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Molecular cloning and sequence analysis of novel cytochrome P450 cDNA fragments from Dastarcus helophoroides.

Wang HD, Li FF, He C, Cui J, Song W, Li ML - J. Insect Sci. (2014)

Bottom Line: In our study, for the better understanding of P450 enzymes in D. helophoroides, 100 novel cDNA fragments encoding cytochrome P450 were amplified from the total RNA of adult D. helophoroides abdomens using five pairs of degenerate primers designed according to the conserved amino acid sequences of the CYP6 family genes in insects through RT-PCR.Ninety-six fragments were determined to represent CYP6 genes, mainly from CYP6BK, CYP6BQ, and CYP6BR subfamilies, and four fragments were determined to represent CYP9 genes.Twenty-two fragments, submitted to GenBank, were selected for further homologous analysis, which revealed that some fragments of different sizes might be parts of the same P450 gene.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Forestry Pests Biological Control, College of Forestry, Northwest A&F University, Yangling, Shaanxi, 712100, P. R China These authors contributed equally to this work wanghd2826@gmail.com.

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Deduced amino acid alignments of products E and G. High quality figures are available online.
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f03_01: Deduced amino acid alignments of products E and G. High quality figures are available online.

Mentions: The deduced amino acid identity of the 22 sequences varied from 38.4 to 100% (Table 5). Sequences from the same contig shared a high amino acid identity, such as A3 and A5, F2 and F6, H1 and H2, and E2 and E8 (Table 5). According to Figure 2 and Figure 3, the heme-binding sequence motif FxxGxxxCxG (residue 77–86; 135–140) that is universal among P450 enzymes was found in all the 22 deduced amino acid sequences. As is shown in Figure 2, with the exception of A1, A3, A5, and A6, the primer corresponding motif ETLRKYPP (residue 2–9) was found in the upstream of the other 11 deduced amino sequences. Moreover, the characteristic P450 motif PERF (residue 58–61) was found in all the other 12 deduced amino acid sequences except H3 and F6. In addition, compared with the other 13 deduced amino acid sequences, A6 had a particular sequence PNEKP (residue 28–32), which may be one of the features to distinguish CYP6 and CYP9 families. Judging from Figure 3, the primer corresponding motif AG(Y/F)ETSST (residue 1–8) was found in all eight deduced amino acid sequences. Furthermore, the particular P450 sequence motifs ExxR (residue 59–62) and PERF (residue 115–118) were also found.


Molecular cloning and sequence analysis of novel cytochrome P450 cDNA fragments from Dastarcus helophoroides.

Wang HD, Li FF, He C, Cui J, Song W, Li ML - J. Insect Sci. (2014)

Deduced amino acid alignments of products E and G. High quality figures are available online.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206240&req=5

f03_01: Deduced amino acid alignments of products E and G. High quality figures are available online.
Mentions: The deduced amino acid identity of the 22 sequences varied from 38.4 to 100% (Table 5). Sequences from the same contig shared a high amino acid identity, such as A3 and A5, F2 and F6, H1 and H2, and E2 and E8 (Table 5). According to Figure 2 and Figure 3, the heme-binding sequence motif FxxGxxxCxG (residue 77–86; 135–140) that is universal among P450 enzymes was found in all the 22 deduced amino acid sequences. As is shown in Figure 2, with the exception of A1, A3, A5, and A6, the primer corresponding motif ETLRKYPP (residue 2–9) was found in the upstream of the other 11 deduced amino sequences. Moreover, the characteristic P450 motif PERF (residue 58–61) was found in all the other 12 deduced amino acid sequences except H3 and F6. In addition, compared with the other 13 deduced amino acid sequences, A6 had a particular sequence PNEKP (residue 28–32), which may be one of the features to distinguish CYP6 and CYP9 families. Judging from Figure 3, the primer corresponding motif AG(Y/F)ETSST (residue 1–8) was found in all eight deduced amino acid sequences. Furthermore, the particular P450 sequence motifs ExxR (residue 59–62) and PERF (residue 115–118) were also found.

Bottom Line: In our study, for the better understanding of P450 enzymes in D. helophoroides, 100 novel cDNA fragments encoding cytochrome P450 were amplified from the total RNA of adult D. helophoroides abdomens using five pairs of degenerate primers designed according to the conserved amino acid sequences of the CYP6 family genes in insects through RT-PCR.Ninety-six fragments were determined to represent CYP6 genes, mainly from CYP6BK, CYP6BQ, and CYP6BR subfamilies, and four fragments were determined to represent CYP9 genes.Twenty-two fragments, submitted to GenBank, were selected for further homologous analysis, which revealed that some fragments of different sizes might be parts of the same P450 gene.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Forestry Pests Biological Control, College of Forestry, Northwest A&F University, Yangling, Shaanxi, 712100, P. R China These authors contributed equally to this work wanghd2826@gmail.com.

Show MeSH