Trypsinogen-like cDNAs and quantitative analysis of mRNA levels from the Indianmeal moth, Plodia interpunctella

Two cDNA fragments encoding full-length trypsinogen-like proteins were clon ed from larvae of two strains (RC688s and HD198r) of the Indianmeal moth, P lodia interpunctella (Hubner), which differed in their sensitivity to Bacil lus thuringiensis protoxins. One cDNA fragment contained 874 nucleotides, i ncluding a 780-nucleotide open reading frame that encoded a trypsinogen-lik e protein (PiT2b). Another cDNA fragment amplified from both P. interpuncte lla strains contained 864 nucleotides including a 780 bp open reading frame encoding a second trypsinogen-like protein (PiT2c). The cDNA sequence of P iT2b shared 89% sequence identity with PiT2a, a trypsinogen-like protein cl oned previously from this species. The cDNA sequences of PiT2a and PiT2c sh ared 83% identity. The cDNA sequence identity between PiT2b and PiT2c was 8 0%. The cDNA for PiT2b from strain RC688s was different at six nucleotide p ositions from that of PiT2b from strain HD198r. Five nucleotide replacement s occurred in the open reading frame leading to amino acid changes at all f ive positions. There were five nucleotide differences in the cDNAs for PiT2 c trypsinogen-like proteins from the two strains. Two nucleotide substituti ons in the open reading frame resulted in replacements of two amino acid re sidues in the deduced protein sequences. Amino acid sequences for PiT2a and PiT2b shared 84% identity, but only 50% identity was observed between PiT2 c and the other two trypsinogen-like proteins. The deduced amino acid seque nces for PiT2b and PiT2c included both signal and zymogen activation peptid es and amino acid sequence motifs which are conserved in seven homologous t rypsinogen-like proteins from other insects. Typical features of the putati ve trypsinogen-like proteins from P. interpunctella included the serine pro teinase active site triad (His(81), Asp(133), and Ser(233)), three pairs of cysteine residues for disulfide bridges, and three residues, Asp(227), Gly (250), and Gly(260): that help to confer trypsin-like specificity to the en zymes. Quantitative RT-PCR analyses showed that, in fourth instar larvae, R C688s had 1.6-fold higher PiT2a trypsinogen-like mRNA than did HD198r. Expr ession of PiT2b mRNA was 3.4-fold higher in HD198r than in RC688s. Expressi on of PiT2c mRNA was 2.8-fold higher in RC688s than in HD198r. Mean accumul ation levels of mRNAs for all three trypsinogen-like proteins were slightly higher in RC688s than in HD198r based on total RNA, and 1.3-fold higher in RC688s than in HD198r based on wet weight of larval body tissues. Publishe d by Elsevier Science Ltd.