关键词: 菜粉蝶, 丝氨酸蛋白酶, 先天免疫, 基因克隆, 表达谱

Abstract: Insects mainly rely on innate immune responses to resist foreign invasion. Activation of the serine proteinase cascades, involved in hemolymph melanization and antimicrobial peptide synthesis, plays an important role. To define the role of serine proteinases in the innate immune responses of Pieris rapae, a cDNA fragment of a serine proteinase gene, Pr-SP1 screened by RT-PCR using degenerate primer, was sorted out in this article. The full-length cDNA of Pr-SP1 was cloned using RACE strategy. The cDNA length was found to be 1 489 bp, including a 1 059 bp open reading frame, which encodes 353 amino acids with a 20-amino-acid signal peptide. The predicted protein molecular mass and pI are 36.85 kDa and 6.41, respectively. Multiple sequence alignment result revealed that Pr-SP1 shares high identities with its homologs in other insect species, which also possesses a clip and active catalytic domain at N-terminus and C-terminus, respectively. Real-time quantitative PCR and Western blotting results indicated that Pr-SP1 was primarily transcribed in granulocytes in the pupal stage and the protein product of Pr-SP1 was detected mainly in plasma. Pr-SP1 was transcribed and its protein product expressed in different developmental stages and instars. The highest and lowest transcript and expression levels appeared in 5th instar larvae and egg stages, respectively. The transcript level of Pr-SP1 and expression level of Pr-SP1’s protein product could be induced by Escherichia coli, Micrococcus luteus and Pichia pastoris. According to these results, Pr-SP1 could be considered as a candidate of Spätzle processing enzyme, which may participate in the innate immune responses in P. rapae.

Key words: Pieris rapae, serine proteases, innate immunity, gene cloning, expression profile