Preparation And Control Effect Of Plant Immune Proteins From Lonsdalea Quercina Subsp. Populi

Lonsdalea quercina subsp.populi is a major branch disease of populus×euramericana that caused serious disease in recent years,which serious threat to the health and sustainable development of poplar forests in China.Harpin proteins are a class of proteins with elicitor function secreted by the Gram-negative type III secretion system,which can stimulates multiple signal pathways in plants,induces plants to generate defense responses,improves plant disease resistance and stress resistance,promotes plant growth and development,and has broad application prospects.The purpose of this study was to induce the expression of harpin protein and compare the control effects of different proteins on Lonsdalea quercina subsp.populi,in order to screen out harpin proteins with excellent control effects,and provide a theoretical basis for the application of plant immune protein.The main findings are as follows:1.Acquisition of Harpin encoding genes and analysis of their physicochemical properties:Design specific primers based on the gene sequence to amplify Hrp W,Hrp W₂,Hrp N,and Hrp N₂ from the highly pathogenic strain N-5-1 and the attenuated strain 15572.According to bioinformatics analysis,the harpin proteins encoded by the four genes are rich in glycine and serine,and do not contain cysteine.They are stable and hydrophilic proteins.Analysis of the signal peptide and transmembrane structure of the harpin protein revealed that no signal peptide or transmembrane region existed in any of the four proteins.2.Acquisition of Harpin proteins:Prokaryotic expression vectors p ET-Hrp W,p ET-Hrp W₂,p ET-Hrp N,and p ET-Hrp N₂ were successfully constructed for four genes.After BL21chemically competent cells were transformed by heat shock,IPTG induced the expression of recombinant strains to obtain recombinant proteins.After SDS-PAGE gel electrophoresis and Western Blot detection,bands appeared at 46.8k Da,46.8k Da,33.3k Da,and 33.3k Da.The size of the obtained recombinant protein was consistent with the predicted protein size.Induced expression of recombinant harpin protein in tobacco leaves was tested for protein activity.The results showed that all four proteins could induce tobacco allergic reactions,but the degree of allergic reactions was different.The allergic reactions caused by the Hrp W treatment group were the most obvious.3.Screening agent selection:Three types of spreading agents are used:orange peel essential oil,high-performance spreading agent,and Silwet L-77 silicone surfactant.The spreading effect,the relative spreading area,and the drying time are three indicators of spreading effect.By comparison,the results show that Silwet L-77 silicone surfactant has the best effect,the average diffusion area is 5.5 times that of the control group,and the average drying time is shortened by 16 minutes.4.Comparison of Harpin protein’s control effect:4 aqueous solutions of the target protein were mixed with the spreading agent,and sprayed on 107 branches and leaves.Observe the plant changes and evaluate the safety of the protein.The results are not significantly different from the control group,and neither will cause plant disease.Then the pathogenic bacteria were inoculated by stabbing,and the spraying effects of the four proteins were compared from three aspects:bacterial content,diseased area,and relative control effect.The results of the determination of the bacteria content showed that the bacteria content of the four treatment groups was significantly lower than that of the control group,among which the Hp W treatment group had the lowest bacteria content and the Hrp N treatment group had the highest bacteria content.The measurement of the lesion area showed that the average lesion area of the Hrp N-treated group was the largest,and the average lesion area of the Hrp W-treated group was the smallest.This is consistent with the determination of the bacteria content,indicating that the plants can effectively inhibit the infection of pathogenic bacteria after spraying Hrp W.Calculate the disease index of branches and calculate the relative control effect.Finally,Hrp W and Hrp N₂ two protein elicitors were selected as immunostimulants.After spraying with the spreading agent,the relative control effects can reach 41.2%and 36.2%,which can significantly enhance effect of Populus×euramericana cv.‘74/76’on prevention and treatment of Lonsdalea quercina subsp.populi.