RNA interference mediated serine protease gene (Spbtry1) knockdown affects growth and mortality in the soybean pod borer (Lepidoptera: Olethreutidae)

  • Fan Li Meng Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
  • Rui Xue Ran 1Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China;
  • Yang Li Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
  • Na Li Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
  • Han Zhe Li Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
  • Zhi Kun Wang Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
  • Wen Bin Li Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China Northeast Agricultural University, Key Laboratory of Biology and Genetics and Breeding for Soybean in Northeast China, Harbin 150030, China
Keywords: Leguminivora glycinivorella, gene expression, RNAi, larval development,

Abstract

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.

 

Resumen

El barrenador de la vaina de la soja, Leguminivora glycinivorella Matsumura (Lepidoptera: Tortricidae), es una plaga económicamente significativa de la soja en el noreste de Asia. Las serinas proteasas son enzimas cruciales responsables de la digestión de proteínas en los lepidópteros herbívoros. En este estudio, se clonó un gen (Spbtry1) que codifica una serina proteasa del intestino medio del organismo del barrenador de vaina de la soja. El marco de lectura abierto Spbtry1 codificó una proteína de 269 aminoácidos con una masa molecular predicha de aproximadamente 29 kDa. La alineación de Spbtry1 con tripsinas y quimotripsinas de otros insectos reveló un alto grado de conservación en la región del dominio catalítico putativo. El análisis por la reacción en cadena de la polimerasa de la transcriptasa inversa indicó que Spbtry1 se expresó específicamente en el intestino medio, su transcripción existió constitutivamente en la fase larvaria, y su expresión fue más alta en la fase larvaria del 3er instar. La interferencia de ARN indicó que los niveles de expresión de Spbtry1 disminuyeron en dietas que contenían ARN bicatenario Spbtry1 (dsRNA). Las larvas tuvieron significativamente menor peso corporal (18,7 ± 0,25 mg en el grupo alimentado con dsRNA de Spbtry1frente a 30,1 ± 0,78 mg y 29,9 ± 0,88 mg en los grupos alimentados con dsRNA de PBS y GFP, respectivamente (prueba t de Student, P <0,01) y una mayor mortalidad (43,8%) que los grupos control (20,8% en PBS tratados y 19,5% en GFP dsRNA tratados) después de 15 d, lo que sugiere que Spbtry1es importante para la vaina de la soja barrenador de larvas de crecimiento y desarrollo.

 

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Author Biographies

Fan Li Meng, Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural University
Rui Xue Ran, 1Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China;
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural University
Yang Li, Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural Universit
Na Li, Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural Universit
Han Zhe Li, Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural University
Zhi Kun Wang, Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural University
Wen Bin Li, Northeast Agricultural University, Key Laboratory of Soybean Biology in Chinese Ministry of Education, Harbin 150030, China Northeast Agricultural University, Key Laboratory of Biology and Genetics and Breeding for Soybean in Northeast China, Harbin 150030, China
Key Laboratory of Soybean Biology in Chinese Ministry of Education, Northeast Agricultural University
Published
2017-10-10
Section
Research Papers