Kom.

Caragana korshinskii Kom.,the xerophytic shrubs of legume,is widely distributed in the northwest of China,which has important ecological and economic value not only in windbreak and sand-fixation,soil and water conservation,as well as in utilization as animal feed.On the other hand,C.korshinskii Kom.shows strong stress tolerance,therefore it has great significance to study the stress-tolerance mechanism and to explore stress-related genes.Trehalose is a kind of non-reducing disaccharide,which can be used as a stress protectant for proteins and membranes.Trehalose-6-phosphoric acid phosphatase(TPP)is the key enzyme in trehalose biosynthesis pathway,which catalyzes the reaction of trehalose-6-phosphoric acid to phosphorylation.The expression abundance of TPP directly affects the trehalose biosynthesis.Previous studies have shown that overexpression of TPP in Arabidopsis enhanced the stress resistance of transgenic lines.Therefore,it is very important to study the expression mechanism of the CkTPP and its function in response to abiotic stress.In this study,the expression feature and function of the CkTPP were analyzed on the basis of the cloned CkTPP sequence.The main results are as follows:(1)The results of real-time fluorescence quantitative PCR analysis showed that CkTPP was expressed in the root,stem and leaf of C.korshinskii,and the expression in stem was higher than root and leaf,its expression was induced by methyljasmonate(MeJA)and PEG-simulated drought stress.(2)Using the method of genomic walking,1912 bp of Ck TPP promoter sequence was cloned,and sequence analysis revealed that the promoter region contains some cis-elements responding to plant hormones including gibberellin,ethylene,methyl-jasmonate and abscisic acid,photoinducible elements and stress response elements.(3)Heterologous expression of GUS reporter gene driven by CkTPP promoter in Arabidopsis thaliana showed that CkTPP:: GUS expressed in root,stem,leaf and flower of transgenic plants,its expression activity was induced by methyl jasmonic acid,gibberellin,ethylene,abscisic acid,salt,and mannitol.(4)The phenotypic analysis of transgenic A.thaliana showed that the overexpression of CkTPP increased the drought tolerance of transgenic plants.These results provided an experimental basis for further study on expression and regulation mechanism of the CkTPP and genetic improvement of economic crops,forage grass and trees by using this gene.