Bioinformatics Analysis And Prokaryotic Expression Of Metalloproteinase Gene From Puccinia Helianthi Schw.

Puccinia helianthi Schw.is one of the important diseases of sunflower,which severely harm the quality and oil content of sunflower.Extracellular secretory proteases are an important component of extracellular pathogenic factors,which can destroy the external protective barriers of plant cells,and make for invasion and cause host disease.In this study,based on the screening of the secretory proteins from the transcriptome data of P.helianthi Schw.,the high-expression gene(KU994904)was selected for analysis.Firstly,we analyzed the gene by bioinformatics to determine its main function.It was named as metalloproteinase(mep),and secondly cloned the gene of this metalloproteinase,and constructed prokaryotic vector for expression in vitro.At the same time,its subcellular localization was preformed to determined its acting site.This search laid the foundation for future understanding of the pathogenesis of this enzyme.Main results:1.Bioinformatics analysis.This gene sequence of full-length mature protein was 1335bp,which coded 445 amino acids.Using the online software SignalP4.1 Server,TMpred,big-PI predictor and TargetP 1.1,this protease was predicted to be an extracellular secreted protein.Based on NCBI database,it had high similarity to the five sequences in the M36 family.Based on the homology modeling method using SWISS-MODEL,it was searched in the PDB(protein data bank),and found that had 53.77%similarity to the extracellular metalloproteinase.Therefore,it was inferred to be an extracellular secretory metalloproteinase.2.Prokaryotic expression.The sequence was performed by the codon optimization software MaxCodonTM Optimization Program(V13).Using the whole gene cloning method to connected to an expression vector pET30a,the prokaryotic expression vector pET30a-metalloproteinase was successfully constructed.The recombinant plasmid pET30a-metalloproteinase was transferred into the BL21 strain and recombinant protein was induced to express with 0.1 mM IPTG.The results showed that the recombinant protein could be substantively expressed at 15? and 37? for 16h,and the protein was elluted by different concentrations of imidazole.Protease existed in inclusion bodies.The protein was further purified by Ni-IDA affinity chromatography.And the concentration of protein was 0.558mg/mL with Bradford method.Finally,the protein was verified to be the target protein with Western-blot and its molecular weight was about 49.5kDa.3.Subcellular localization.We constructed the expression vector pCambia1302-eGFP by T4 ligase,and transferred the expression vector to Agrobacterium GV3101,and then transformed Agrobacterium GV3101 which carried the expression vector into Nicotiana benthamiana,and observed results of the subcellular localization.It was found that the metalloproteinase of P.helianthi Schw.localized to the barrier of the tobacco cell(cell wall or cell membrane),which indicted that it acted on the external barrier of the cell in the plant,and destroyed the external cells of the host to play pathogenic role.