Screening And Expression Analysis Of Dirigent1-interacting Proteins Of Eucommia Ulmoides Oliver

Eucommia ulmoides Oliver(E.ulmoides)has a long history of medicinal use and is a valuable nourishing medicinal material in China.Phenylpropanoids,iridoids and lignans are the main medicinal components of E.ulmoides.Studies have found that DIR proteins are involved in the biosynthesis of lignans and lignin,and some DIR proteins can regulate the coupling of coniferyl alcohol to form(+)-pininol,thereby promoting the biosynthesis of lignans,involving in cell wall modification,lignifying plants to resist pathogenic microbial infection and abiotic stress.Previous studies have found that the overexpression of Eu DIR1 gene can improve the resistance to Botrytis cinerea and Fusarium oxysporum in transgenic tomato and tobacco,and it is not only related to the thickening of the xylem cell wall but also responds to abiotic stresses such as high salt.In this study,in order to further understand the regulatory mechanism of E.ulmoides under stress by studying Eu DIR1 interacting proteins,the resultant Eu DIR1 was used as a bait protein to screen the library for Eu DIR1 interacting proteins.The main research results obtained are as follows:1)Screening and classification of interacting proteins of Eu DIR1A total of 32 positive cloned proteins of Eu DIR1 were obtained by yeast two-hybrid screening assay,and these interacting proteins were classified based on their functions.Among them,13 proteins had the function response to stress.In addition,19 other proteins may be involved in diverse physiological and biochemical processes,including photosynthesis,glycolysis and the tricarboxylic acid cycle.2)Cloning and bioinformatics analysis of Eu MLPThe main latex protein genes were obtained based on the interaction protein screening.The full-length gene sequences were obtained from the transcriptome of E.ulmoides,and the Eu MLP gene was successfully cloned by RT-PCR.This gene is a stress response gene in response to powdery mildew stress and abiotic stress such as high salinity and drought.The Eu MLP gene,belonging to the SRPBCC superfamily and Bet v1 family,is the length of 483 bp and encodes a total of 161 amino acids.Subcellular localization analysis shown that Eu MLP is located in the cytoplasm.The secondary structure predictions for Eu MLP indicated that it is a protein with 27.50%,43.12% β-sheet structure,and 29.38% random coil.The prediction results of the three-dimensional structure of Eu MLP found that its 2-160 amino acids have the crystal structure of the Q64 W mutant,including the main allergen Fra 1-2 functional domain.Prediction analysis of transmembrane domain and signal peptide shown that Eu MLP had no transmembrane domain and signal peptide.Phosphorylation site and glycosylation site of Eu MLP were predicted,it was found that Eu MLP has 8 Ser,6Thr and 4 Tyr which may become the phosphorylation sites of protein kinases,and there is no potential glycosylation site.The results of Eu MLP codon bias analysis shown that it had obvious codon bias within species,but a low degree of inter-synonymous codon bias.3)Cloning and bioinformatics analysis of Eu NATThe N-acetyltransferase gene was further screened on the basis of the interacting protein and successfully cloned from the E.ulmoides plant c DNA library.The gene may be involved in temperature,drought and salt stress response,and can enrich and detoxify fluorine.The full length of Eu NAT gene is 927 bp,encoding a protein of 309 amino acids.Domain analysis of Eu NAT belongs to the Rim I superfamily,and its subcellular localization analysis demonstrated that Eu NAT is located in the chloroplast.The secondary structure predictions for Eu NAT indicated that it is a protein with25.00%,19.81% β-sheet structure,and 55.19% random coil.The three-dimensional structure prediction results of Eu NAT have shown that its 111-293 amino acids have the crystal structure of NAA60 complexed with acetyl-Co A,including the acyl-Co A N-acyltransferase(NAT)superfamily protein domain.The prediction analysis of transmembrane domain and signal peptide found that Eu NAT had no transmembrane domain and signal peptide;The phosphorylation site and glycosylation site of Eu NAT were predicted,indicating that the phosphorylation sites of protein kinases for Eu NAT was detected at 28 Ser,11 Thr and 4 Tyr which may become the phosphorylation sites of protein kinases,but there is no potential glycosylation site.The results of Eu NAT codon bias analysis shown that it had obvious codon bias within species,but a low degree of inter-synonymous codon bias.4)Validation of protein interactions by Bi FCEu DIR1 was fused to the N-terminus of YFP,and Eu MLP and Eu NAT were fused to the C-terminus of YFP,respectively.There is no yellow fluorescence in the co-transformation results of p UC-SPYNE and p UC-SPYCE empty-loaded,indicating that there is no protein interaction in the empty-loaded.There is obvious yellow fluorescence in the results of p UC-SPYNE-DIR1 and p UC-SPYCE-MLP co-transformation,,indicating that the C-terminus and N-terminus of YFP are close to each other to re-excite fluorescence,and Eu MLP interacts with Eu DIR1.Similarly,co-transformation of p UC-SPYNE-DIR1 and p UC-SPYCE-NAT can also excite yellow fluorescence,proveing that Eu NAT can interact with Eu DIR1.5)Subcellular localization and gene expression analysis of Eu MLP and Eu MLPThe fusion expression of Eu MLP and GFP found that GFP was expressed in the chloroplast and in the cytoplasm,indicating that the main latex proteins were localized in the cytoplasm and chloroplast.The fusion expression of Eu NAT and GFP demonstrated that only GFP was expressed in the chloroplast,indicating that the acetyltransferase was localized in the chloroplast.The spatiotemporal expression results of Eu MLP shown that the expression of Eu MLP was the highest in leaves,followed by stems and the lowest in roots.In addition,the expression levels in stems were hardly affected by months,and the expression levels in roots increased significantly in June.Under the treatments of both high-salt and Me JA,the expression of Eu MLP was significantly increased,indicating that it responded to high-salt and Me JA stress.The spatiotemporal expression analysis shown that expression level of Eu NAT in leaves was also significantly higher than that in roots and stems.The expression level in stems was hardly affected by the month,but the expression level in roots increased with the month.Under high-salt treatment,Eu NAT expression first decreased and then increased significantly.Under Me JA treatment,Eu NAT expression increased significantly,indicating that it responded to high-salt and Me JA stress.Taken togather,this study screened two Eu DIR1-interacting proteins via yeast two-hybrid and Bi FC technology.Eu MLP and Eu NAT were successfully cloned,their subcellular localization was performed,and sequence characteristics and expression patterns of these two genes were analyzed.This provides a theoretical basis for further research on the molecular mechanism of E.ulmoides in response to stress.