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Genomic organization and developmental expression of glutathione S-transferase genes of the diamondback moth, Plutella xylostella.

Sonoda S, Ashfaq M, Tsumuki H - J. Insect Sci. (2006)

Bottom Line: Based on amino acid identity with GST genes from other insects, this gene was classified as a member of the Sigma class.Southern blot analysis showed that PxGSTs was a single copy gene, whereas there were homologous members of the PxGSTe gene in the P. xylostella genome.RNA gel blot analysis indicated that the expression levels of both genes changed with the developmental stage of P. xylostella.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan. sonodas@rib.okayama-u.ac.jp

ABSTRACT
In the present study, we cloned and sequenced the entire coding regions of two glutathione S-transferase (GST) genes encoding PxGSTs and PxGSTe from the diamondback moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae), along with their respective 5' and 3' flanking regions. The PxGSTs gene was composed of four exons and three introns. Based on amino acid identity with GST genes from other insects, this gene was classified as a member of the Sigma class. The gene encoding PxGSTe had an intron in the 5' flanking region. Southern blot analysis showed that PxGSTs was a single copy gene, whereas there were homologous members of the PxGSTe gene in the P. xylostella genome. RNA gel blot analysis indicated that the expression levels of both genes changed with the developmental stage of P. xylostella.

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Nucleotide and deduced amino acid sequences of PxGSTs gene from Plutella xylostella. Putative TATA boxes are underlined. The asterisk indicates the translational termination codon. The coding sequences are shown in red. Nucleotides in lower case letters are intron sequences.
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f03: Nucleotide and deduced amino acid sequences of PxGSTs gene from Plutella xylostella. Putative TATA boxes are underlined. The asterisk indicates the translational termination codon. The coding sequences are shown in red. Nucleotides in lower case letters are intron sequences.

Mentions: Genomic DNA extraction was described previously (Sonoda and Tsumuki 2005). The coding region of PxGSTe gene was amplified by PCR using a primer set, PxGSTe-5′-1, 5′-CTCACGAGCAATGAAAAGGTTCCAGTG-3′, (nucleotides 1,735–1,761 in Figure 1) and PxGSTe-3′-1, 5′-CAGCAGAATAATCCTTCCGCTTC-3′, (complementary to nucleotides 2,714–2,736 in Figure 1). Both primers were designed based on the published GST cDNA sequence (Huang et al. 1998) (GenBank/EMBL/DDBJ accession no. U66342). The coding region of PxGSTs gene was amplified by PCR using forward primer, PxGSTs-5′-1, 5′-GGCATATGGCCAAGAAACTACACTACTTC-3′, and reverse primer, PxGSTs-3′-1, 5′-CCGGATCCTTATAGCGCGTAGACCTTCCTC-3′. Both primers were designed based on the published GST cDNA sequence (Eum et al., unpublished) (accession # AB180447). On the 5′ ends, a restriction site for NdeI was added to PxGSTs-5′-1 and for BamHI to PxGSTs-3′-1 as a requirement of another study. The coding sequences of PxGSTs-5′-1 (nucleotides 259–282 in Figure 3) and PxGSTs-3′-1 (complementary to nucleotides 2,574–2,595 in Figure 3) are shown as bold letters.


Genomic organization and developmental expression of glutathione S-transferase genes of the diamondback moth, Plutella xylostella.

Sonoda S, Ashfaq M, Tsumuki H - J. Insect Sci. (2006)

Nucleotide and deduced amino acid sequences of PxGSTs gene from Plutella xylostella. Putative TATA boxes are underlined. The asterisk indicates the translational termination codon. The coding sequences are shown in red. Nucleotides in lower case letters are intron sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2990328&req=5

f03: Nucleotide and deduced amino acid sequences of PxGSTs gene from Plutella xylostella. Putative TATA boxes are underlined. The asterisk indicates the translational termination codon. The coding sequences are shown in red. Nucleotides in lower case letters are intron sequences.
Mentions: Genomic DNA extraction was described previously (Sonoda and Tsumuki 2005). The coding region of PxGSTe gene was amplified by PCR using a primer set, PxGSTe-5′-1, 5′-CTCACGAGCAATGAAAAGGTTCCAGTG-3′, (nucleotides 1,735–1,761 in Figure 1) and PxGSTe-3′-1, 5′-CAGCAGAATAATCCTTCCGCTTC-3′, (complementary to nucleotides 2,714–2,736 in Figure 1). Both primers were designed based on the published GST cDNA sequence (Huang et al. 1998) (GenBank/EMBL/DDBJ accession no. U66342). The coding region of PxGSTs gene was amplified by PCR using forward primer, PxGSTs-5′-1, 5′-GGCATATGGCCAAGAAACTACACTACTTC-3′, and reverse primer, PxGSTs-3′-1, 5′-CCGGATCCTTATAGCGCGTAGACCTTCCTC-3′. Both primers were designed based on the published GST cDNA sequence (Eum et al., unpublished) (accession # AB180447). On the 5′ ends, a restriction site for NdeI was added to PxGSTs-5′-1 and for BamHI to PxGSTs-3′-1 as a requirement of another study. The coding sequences of PxGSTs-5′-1 (nucleotides 259–282 in Figure 3) and PxGSTs-3′-1 (complementary to nucleotides 2,574–2,595 in Figure 3) are shown as bold letters.

Bottom Line: Based on amino acid identity with GST genes from other insects, this gene was classified as a member of the Sigma class.Southern blot analysis showed that PxGSTs was a single copy gene, whereas there were homologous members of the PxGSTe gene in the P. xylostella genome.RNA gel blot analysis indicated that the expression levels of both genes changed with the developmental stage of P. xylostella.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan. sonodas@rib.okayama-u.ac.jp

ABSTRACT
In the present study, we cloned and sequenced the entire coding regions of two glutathione S-transferase (GST) genes encoding PxGSTs and PxGSTe from the diamondback moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae), along with their respective 5' and 3' flanking regions. The PxGSTs gene was composed of four exons and three introns. Based on amino acid identity with GST genes from other insects, this gene was classified as a member of the Sigma class. The gene encoding PxGSTe had an intron in the 5' flanking region. Southern blot analysis showed that PxGSTs was a single copy gene, whereas there were homologous members of the PxGSTe gene in the P. xylostella genome. RNA gel blot analysis indicated that the expression levels of both genes changed with the developmental stage of P. xylostella.

Show MeSH