Cloning,Expression And Function Analysis Of Non-specific Lipid Transfer Protein Gene (nsLTP) In Chinese Flowering Cabbage (Brassica Parachinensis L.)

In recent years,contamination of phthalate esters(PAEs)in agricultural soils has become increasingly serious,which directly affects the quality safety of agricultural products and thus become great risk to human health.The transcriptomic and proteomic analysis of the high-and low-PAEs accumulators of Chinese flowering cabbage(Brassica parachinensis L),was conducted in our previous study.It was found that the expression of non-specific lipid transfer protein(nsLTP)genes was significantly increased with more PAEs accumulating in Chinese flowering cabbage cultivars.Therefore,in order to investigate the role of nsLTP in forming high-or low-PAEs accumulation charater,the full length of nsLTP gene was cloned by RACE technology Gene sequence analysis,prokaryotic expression,subcellular localization,and molecular simulation experiments were conducted to reveal the biological mechanism of PAEs cumulative traits in different genotypes of Chinese flowering cabbage.The main results of this paper were obtained as follows:(1)Full-length cloning and sequence analysis of nsLTP geneBased on the transcriptome data of Chinese flowering cabbage(B.parachinensis L),the full-length cDNA of nsLTP gene was cloned by RACE.The full-length of this gene was 587 bp and the open reading frame(ORF)was 297 bp,encoding 97 amino acids with a molecular weight of 10.36 KDa.According to the protein molecular weight,it belongs to the nsLTPI type.The gene sequence alignment analysis showed that nsTLP gene shared the highest homology with Brassica nupus,as high as 97%.Protein multiple alignment and phylogenetic tree analysis revealed that B.parachinensis L was rather high nucleotide sequence identity from B.rapa and B.napus,but differed with Camelina sativa.By analyzing the amino acid sequence,this hydrophobic protein has a transmembrane structure that overlaps with the signal peptide composed of 29 amino acids.The prediction of the secondary and tertiary structure of nsLTP protein in Chinese flowering cabbage showed that the nsLTP had hydrophobic cavities formed by the formation of 8 cysteines and 4 pairs of disulfide bonds.(2)Prokaryotic expression and protein purification of nsLTPAfter constructing the prokaryotic expression vector of pET-32a-nsLTP and transferring it into Escherichia Coli,a large amount of recombinant protein could be obtained in a short time(2h)under the induction of IPTG.Through the analysis of soluble protein and optimization of expression conditions,the target protein is a soluble protein,and low temperature(25℃)and high concentration of the inducer(1mM)could facilitate the soluble expression.Recombinant protein was obtained by His-tag purification,and Western Blot analysis was conducted to identity the target recombinant protein.The prokaryotic expression and protein purification of nsLTP provided the based informations for further study of nsLTP.(3)Subcellular localization of nsLTPThe constructed pBI-121-GFP-nsLTP vector was transformed into E.coli BL21(DE3)and cultured for 12 h,the recombinant plasmid was extracted and transformed into Agrobacterium tumefaciens EHA105 competent cells.The infection onion epidermal cells were infiltrated with nuclear staining agent DAPI.The distribution of green fluorescence signal of pBI-121-GFP-nsLTP fusion protein in the cells was observed by laser confocal microscopy.The results indicate that the green fluorescent signal of GFP-nsLTP fusion protein was mainly distributed on the cell wall,and instead of on the nucleus,cytoplasm and cell membrane.To further confirm the distribution of the fusion protein fluorescence signal,the onion cells were plasmolysed through a sucrose solution.The result show that the protoplasts were significantly shrunken,and there was no fluorescence signal in the cytoplasm and cell membranes,while a clear fluorescent signal was found on the cell wall.It indicated that nsLTP protein was located on the cell wall,which consistent with bioinformatics predictions.(4)Molecular simulation of nsLTP binding with DBPThe nsLTP protein has the ability to bind lipids in vitro,and its hydrophobic cavity can entrap lipids.The interaction of nsLTP with DnBP was further analyzed to investigate the binding affinity of DBP ligands with nsLTP.The result showed that,there was a binding site located at the H3 and H5 alpha helices of the nsLTP protein hydrophobic pockets.The complex connected with cysteine(Cys-37)amino acid residue by hydrogen bonding to form a Cys-37:HN:DBP structure,with a bond length of 1.784 A.The DBP molecule was surrounded by amino acid residues such as Cys-65、Leu-36、Asn-64、Pro-63、Pro-34、Ser-62、Ala-61、Lys-33、etc.Since the ligand had a rotatable atom,the torsional energy in molecular docking was 2.98 kcal/mol,and the intermolecular energy between the ligand and the protein was-7.44 kcal/mol.Therefore,the binding energy of the complex was-4.46 kcal/mol(binding energy = intermolecular energy + torsional energy).nsLTP protein can combine with DBP to form a stable complex,which plays an important role in the absorption and accumulation of PAEs in Chinese flowering cabbage(B parachinensis L).