Isolation And Functional Analysis Of ZmGLY?-2 Gene From Maize

Methylglyoxal?MG?is a cytotoxic byproduct of metablism of sugar,amino acids,acetone.The glycation modification and irreversible cross-link of proteins by MG results in change of structure and function of proteins,nucleic acids,lipids and consequent cell injury and even cell death.Glyoxalase system is the major pathway of detoxication in plants by converting MG to lactate.It plays an important role in regulating the plant growth and development or alleviate the adverse effect of abiotic stresses.According to the transcriptome of maize responding to MG treatment,a glyoxalase II gene was screened from the up-regulated gene profile.The candidate gene in genome of maize inbred line B73,designated as ZmGLY?-2,was cloned by RT-PCR method.The main results of structural characteristics,expression pattern and function analyses are as follows:Sequence analysis showed that the full-length CDS of 777bp encodes a protein of 258amino acids,molecular weight of which is 28755.8Da and the isoelectric point is 5.67.The ZmGLY?-2 protein contains the conserved metal binding motif?THXHXDH?and active site?C/GHT?of metallo-?-lactamase.Sequences alignment and phylogenetic analysis indicated that ZmGLY?-2 and its orthologues are the ubiquitious member of GLY? subfamily in plant genomes and share the highest similarity with homologous GLY? of Sorghum.The fusion expression vector of 35S::ZmGLY?-2-GFP was contructed and transferred into tobacco leaves by agroinfiltration.The laser confocal microscopic investigation of green fluorescence in the leaf epidermal cells revealed the distribution of fusion protien in cytoplasm and nucleus.Analysis of expression pattern of ZmGLY?-2 gene by qRT-PCR showed tissue-specific expression in different organs.Higher transcription level was detected in the leaves than that in root and stem.Multiple stresses of drought,salt and heat treatment can induced the expression of ZmGLY?-2 gene.The up-regulation of transcription level was also revealed under treatments of MG,abscisic acid,methyl jasmonate and salicylic acid.While it was down regulated by ethylene.Overexpression?OE?of ZmGLY?-2 gene enhanced the stress tolerance to MG in N.benthamiana.Under treatment of different concentration of MG,seeds of OE lines germinate faster than that of wild type.According to the leaf-disk incubation of MG,the OE lines showed higher chlorophyll content than that of wild type.Overexpression of ZmGLY?-2 gene enhanced the tolerance to stresses of drought and salt in N.benthamiana.Comparing with wild type,seedlings of OE lines showed longer roots under both stress conditions.Upon incubation in mannitol or NaCl solution,leaf disks of OE lines retained higher content of chlorophyll than wild type.According to histochemical staining of leaves using DAB and NBT,the lighter color in leaves of OE lines showed lower accumulation of H₂O₂ and O^2-than that of wild type.Activites of antioxidant enzymes and the expression of corresponding genes represented higher level in OE lines.Accumulation of MDA and MG were also inhibited by overexpression of ZmGLY?-2 gene.Higher increasement of proline in OE lines than wild type suggested smaller osmotic damage under stress of drought.Comparing with wild type,overexpression of ZmGLY?-2 gene reduced the stomatal aperture of tabacco leaves under both stresses.In conclusion,the overexpression of ZmGLY?-2 gene can enhance stress tolerance of plant by improving abilities of antioxidation and water conservation.