Isolation And Function Analysis Of NtLTP4 From Tobacco

Tobacco is an important economic crop in China and also an important economic source of state.But adversity stresses such as drought,salinity,diseases and insect pests seriously affected the production and quality of tobacco,which restricted the agricultural development of tobacco.How to improve the biological and abiotic stress resistance of tobacco has become an important topic in today's research.In order to deal with this kind of bad stimulation,plant itself had formed a set of precise and complicated defense mechanism.The expression regulation of stress related genes played an important role in the process of environmental signals' stimulus.In the present study,common tobacco?N.tabacum cv.NC89?was selcted as the experimental material,and the NtLTP4 gene was isolated.The multiple sequences alignment and phylogenetic analysis was performed,also did the biological function analysis.The main results are as follows:?1?Sequence analysis revealed that the full-length cDNA consisted of 354 nucleotides,which encoded a protein of 116 amino acid residues with a putative molecular weight of 9 kDa,and contained five conservative peptide structure domain: T/S-X-X-D-R/K and P-Y-X-I-S.There were eight position conservative cysteine.NtLTP4 had typical nsLTP protein's tertiary structure,which were 4 alpha helix,4 disulfide bond,one can combine and accommodate lipid molecules of hydrophobic cavity,played an important role in lipid molecules.The gene cluster analysis showed that NtLTP4 had high similarities with NgLTP1,AtLTP7 and AtLTP5 homology is higher,and these genes all belong to the Type I group of LTP family.?2?A fusion protein 35S-NtLTP4::GFP was constructed and instantaneous infected the leaf of N.benthamiana.The obtained fluorescence pattern was observed using a confocal laser confocal scanning microscope.The result showed that the Nt LTP4-GFP fusion protein emitted green fluorescence predominantly in around the cell wall whereas the fluorescence of 35s-GFP was detected almost around all cell.These results revealed that the Nt LTP4 protein was localized in the cell wall,which provided a theoretical basis for its function.?3?The expression patterns analysis showed that the expression level of NtLTP4 was used the qRT-PCR method.The results showed that NtLTP4 was induced by wounding,salt and PEG and other abiotic stress.The biotic stress including R.Solanacearum and Potato virus Y also induced the gene.Signaling molecules,such as MeJA,ABA and SA can strongly increase the transcripts level of NtLTP4.All these results revealed that NtLTP4 might be involved in multiple signaling pathways and play an important role in development and defense processes.?4?The NtLTP4 cDNA was inserted into the binary vector pROKII for the construction of the recombinant vector pROKII-NtLTP4,and then the recombinant vector was transformed to the N.Tabacumusing A.tumefaciens-mediated leaf disk method.Result of RT-PCR analysis showed that NtLTP4 was successfully expressed in transgenic plants.Using qRT-PCR analysis for part of the transgenic plants,selected the high,medium and low three different expression level for the functional analysis.?5?After salt stress treatment,compared with wild type plant,the transgenic plants can improve the resistance to salt tolerance.On the MS culture medium containing different concentrations of NaCl,the transgenic tobacco had the higher seed germination rate.Their root length was longer than that of the wild type,and the growth potential was aslobetter under the salt tolerance.After salt treatment,the content of malondialdehyde in transgenic plants was lower than those of the wild type,which explained the cell membrane lipid peroxidation level of transgenic plantswas lower,and plant cell damaged to a lesser degree.The expression level of the genes related to the ROS production was lower in transgenic plants,and the expression level of the genes related to the ROS eliminating was higher in transgenic plants.The activities of antioxidases were also higher in transgenic plants after salt treatment.The content of H₂O₂ and O^2-were lower in the transgenic plants.From all these,we can suggest that NtLTP4 can increase the genes related to ROS eliminating,which can increase the activities of antioxidases.All of these results suggested that NtLTP4 increased the resistance of salt stress.?6?After drought stress,overexpression of NtLTP4 in transgenic plants enhanced drought tolerance.The expression level of the genes related to the ROS in transgenic plants was changed similar to the salt stress.This result revealed that NtLTP4 might act as a positive regulator in regulating the drought stress.?7?Comparison with the wild type plant,NtLTP4-overexpressing plants enhanced the resistance to the infection of R.solanacearum.After infection,the expression level of the genes related to the SA signaling pathway?PR1a?PR5?and level of the genesrelated to the MeJA signaling pathway?PR4?were higher in transgenic plants.The results revealed that the cross talk between SA-and JA-mediated signaling pathway might contribute to the enhanced pathogen resistance of the NtLTP4-overexpressing plants.?8?The NtLTP4-overexpressing plants enhanced the resistance to the infection of PVY-O.After infection,the expression level of the PR genes?PR1a?PR5?were enhanced.The results revealed that some signaling pathways might contribute to the enhanced virus resistance of the NtLTP4-overexpressing plants.Taken together,these results showed that overexpression of NtLTP4 enhanced the biotic and abiotic stress.This study will broaden our knowledge about the function of the type I LTP genes,and provide the basis for the construction of the transgenic tobacco with the stress and pathogen resistance.