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RNA Interference of Chitinase Gene in Spodoptera litura

담배거세미나방(Spodoptera litura) Chitinase gene의 RNA interference

  • Jeon, Mi Jin (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Seo, Mi Ja (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Youn, Young Nam (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University) ;
  • Yu, Yong Man (Department of Applied Biology, College of Agriculture and Life Sciences, Chungnam National University)
  • 전미진 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 서미자 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 윤영남 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 유용만 (충남대학교 농업생명과학대학 응용생물학과)
  • Received : 2014.09.01
  • Accepted : 2014.09.23
  • Published : 2014.09.30

Abstract

RNA interference (RNAi) is the method which controls phenotypes of gene in live cells. Chitinase is the enzyme helping digestion and absorption of old cuticles during the ecdysis of insects. In order to investigate molting-inhibition effect with the chitinase related gene in Spodoptera litura, RNA was extracted from the $5^{th}$ instars. cDNA was synthesized and then we obtained about 700 bp size chitinase. After PCR products were cloned into a pGEM T-easy vector, colonies were picked. DNA was extracted from the colony cultures. EcoR I enzyme was used to check whether PCR products were inserted or not. And then we confirmed vector band of about 3 kb and insert band of about 700 bp. To synthesize the dsRNA, each DNA was cut with Spe I and Nco I enzymes (Circular DNA became lineared DNA). After synthesis of dsRNA, approximately 5 ul dsRNA was injected into the $3^{rd}$ abdominal segment of S. litura $4^{th}$ larvae. The concentration of dsRNA was about $10{\mu}g/{\mu}l$. We confirmed larval-larval molting : there were phenotypically abnormal individuals - for instance malformation, molting inhibition and change of integument color. Pupaadult molting : there were phenotypically abnormal individuals - for instance molting inhibition, change of wings and malformation. Also we could investigate the pupation, emergence and variation about noninjection, treated with DW and dsRNA. Each pupation was non-injection 83.3%, DW 78.3% and dsRNA 66.7%. Each emergence was non-injection 90.0%, DW 72.3% and dsRNA 65.0%. So we considered that chitinase dsRNA induced molting inhibition effect. But each variation was non-injection 8.9%, DW 2.9% and dsRNA 19.2%. Therefore dsRNA group showed the highest variation value. When 18 hours after injecting dsRNA, we could obtain abnormal individual.

Acknowledgement

Grant : 담배가루이 제어용 RNAi 발현 토마토 유묘 개발 및 대량 생산 기술 확립

Supported by : 농림수산식품기술기획평가원

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