Sequence And Expression Analysis Of Beta-1,3-glucanase Related Genes In Antheraea Pernyi

Insect ?-1,3-glucanase family,include ?-1,3-glucanase,?-1,3-glucan recognition protein and gram negative bacteria-binding protein,play an important role in insect immunity.In order to study the phylogenetic relationship of ?-1,3-glucanase gene family in Antheraea pernyi,bioinformatics analysis was carried out in the scope of Bombycoidea and Lepidoptera.For study the adaptive evolution of insect ?-1,3-glucanase and chitinase genes in aspect of codon usage,multivariate statistical methods were used to analyze the codon usage patterns.In order to study the immune functions of ?-1,3-glucanase and ?-1,3-glucan recognition protein genes in Antheraea pernyi,the expression profiles were analyzed by semi-quantitative method,and the microbial stressed expression profiles were analyzed by real-time quantitative method.The coding sequences of ?-1,3-glucanase,?-1,3-glucan recognition protein and gram negative bacteria-binding protein genes contain signal peptide sequence and domain of Glycosylhydrolase family 16,but not contain transmembrane region.For the four Bombycoidea insects,Anthereaea pernyi ?-1,3-glucanase gene and Manduca sexta ?-1,3-glucanase gene,Anthereaeapernyi 1,3-glucanase recognition protein gene and Bombyxmori ?-1,3-glucan recognition protein gene,Anthereaea pernyi gram negative bacteria-binding protein gene and Manduca sexta gram negative bacteria-binding protein gene had the highest sequence identity and similarity.The phylogenetic analysis using amino acid sequence of the three proteins of the four Bombycoidea insects showed that the clade of ?-1,3-glucanase stranded alone,and the clades of ?-1,3-glucan recognition protein and gram negative bacteria-binding protein formed a larger clades.In codon usage patterns,there were no significant differences in GC and GC3 s content and the ENc value between insect ?-1,3-glucanase and chitinase genes.However,the GC and GC3 s content of insect ?-1,3-glucanase and chitinase genes were significantly lower than those of Salmonella glucanase and chitinase genes,and the ENc values of insect ?-1,3-glucanase and chitinase genes were significantly higher than those of Salmonella glucanase and chitinase genes.The codon bias of insect ?-1,3-glucanase and chitinase genes was weaker than that of Salmonella glucanase and chitinase genes.The codon usage similarity between genes within species was higher than that between homologous genes between species.The codon usage of insect ?-1,3-glucanase and chitinase genes were affected mainly by selection.Expression profile of Anthereaea pernyi showed that ?-1,3-glucanase gene expressed the highest level in the fifth instar larva and expressed the second level in pupa.No expression was detected in eggs,newly hatched larva,the third instar larva and adult.?-1,3-glucan recognition protein gene expressed the highest level in pupa and adult and expressed the second level in newly hatched larva.The expression was relatively low in eggs and in the fifth instar larva,and no expression was detected in the third instar larva.Beta-1,3-glucanase gene expressed the highest level in midgut of the fifth instar larva of Anthereaea pernyi and expressed the second level in brain and fat body.No expression was detected in hemolymph,body wall,silk gland,malpighian tubule and testis.Beta-1,3-glucan recognition protein gene expressed the highest level in the body wall and testis of 5th instar larva.However,the expression levels in midgut,hemolymph,malpighian tubule and brain were low and the expression in silk gland and fat body was hardly detectable.When induced by five microorganisms(Nosema pernyi,Anthereaea pernyi nucleopolyhedrovirus,Escherichia coli,Enterococcus pernyi,and Beauveria bassiana),the expression of ?-1,3-glucanase and ?-1,3-glucan recognition protein genes changed(up or down).Beauveria bassiana induced the highest expression increase of ?-1,3-glucan recognition protein gene in Anthereaea pernyi.