关键词: 大豆蚜, 丝氨酸蛋白酶抑制剂, RNA干扰, 酶活性, 蜡蚧刺束梗孢菌

Abstract:

 【Aim】 The objective of this study is to explore the role of Kazal-type serine protease inhibitor KaSPI in the development, digestion, and immune defense processes of the soybean aphid, Aphis glycines. 【Methods】 The cDNA sequence of Kazal-type serine protease inhibitor gene from A. glycines was cloned by PCR based on the transcriptome data of A. glycines. qRT-PCR was used to detect the expression levels of AgKaSPI in the 1st-4thinstar nymphs and adults of A. glycines, and in A. glycines adults at 3, 6, 12, 24, 48 and 72 h after infection by Akanthomyces lecanii. After RNAi of AgKaSPI with siRNA for 3, 6, 12, 24 and 48 h, the RNAi efficiency was detected by qRT-PCR, and the mortality and fecundity of A. glycines adults at 12, 24, 48 and 96 h post RNAi were recorded. The AgKaSPI content and the activities of serine protease, trypsin and chymotrypsin in A. glycines adults were detected by double antibody sandwich method after infection by A. lecanii and RNAi of AgKaSPI. The mortality of A. glycines adults at 12, 24, 48 and 96 h after infection by A. lecanii following RNAi of AgKaSPI for 6 h was observed and recorded. 【Results】 The cDNA sequence of a serine protease inhibitor gene AgKaSPI (GenBank accession no. MK440557) was cloned from A. glycines. AgKaSPI is 1 019 bp in length, with the open reading frame of 324 bp, encoding 107 amino acids. AgKaSPI has the Kazal domain, with the molecular weight of 11.43 kD and isoelectric point of 9.24. AgKaSPI was expressed at different developmental stages of A. glycines. At 24 h after infection by A. lecanii, the expression level of AgKaSPI and the corresponding AgKaSPI content in A. glycines adults were significantly up-regulated, being 4.31-fold and 1.69-fold as high as those of the control group, respectively, while the activities of serine protease, trypsin and chymotrypsin were inhibited. At 6 h after RNAi of AgKaSPI, the expression level of AgKaSPI in A. glycines adults decreased by 71.05%. The KaSPI content decreased by 51.11% and the activities of serine protease and chymotrypsin increased significantly at 12 h after RNAi of AgKaSPI. The fecundity per one hundred individuals decreased by 49 born aphids and the mortality of A. glycines adults increased by 10.12% at 96 h after RNAi of AgKaSPI. 【Conclusion】 AgKaSPI is expressed at different developmental stages of A. glycines, and the expression level of AgKaSPI is significantly up-regulated at 24 h after infection by A. lecanii. AgKaSPI may participate in the immune response of A. glycines to A. lecanii infection by regulating the serine protease activity.

Key words: Aphis glycines, serine protease inhibitor, RNA interference, enzyme activity, Akanthomyces lecanii