Study On C-type Lectin ApCTL4-X1 Gene Expression And Its Function In Antheraea Pernyi

C-type lectin is one of the pattern recognition receptors in the innate immune defense system of insects to recognize pathogenic microorganisms,and has various functions such as participating in the recognition and clearance of bacteria,regulating blood encapsulation and blackening.Along with the development of Antheraea pernyi industry,at present,a series of health problems caused by the invasion of pathogenic microorganisms appear,which seriously affects the yield of Antheraea pernyi.Mining the key factors in Antheraea pernyi in response to pathogenic microbial stress can provide reliable data to support the study of immune defense mechanism of Antheraea pernyi.In this study,an immune-related factor,C-type lectin,named ApCTL4-X1(Gen Bank accession number: MZ851153),was firstly screened from the Antheraea pernyi transcriptome database,and the cDNA sequence of ApCTL4-X1 was found to be composed of 669 The cDNA sequence of Antheraea pernyi ApCTL4-X1 consisted of 669 bases and encoded 228 amino acids.The online website predicted the protein properties and found that lectin ApCTL4-X1 is a secreted protein containing a signal peptide with hydrophilic and no transmembrane region.Real-time quantitative PCR detected that ApCTL4-X1 gene was much more expressed in the body wall of wild Antheraea pernyi pupae than in the hemolymph,fat body and genitalia.After stimulation of Quercus nymphs by different pathogenic microorganisms,it was found that in the fat body of Quercus nymphs,the stimulation of E.coli led to a large expression of ApCTL4-X1 gene,which was much more up-regulated than the stimulation of other tissues of Quercus nymphs by other pathogenic microorganisms,indicating that the lectin ApCTL4-X1 responded rapidly and precisely immunized against the invasion of E.coli in the fat body.The lectin ApCTL4-X1 was prokaryotic expressed and purified by constructing the p ET28a-ApCTL4-X1 plasmid.The results of agglutination assay showed that agglutinin ApCTL4-X1 is a calcium ion-dependent protein,which can agglutinate Staphylococcus aureus 、 Micrococcus lysodeikticus 、 Bacillus cereus 、 Candida albicans 、 Pseudomonas aeruginosa、Salmonella Enteritidis、Azotobacter sp.、Escherichia coli and blood cells,and has the best agglutination effect on Bacillus cereus and Salmonella Enteritidis.The results of the inhibition experiments showed that the agglutinin ApCTL4-X1 inhibited Staphylococcus aureus 、 Pseudomonas aeruginosa 、 Salmonella Enteritidis 、 Bacillus methylotrophicus 、Escherichia coli,,and had the most obvious inhibition effect on Escherichia coli.Using ELISA enzyme-linked immunosorbent assay and sugar competitive inhibition agglutination assay,it was detected that lectin ApCTL4-X1 could bind to peptidoglycan,inulin,lipopolysaccharide and D-galactose,among which it had the strongest binding ability to D-galactose.The addition of lectin ApCTL4-X1 to Antheraea pernyi hemolymph may lead to the activation of phenol precursor oxidase,resulting in an accelerated rate of hemolymph blackening,and when ApCTL4-X1 lectin bound to PGN and D-galactose,the rate of hemolymph blackening also increased.In conclusion,the sericulture lectin ApCTL4-X1,as a pattern recognition receptor,plays an indispensable role in the immune defense of sericulture.The results of the study provide some theoretical support for an in-depth understanding of the intrinsic immune defense mechanism of sericulture and new ideas for the protection of sericulture and the development of new pest control strategies.