Expression of mRNA for the t-complex polypeptide-1, a subunit of chaperonin CCT, is upregulated in association with increased cold hardiness in Delia antiqua

Abstract

Summer-diapause and winter-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), were significantly more cold hardy than nondiapause, prediapause, and postdiapause pupae. Moreover, cold acclimation of nondiapause pupae conferred strong cold hardiness comparable with that of diapause pupae. Differential display analysis revealed that the expression of a gene encoding TCP-1 (the t-complex polypeptide-1), a subunit of chaperonin CCT, in D antiqua (DaTCP-1) is upregulated in the pupae that express enhanced cold hardiness. Quantitative real-time polymerase chain reaction analyses showed that the levels of DaTCP-1 messenger RNA in pupal tissues, brain, and midgut in particular, are highly correlated with the cold hardiness of the pupae. These findings suggest that the upregulation of DaTCP-1 expression is related to enhanced cold hardiness in D antiqua. The upregulation of CCT in response to low temperature in an organism other than the yeast is newly reported.

Fig 1.

Cold hardiness of Delia antiqua pupae assessed by survival after a 5-day treatment at −20°C. After the −20°C treatment, pupae were maintained at 17°C. (A) Nondiapause pupae acclimated at 5°C for 0, 2, 8, and 32 days (CA0, CA2, CA8, and CA32) (n = 150). (B) The pupae obtained under winter diapause–inducing conditions were maintained under the same conditions for 3, 10, and 50 days (WD3, WD10, and WD50) (n = 200). (C) Pre–summer diapause (SD) pupae (SD3), SD pupae (SD20), and pupae that had terminated summer diapause (SDT) (n = 150). Bars with the same letter are not significantly different (Ryan's multiple comparison test of proportions, P < 0.05)

Fig 2.

Differential display of messenger RNA derived from the brain of CA0, CA2, CA32, and WD50 pupae. The arrow indicates a band corresponding to the DaTCP-1 complementary DNA fragment, the size of which is 1.5 kbp

Fig 3.

Nucleotide and deduced amino acid sequences of DaTCP-1 complementary DNA. The underlined amino acids represent adenosine triphosphate–binding motifs. Double-underlined nucleotides represent a presumable polyadenylation signal (AATAAA) and RNA instability motif (ATTTA). This sequence has been deposited in the GenBank database (Accession no. AB194475)

Fig 4.

Quantitative real-time PCR analysis of DaTCP-1 messenger RNA (mRNA) levels. The levels of DaTCP-1 mRNA were normalized to those of the internal standard, 18S ribosomal RNA (3 replicates). DaTCP-1 mRNA levels in cold-acclimated nondiapause pupae (A), winter-diapause pupae (B), and summer-diapause pupae (C). Br, brain; MG, midgut; MP, Malpighian tubules; FB, fat body