关键词: 斜纹夜蛾, SlGNBP2基因, 革兰氏阴性菌结合蛋白, 病原微生物诱导, 组织表达

Abstract: 【Objective】To reveal the action pattern of the SlGNBP2 gene in the immune response of Spodoptera litura against pathogenic microorganisms.【Methods】The full-length cDNA sequence of SlGNBP2 was obtained by homologous cloning and rapid amplification of cDNA ends （RACE）. The structure and evolutionary characteristics of SlGNBP2 were analyzed using bioinformatics software. The relative mRNA expression levels of SlGNBP2 at different developmental stages,in various tissues,and after induction by pathogenic microorganisms in S. litura were detected by quantitative real-time PCR （qRT-PCR）.【Results】The cloned SlGNBP2 gene was 1 731 bp in full-length,with an open reading frame （ORF） of 1 431 bp,encoding 476 amino acids. The results of bioinformatics analysis showed that the predicted protein had a conserved glycoside hydrolase domain （Glyco_hydro_16） of the GNBPs family. The results of homology analysis showed that the conserved domain of SlGNBP2 showed high similarity to those of GNBP2 from other Lepidoptera insects,and was likely to possess similar functions. The sequence similarity between SlGNBP2 and TnGNBP2 of Trichoplusia ni was the highest,with a similarity of 56%. The phylogenetic tree indicated that SlGNBP2 was highly homologous to Lepidoptera insects such as T. ni,Manduca sexta and Plutella xylostella,and was in the same evolutionary branch. Among them,SlGNBP2 showed the closest relationship with TnGNBP2 of T. ni,and the two were clustered into one branch. The results of qRT-PCR showed that the expression of the SlGNBP2 gene was detected at different developmental stages of S. litura,and the highest relative expression level was the 4th instar larvae,which was 64.67 times higher than the relative expression level of eggs. The SlGNBP2 gene was expressed in the hemocytes,fat body,midgut,epidermis,head and malpighian tubules of the 4th instar larvae,with predominant expression in the midgut and fat body,which were up-regulated by 12.74-fold and 11.89-fold compared with the relative expression level of the head. The results of pathogenic microorganism induction showed that SlGNBP2 could be up-regulated by Gram-positive bacteria,Gram-negative bacteria and fungi. Among them,compared with the control,the expression of SlGNBP2 in fat body was up-regulated by 14.47-fold after induction by Gram-positive bacteria Bacillus thuringiensis for 24 h.【Conclusion】SlGNBP2 is most sensitive to the induction of Gram-positive bacteria B. thuringiensis,and plays an important role in pattern recognition in the process of immune defense against pathogenic microorganisms in S. litura.

Key words: Spodoptera litura, SlGNBP2 gene, Gram-negative bacteria binding protein, Induction of pathogenic microorganisms, Tissue expression