RNA Interference Mediated Serine Protease Gene (Spbtry1) Knockdown Affects Growth and Mortality in the Soybean Pod Borer (Lepidoptera: Olethreutidae)

Abstract

The soybean pod borer, Leguminivora glycinivorella Matsumura (Lepidoptera: Tortricidae), is an economically significant soybean pest in northeastern Asia. Serine proteases are crucial enzymes responsible for protein digestion in herbivorous lepidopterans. In this study, a gene (Spbtry1) encoding a soybean pod borer serine protease was cloned from the organism's midgut. The Spbtry1 open reading frame encoded a 269 amino acid protein with a predicted molecular mass of approximately 29 kDa. Alignment of Spbtry1 with trypsins and chymotrypsins from other insects revealed a high degree of conservation in the putative catalytic domain region. Analysis by reverse transcriptase polymerase chain reaction indicated that Spbtry1 was specifically expressed in the midgut, its transcript existed constitutively in the larval stage, and its expression was highest in the 3rd instar larval stage. RNA interference indicated that Spbtry1 expression levels decreased on diets containing Spbtry1 double-stranded RNA (dsRNA). Larvae had significantly lower body weight (18.7 ± 0.25 mg in the Spbtry1 dsRNA-fed group versus 30.1 ± 0.78 mg and 29.9 ± 0.88 mg in the phosphate buffered saline (PBS) and green fluorescent protein (GFP) dsRNA-fed groups, respectively (Student's t-test, P < 0.01) and higher mortality (43.8%) than the control groups (20.8% in PBS-treated and 19.5% in GFP dsRNA-treated) after 15 d, suggesting that Spbtry1 is important for soybean pod borer larval growth and development.