Molecular Cloning, Characterization, and mRNA Expression of Two <I>Cryptochrome</I> Genes in <I>Helicoverpa armigera</I> (Lepidoptera: Noctuidae)

Abstract

Light is a major environmental signal for insect circadian. In this study, we isolated two cryptochrome (cry) genes from Helicoverpa armigera (Hübner) by reverse transcription polymerase chain reaction and RACE-PCR strategies, designated as Ha-cryl (GenBank accession GQ896502) and Ha-cry2 (GenBank accession GQ896503). Ha-CRY1 encoded a fly-like protein of 548 amino acids, while Ha-CRY2 encoded a mammal-like protein of 657 amino acids. Both of these proteins had two conserved domains: a DNA photolyase domain and a flavin adenine dinucleotide (FAD) binding seven domain, and alignment of the amino acid sequence indicated that there was a high degree of homology between the CRYs of H. armigera and other insects. Real-time polymerase chain reaction revealed that: 1) Ha-cry1 and Ha-cry2 mRNA expressions were neither organ-specific nor developmental-stage-specific. 2) Under the light-dark cycle (16:8 L:D), Ha-cry1 abundance tended to increase during the day, then decrease in the night, whereas the expression pattern of Ha-cry2 was opposite. 3) The cyclings of Ha-cry1 and Ha-cry2 expression were disturbed by constant light and darkness. Our study has significant importance for the further study of the functions of the Ha-cry genes and potential control of the cotton bollworm.

Phylogeny and oscillating expression ofperiodandcryptochromein short and long photoperiods suggest a conserved function inNasonia vitripennis

“…This has been observed in the hymenopterans A. mellifera (Rubin et al, 2006), A. cerana (Shimizu et al, 2001), and Solenopsis invicta (Ingram et al, 2012); in the dipterans Drosophila melanogaster (Qiu & Hardin, 1996), A. aegypti, Culex quinquefasciatus (Gentile et al, 2009), Protophormia terranovae (Muguruma et al, 2010), Sarcophaga crassipalpis (Goto & Denlinger, 2002; Kostál et al, 2009), Chymomyza costata (Kobelková et al, 2010), and Sarcophaga bullata (Goto et al, 2006); in the cricket Modicogryllus siamensis (Sakamoto et al, 2009), the cockroach Rhyparobia maderae (Werckenthin et al, 2012), and in the lepidopterans Bombyx mori (Iwai et al, 2006), Danaus plexippus (Zhu et al, 2008), and Spodoptera littoralis (Merlin et al, 2007). In species where cry2 (or “vertebrate-like” cry) is present, its oscillation matches that of per , as found in our study, with a trough in the light phase and a peak in the dark phase (Gentile et al, 2009; Ikeno et al, 2008; Ingram et al, 2012; Merlin et al, 2007; Rubin et al, 2006; Werckenthin et al, 2012; Yan et al, 2013; Zhu et al, 2008). Therefore, oscillation of per and cry2 in the heads of N. vitripennis females is similar to what has been found in other insect species.…”

Daily expression of two circadian clock genes in compound eyes of Helicoverpa armigera: evidence for peripheral tissue circadian timing

“…RNA samples were collected immediately after 6 h of 500‐lux illumination, and samples were collected at Zeitgeber time (ZT)20 from the moths reared under 14 : 10 L : D to serve as a control (C). The amount of each gene expression was normalized to the abundance of two reference genes, EF‐1α and RPS15 (Fuller & Claricoates, ; Yan et al ., , , ). Each value is the mean ± SE of three collections.…”

“…The qPCR conditions were one cycle at 95°C for 10 min, followed by 40 cycles at 94°C for 15 s, 55°C for 40 s, and 72°C for 35 s and a melting curve ramp was constructed to confirm that no reaction produced nonspecific amplification. The amount of each gene expression in all treatments was normalized to the abundance of two reference genes, EF‐1α and RPS15 (Fuller & Claricoates, ; Yan et al ., , , ), using the 2 −ΔΔCt method described by Livak and Schmittgen (), and the geometric mean of the two reference genes was applied as a normalization factor (Gharbi et al ., ). Triplicate assays per cDNA sample were carried out independently to determine average values, and all results were obtained from three independent RNA samples.…”

“…The fluctuation of circadian clock gene transcript levels is usually regulated by multiple endogenous and exogenous factors, including light condition, environmental temperature and physiological status (Boothroyd et al ., ; Fan et al ., ; Gegear et al ., ; Ni et al ., ; Yan et al ., ; Liu et al ., ). Daily changes in the expression levels of circadian clock genes are typically observed within and outside the central nervous system (Giebultowicz et al ., ; Dolezel et al ., ; Weigl et al ., ; Yan et al ., , ), and the rhythmic expression of circadian clock genes in peripheral tissues was previously observed in songbirds (Singh et al ., ), rats (Yamazaki et al ., ; Buonfiglio et al ., ), chickens (Chong et al ., ) and moths (Merlin et al ., ; Yan et al ., ). The circadian clock is commonly assumed to be involved in photoperiodism, and a retinal clock may play an important role in shaping retinal functions into high acuity “day” and high sensitivity “night” states (Cashmore, ; Lin & Todo, ; Hirayama et al ., ; Saunders, ; Goto, ; Buonfiglio et al ., ).…”

Genomewide transcriptional signatures of migratory flight activity in a globally invasive insect pest

“…2013; Yan et al . 2013), were used for internal normalization. The specificity, dynamic range and PCR efficiency of each primer set were determined by testing against a fivefold serial dilution of cDNA (from 1/10th to 1/6000th).…”