Isolation And Functional Identification Of ZmLTPL63 Gene Of Maize

Plants is often affected by various abiotic stresses such as high temperature,dought and salt during the growth and development.These stress conditions are damage to plant physiological and metabolic activities and severely disrupt yield and quality of crops.In response to these environmental stresses,plants have formed a set of sophisticated defense mechanisms in their long-term evolutionary history,including multiple signaling pathways,transcription factors and functional genes.Recent studies showed that lipid transfer proteins?Lipid Transfer Protein,LTPs?play an important role in regulation of plant development and abiotic stress response.The detailed mechanisms for most LTPs are not clear.According to the response of tropical maize germplasm POB21 to stresses of high temperature and drought,a function unknown LTPs-like gene was screened from the up-regulated gene profile.The candidate homologous to the reference gene DRMZM2G05208 in B73 genome from MaizeGDB database,designated as ZmLTPL63,was cloned by RT-PCR method.Tthe main analysis results of structure,phylogenetic relationship,expression pattern and function are as follows:?1?Sequence analysis showed that the full-length CDS of 759 bp encods a protein of 252 amino acids,molecular weight of which is about 25.9kD.The predicted signal peptide of 23 amino acids is localized at the N terminus of the mature protein.The tertiary structure of ZmLTPL63 protein was charaterized by 4 alpha helices stablized by the 8 cysteine residues motif?8CM?linked by 4 disulfide bonds to form a three-dimensional hydrophobic cavity binding to lipid molecules.However,ZmLTPL63 peptide chain was much longer than typical nsLTPs.Proline was rich at the region between signal peptide and 8CM motif.Sequence alignment analysis revealed homologous proteins of ZmLTPL63 in multiple plant species.Phylogenetic analysis indicated ZmLTPL63 was highly homologous to XP₀12699184.1 of Setaria italica XP₀02439994.1 of Sorghum bicolor,OEL21002.1 of Dichanthelium oligosanthes,EMT12664.1 of Aegilops tauschii.,KQK15914.1 of Brochypodium distachyonand BAC65920.1 of rice rather than AT3G43720 of Arabidopsis thaliana.?2?Expression pattern assay by qRT-PCR method showed ZmLTPL63 gene was reponsive to multiple streeses and treatments by hormores and signal melecules.According to the tissue-specific assy,transcript level in leaves was higher than that in stem root and lowest in roots.Gene expression can be up-regulated by stresses of high temperature,PEG,and NaCl,as well as plant hormone of abscisic acid?ABA?and methyl jasmonate?MeJA?,ethene?ET?and chemical compond of hydrogen peroxide?H₂O₂?rather than gibberellin?GA?treatment.?3?To study the subcellular localization of ZmLTPL63 protein,the recombinant expression vector of pROKII-35S:: ZmLTPL63-GFP was constructed and transferred into tobacco leaves by agroinfiltration.Confocal microscopic investigation of GFP fluorescence in leaf epidermal cells showed ZmLTPL63-GFP fusion protein distributed only in the cell periphery.This is consistent with the result from bioinformatic prediction.?4?With reference to genomic DNA sequence from B73 genome,the upstream1500 bp DNA sequence of ZmLTPL63 was cloned to construct GUS expression vector pBI121-PZmLTPL63::GUS.Sequence analysis of promoter revealed elements responsive to fugus,heat,drought,cold and so on.Histochemical staining of GUS protein in transgenic Arabidopsis seedlings indicated the wide gene expression,especially strong expression in the shoot apexes and root tip.Treatment of ABA and H₂O₂ enhanced the GUS expression drived by promoter of ZmLTPL63 gene,which is concert with the gene expression pattern.?5?Overexpression of pBI121-35S:: Zm LTPL63 in Arabidopsis enchanced stress resistance to drought and salt.Transgenic plants had longer roots and higher survival rate than the wild-type?WT?under abiotic stress conditions.Moreover,slower chlorosis and water loss were also investigated in transgenic Arabidopsis leaf discs in vitro.Compared with control,ZmLTPL63 overexpression alleviated the accumulation of ROS and MDA and increased antioxidant enzyme activities in transgenic plants under stress conditions.These results implied overexpression of ZmLTPL63 gene could improved stress tolerance to drought and salt through increasing antioxidant activities.?6?Lipid acids were detected by gas chromatography in tobacco leaves transientlyexpressing ZmLTPL63 gene.The result showed content of multiple C16-C18 unsaturated fatty acids,such as linoleic acid and linolenic acid,was much higher than control.It indicated that ZmLTPL63 is involved in the metabolism of fatty acids.?7?The ZmLTPL63 gene participates in regulation of plant growth and development.Overexpression of this gene in Arabidopsis plants resulted in late-flowering phenotype.qRT-PCR assay showed that the expression of key genes involved in positive regulation of flowering were significantly reduced,suggesting that ZmLTPL63 may inhibit the expression of key flowering genes so as to delay flowering.