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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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Southern blot analyses of PxGSTe and PxGSTs genes from Plutella xylostella. Total DNA digested with KpnI (lane 1) or EcoRV (lane 2) in combination with PstI was size-fractionated on an agarose gel, transferred to a nylon membrane and hybridized with a PxGSTe probe. HindIII (lane 3), HindIII/ClaI (lane 4), HindIII/KpnI (lane 5), HindIII/PvuII (lane 6) and HindIII/SpeI (lane 7) digests were similarly hybridized with a PxGSTs probe.
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f05: Southern blot analyses of PxGSTe and PxGSTs genes from Plutella xylostella. Total DNA digested with KpnI (lane 1) or EcoRV (lane 2) in combination with PstI was size-fractionated on an agarose gel, transferred to a nylon membrane and hybridized with a PxGSTe probe. HindIII (lane 3), HindIII/ClaI (lane 4), HindIII/KpnI (lane 5), HindIII/PvuII (lane 6) and HindIII/SpeI (lane 7) digests were similarly hybridized with a PxGSTs probe.

Mentions: The presence or absence of genes homologous to PxGSTe or PxGSTs was examined by Southern blot analysis. Genomic DNA digested with KpnI or EcoRV in combination with PstI were hybridized with a PxGSTe gene fragment. In PstI/KpnI digests, a single intense band of 3.0 kb was observed as well as some less intense bands (Figure 5, lane 1). Similarly, a single intense band of 1.2 kb was observed in PstI/EcoRV digests in addition to some less intense bands (Figure 5, lane 2). These results suggest that homologous members of the PxGSTe gene are present. When genomic DNA was digested with HindIII and probed with a PxGSTs fragment, a single intense band of 3.8 kb was observed (Figure 5, lane 3). Single intense bands of 2.0 kb, 3.5 kb, 2.4 kb and 1.9 kb were also detected in HindIII/ClaI, HindIII/KpnI, HindIII/PvuII and HindIII/SpeI digests, respectively (Figure 5, lanes 4 to 7). No additional strong or weak bands were detected in any of the digests (Figure 5 and data not shown), suggesting that PxGSTs is present in a single copy.


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)

Southern blot analyses of PxGSTe and PxGSTs genes from Plutella xylostella. Total DNA digested with KpnI (lane 1) or EcoRV (lane 2) in combination with PstI was size-fractionated on an agarose gel, transferred to a nylon membrane and hybridized with a PxGSTe probe. HindIII (lane 3), HindIII/ClaI (lane 4), HindIII/KpnI (lane 5), HindIII/PvuII (lane 6) and HindIII/SpeI (lane 7) digests were similarly hybridized with a PxGSTs probe.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f05: Southern blot analyses of PxGSTe and PxGSTs genes from Plutella xylostella. Total DNA digested with KpnI (lane 1) or EcoRV (lane 2) in combination with PstI was size-fractionated on an agarose gel, transferred to a nylon membrane and hybridized with a PxGSTe probe. HindIII (lane 3), HindIII/ClaI (lane 4), HindIII/KpnI (lane 5), HindIII/PvuII (lane 6) and HindIII/SpeI (lane 7) digests were similarly hybridized with a PxGSTs probe.
Mentions: The presence or absence of genes homologous to PxGSTe or PxGSTs was examined by Southern blot analysis. Genomic DNA digested with KpnI or EcoRV in combination with PstI were hybridized with a PxGSTe gene fragment. In PstI/KpnI digests, a single intense band of 3.0 kb was observed as well as some less intense bands (Figure 5, lane 1). Similarly, a single intense band of 1.2 kb was observed in PstI/EcoRV digests in addition to some less intense bands (Figure 5, lane 2). These results suggest that homologous members of the PxGSTe gene are present. When genomic DNA was digested with HindIII and probed with a PxGSTs fragment, a single intense band of 3.8 kb was observed (Figure 5, lane 3). Single intense bands of 2.0 kb, 3.5 kb, 2.4 kb and 1.9 kb were also detected in HindIII/ClaI, HindIII/KpnI, HindIII/PvuII and HindIII/SpeI digests, respectively (Figure 5, lanes 4 to 7). No additional strong or weak bands were detected in any of the digests (Figure 5 and data not shown), suggesting that PxGSTs is present in a single copy.

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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