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Title: CHARACTERIZATION OF CDNAS ENCODING THREE TRYPSIN-LIKE PROTEINASES AND QUANTITATIVE ANALYSIS OF MRNA IN BT-RESISTANT AND -SUSCEPTIBLE STRAINS OF OSTRINIA NUBILALIS

Author
item LI, HUARONG - KANSAS STATE UNIV
item Oppert, Brenda
item HIGGINS, RANDALL - KANSAS STATE UNIV
item HUANG, FANGNENG - LOUISIANA STATE UNIV
item BUSCHMAN, LAWRENT - KANSAS STATE UNIV
item GAO, JIAN-RONG - CORNELL UNIV
item ZHU, KUN - KANSAS STATE UNIV

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/20/2005
Publication Date: 8/15/2005
Citation: Li, H., Oppert, B.S., Higgins, R.A., Huang, F., Buschman, L.L., Gao, J., Zhu, K.Y. 2005. Characterization of cDNAs encoding three trypsin-like proteinases and quantitative analysis of mRNA in Bt-resistant and -susceptible strains of Ostrinia Nubilalis. Insect Biochemistry and Molecular Biology 35: 847-860.

Interpretive Summary: Transgenic corn with genes encoding Bacillus thuringiensis (Bt) toxins is being planted to control the European corn borer, but Bt-resistant corn borer strains developed in the laboratory indicate that resistance can threaten the efficacy of Bt corn. We demonstrated that when Bt protoxins were activated, Bt-resistant corn borers were no longer resistant to the toxins. This was apparently due to a loss of proteinase activity in the Bt-resistant borer larvae. Therefore, proteinase genes were cloned and expression was compared in Bt-susceptible and -resistant corn borer larvae. We demonstrated that one of the genes was expressed at lower levels in the resistant larvae and could explain the loss of proteinase activity in these larvae. This information is useful in the development of effective resistance management strategies to Bt in the European corn borer.

Technical Abstract: Our previous results on resistance mechanisms in a Bacillus thuringiensis (Bt)-resistant strain of European corn borer (Ostrinia nubilalis) suggested that resistance was primarily due to reduced trypsin-like proteinase activity. In this study, we demonstrate that the Bt-resistant strain of O. nubilalis was 254-fold resistant to Cry1Ab protoxin and 12-fold to trypsin-activated Cry1Ab toxin, providing more evidence of proteinase-mediated resistance in this strain. To understand the molecular basis of reduced proteinase activity, three cDNAs, OnT2, OnT23, and OnT25, encoding full-length trypsin-like proteinases, were sequenced and their gene expression levels were compared in the Bt-resistant and -susceptible strains. Although a number of nucleotide differences were found in the three cDNA sequences of Bt-resistant and -susceptible strains, the differences were not consistent with reduced trypsin-like activity in Bt-resistant O. nubilalis. However, Northern blot and real-time quantitative PCR analyses revealed that the mRNA levels of OnT23 were reduced by 2.7- and 3.8-fold in the Bt-resistant strain relative to the Bt-susceptible strain, respectively. Reduced trypsin-like activity may be attributed to reduced expression of at least one trypsin-like gene, OnT23, in Bt-resistant O. nubilalis.