Cloning And Function Analysis Of PeSnRK1a Gene In Phyllostachys Edulis

SNF1(sucrose non-fermenting 1)-related protein kinases 1(SnRK1)plays crucial rolesin carbon metabolism and stress responseinannual herbaceous plants.To explore the function of SnRK1 in Perennial gramineousbamboo,in this study,PeSnRK1ais cloned from Phyllostachys edulis.The relative expression of PeSnRK1ain Phyllostachys edulis seedlingsand in leaf of Phyllostachys edulisunder stress is detected byqRT-PCR.The PeSnRK1a protein is expressed in Escherchiacoli,and transformed PeSnRK1a gene into Arabidopsis for studying the physiological and biochemical function.The main results of the research are as follows:1.SnRK1is cloned from Phyllostachysedulisand named as PeSnRK1a.Sequence analysis indicates that the open reading frame(ORF)of PeSnRK1a is 1509bp in length,encoding a protein of 502 aa and the predicted protein molecular weight is 57.4kDa with the isoelectric point of 8.67.The amino acid sequence alignment shows that PeSnRK1a shares 76.22%-92.29%identity with the homologies of Arabidopsis thaliana,Sorghum bicolor,Zea mays,Oryza sativaandsoon.Phylogenetic tree analysis indicate that PeSnRK1a is highly homologous to ZmSnRK1a,SbSnRK1a and belongs to SnRK1asubfamily.Conservation structure domain analysis indicates that PeSnRK1a contains typical STKc,UBA and KAI domains.2.qRT-PCR analysis ofPeSnRK1a expression pattern indicates that it is expressed in the root,stem,and leave of the two-month Phyllostachys edulis,and with the highest expression level in leave;The dark and salt stress could induce the expression of PeSnRK1a in Phyllostachys edulis seedling leaves.3.A prokaryotic expression vector of PeSnRK1a is constructed to expression the PeSnRK1a protein in vitro.The results show that the PeSnRK1a protein existed in the form of inclusion body by using BL21(DE3)and Rosetta(DE3)as expression strains,pMal-c2x as expression vector,1mM IPTG as the inducer and induced at the 20 ~oC and 37 ~oC respectively.4.The activity of SnRK1 protein kinase is detected byprotein immunoblot test.The SAMS polypeptide expressed in Escherchiacoli is used as the substrate of SnRK1 protein kinase,and the Phospho-Acetyl-CoA Carboxylase Antibody(Ser79)is used to detect the phosphorylation level of SAMS for reflectingthe SnRK1 proteinkinase activity.5.A plant expression vector was constructed by ligating the PeSnRK1a gene to GUS reporter geneand the vector is transformed into wild-type Arabidopsis thalianaby Agrobacterium mediating.Compared with the wild type Arabidopsis thaliana,the transgeneticplants overexpressing PeSnRK1a have no significant difference in the time of seeding germination,leaf morphological characteristics,flowering and senescence when cultured in normal condition.While the detached leaves of transgenetic plants delay senescence in dark conditions,and the seeds display a higher germination on the 1/2MS medium with 100-200mM NaCl.