Cloning Expression And Function Of Galectin In Plutella Xylostella

Plutella xylostella is a worldwide migratory pest and a major pest harmful to cruciferous crops,which is difficult to control due to its fast reproduction and resistance to pesticides.Currently,the application of chemical pesticides in large scale has not only failed to control pests effectively,but has adversely affected crops and local ecosystems.Therefore,to find a safe and efficient biological control method has become an important direction for the control of P.xylostella.Previous studies have found that galectin,as an important component of humoral immunity in the insect immune system,participated in the process of innate immunity in the organism.In this experiment,we cloned two galectin genes PxGals based on P.xylostella database,analyzed their physical and chemical properties,and studied its physiological functions through in vitro recombinant protein technology;further clarified its expression patterns under different pathogenic conditions,elucidated preliminarily the immune function of the galectin protein,which would lay the foundation for the further functional study of the protein and evaluation its potential as biological control targets.The main results are summaried as follows:1.Two PxGal genes（PxGal011187,PxGal015997）were cloned based on the P.xylostella genome database.Bioinformatics analysis proved that the open reading frame（ORF）of PxGal011187 is 1017 bp,encoding a total of 338 amino acids;the ORF of PxGal015997 is 648 bp,co-coding 215 amino acids,both of which do not have signal peptides and transmembrane structures,belong to an atypical secreted protein,and each has a highly conserved galectin domain.Phylogenetic tree analysis showed that it is aggregated in the same branch with other lepidopteran insects.2.Two types of expression vectors,p ET-PxGal and p RB-PxGal,were constructed based on the prokaryotic expression system.p ET22 b,24a,28 a,29a,32 a,28a SUMO in the p ET series and p RB1 K in the p RB series were used for the construction of expression vectors.In the p ET series,only bactiria containing p ET28a-PxGal015997 were induced to express the protein successfully.But the recombinant protein was expressed in the form of inclusion bodies and did not have agglutination activity after denaturation analysis.Bacteria with the weak promoter vector p RB1K-PxGal015997 were induced successfully to express soluble protein,which was verified as the target protein by SDS-PAGE and Western-blotting.The recombinant PxGal015997 protein was extracted and purified by a nickel column for subsequent functional studies.3.The purified PxGal protein p RB-PxGal015997 was subjected to in vitro functional experiments.The results of antibacterial experiments showed that p RB-PxGal015997 protein had no obvious inhibitory effect on bacteria.The bacteria in the control group were evenly distributed on the plate and existed as a single colony.However,due to the presence of PRB-PxGal015997 protein in the experimental group,the bacterial cells were aggregated by PRB-PxGal015997 protein during the growth process,resulting in bacterial adhesion in the plate,which was dose-dependent – the higher the concentration,the higher the degree of bacterial adhesion.The result indicated that PRB-PxGal015997 protein did not have antibacterial effect to reduce the growth of bacteria,but it had certain agglutination effect on bacteria.Bacterial binding and sugar suppression experiments show that PRB-PxGal015997 protein has different strengths of binding to different types of bacteria,and can inhibit different carbohydrates（sucrose,lactose,galactose,maltose,fructose,glucose,xylose）.Later,the ability to bind bacteria decreased,indicating that different carbohydrates would bind to PRB-PxGal015997 protein to inhibit the binding effect of PRB-PxGal015997 protein to bacteria.In order to further visualize the function of the PxGal015997 protein,the green fluorescent protein（GFP）and PxGal015997 were fused and expressed,and the fusion protein was analyzed by bacterial agglutination experiments.The results showed that PRB-PxGal015997 protein has agglutination effect on bacteria under a fluorescence microscope,which is consistent with the results of the plate antibacterial experiment,and the PRB-PxGal015997 protein has stronger agglutinating ability for Gram-positive bacteria than Gram-negative bacteria.It is speculated that the binding ability of PRB-PxGal015997 protein to the bacterial cell wall component peptidoglycan is stronger than that of the binding capacity of lipopolysaccharide.4.Gene expression analysis found that PxGal015997 is expressed at different developmental stages of P.xylostella,and its expression is highest in the 3rd instar larvae;The 4th instar larva accounts for about 80% of the total food intake,which is the main hazard to cruciferous crops.Bacillus thuringiensis（Bt）,Serratia marcescens（Sm）and fungal Pichia pastoris（sp）were used to treat the fourth instar larvae of P.xylostella.The results showed that the expression of PxGal015997 decreased significantly after stimulation with B.thuringiensis and S.marcescens,while the expression of PxGal015997 increased significantly after P.pastoris stimulation and then tended to be flat.It is speculated that PxGal015997 may be involved in the immune defense against the invasion of pathogenic microorganisms,but the specific immune mechanism still needs to be further studied in the future.P.xylostella has strong resistance to pesticides and rapid reproductive ability.Ordinary chemical control leads to increased resistance to pesticides,and the use of a large number of chemical pesticides intensifies environmental pollution.This article reveals its innate immune factor galectin of the diamondback moth.The interaction and function with bacteria provide a theoretical basis for the subsequent in-depth study of this gene and the excavation of this gene as a target for biological control of P.xylostella.