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Identification and Expression Patterns of Halloween Genes Encoding Cytochrome P450s Involved in Ecdysteroid Biosynthesis in the Cotton Bollworm Helicoverpa Armigera

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Identification and Expression Patterns of Halloween Genes Encoding Cytochrome P450s Involved in Ecdysteroid Biosynthesis in the Cotton Bollworm Helicoverpa Armigera Bulletin of Entomological Research (2017) 107,85–95 doi:10.1017/S0007485316000663 © Cambridge University Press 2016 Identification and expression patterns of Halloween genes encoding cytochrome P450s involved in ecdysteroid biosynthesis in the cotton bollworm Helicoverpa armigera J. Zheng1,2, K. Tian1,2, Y. Yuan1,M.Li1 and X. Qiu1,2* 1State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China: 2University of Chinese Academy of Sciences, Beijing 100049, China Abstract 20-Hydroxyecdysone (20E) is a key hormone which regulates growth, development and reproduction in insects. Although cytochrome P450 enzymes (P450s) participating in the ecdysteroid biosynthesis of 20E have been characterized in a few model insects, no work has been published on the molecular entity of their orthologs in the cotton bollworm Helicoverpa armigera, a major pest insect in agriculture worldwide. In this study, four cytochrome P450 homologs, namely HarmCYP302A1, HarmCYP306A1, HarmCYP314A1 and HarmCYP315A1 from H. armigera, were identified and evolutional conservation of these Halloween genes were revealed among lepidopteran. Expression analyses showed that HarmCYP302A1 and HarmCYP315A1 were predominantly ex- pressed in larval prothoracic glands, whereas this predominance was not always ob- served for HarmCYP306A1 and CYP314A1. The expression patterns of Halloween genes indicate that the fat bodies may play an important role in the conversion of ec- dysone into 20E in larval–larval molt and in larval–pupal metamorphosis, and raise the possibility that HarmCYP315A1 plays a role in tissue-specific regulation in the steroid biosynthesis in H. armigera. These findings represent the first identification and expression characterization of four steriodogenic P450 genes and provide the groundwork for future functional and evolutionary study of steroid biosynthesis in this agriculturally important pest. Keywords: Cytochrome P450, Ecysteroid, Expression pattern, Halloween genes, Helicoverpa armigera (Accepted 30 June 2016; First published online 22 August 2016) Introduction molting is strictly guided by precisely timed changes of the molting hormone 20-hydroxyecdysone (20E) (Iga & Insect growth and developments are regulated by hor- Smagghe, 2010). Insect metamorphosis from larva over pupa mones. Steroid hormones play key roles in the coordinated to a reproductive adult is under the control of 20E as well regulation of many developmental and physiological events (Iga & Smagghe, 2010). It is generally recognized that insects such as molting, metamorphosis and diapause in insects synthesize ecdysone in the prothoracic gland (PG) from dietary (Niwa & Niwa, 2014). For example, the precise timing of cholesterol or phytosterol, and the synthesized ecdysone (E) is secreted in the hemolymph, and then converted into 20E in various peripheral tissues predominantly in midgut (MG) or *Author for correspondence fat body (FB) (Gilbert et al., 2002). Phone: 86-10-64807231 Ecdysteroids are biosynthesized from the embryonic to the Fax: +86-10-64807099 adult stage. Ecdysone biosynthesis involves a series of enzym- E-mail: [email protected] atic oxidation reactions (fig. S-1) (Niwa & Niwa, 2014). It has 86 J. Zheng et al. demonstrated that Neverland (Nvd) (the [2Fe–2S] Rieske oxyge- Materials and methods nase) catalyzes the first step from cholesterol to 7-dehydro- Insects cholesterol (7-DC) (Yoshiyama-Yanagawa et al., 2011). However, the conversion process from 7-DC to ketodiol re- A colony of cotton bollworm H. armigera (Hübner) was es- mains unclear, commonly referred as the ‘Black Box’. The tablished from a field collection from Henan Province, China ‘Black Box’ is hypothesized to be catalyzed by at least three en- in 2005 and was maintained in the laboratory. Larvae were in- zymes, including the cytochrome P450 monooxygenases dividually reared in glass tubes on wheat germ-based artificial Spook (Spo) and CYP6T3, and the short-chain dehydrogen- diets (Wu & Gong, 1997), at 25 ± 1°C and relative humidity of ase/reductase Shroud (Sro) (Niwa & Niwa, 2014). In addition, 70% with a photoperiod of 16-h light/8-h dark. Adults were it is still possible that other uncharacterized enzymes play a kept under the same temperature and light conditions, and role in ecdysteroid biosynthesis. Recent studies have demon- provided with a 10% honey solution. strated that a glutathione S-transferase encoding gene, noppera-bo (nobo), plays a crucial role in regulating ecdysone biosynthesis in insects (Enya et al., 2014, 2015). Cloning of full-length transcripts of Halloween genes Up to now, four cytochrome P450 enzymes encoded by Total RNA was extracted from entire 2nd-instar larvae of Halloween genes (phm, dib, sad and shd) have been characterized H. armigera by using TRIzol (Invitrogen, CA, USA) according in Drosophila melanogaster, Bombyx mori and Manduca sexta.Phm to the manufacture’s protocol. First-strand cDNA was synthe- (CYP306A1), Dib (CYP302A1) and Sad (CYP315A1) sequential- sized from total RNA (1 µg) using PrimeScript First Strand ly convert the precursor of ecdysone, 2, 22, 25-trideoxyecdysone Synthesis Kit according to the manufacturer’s instructions (ketodiol), to 22, 2-dideoxyecdysone (ketotriol), 2-deoxyecdy- – (Takara, Dalian, China). sone and E. The product of shd (CYP314A1) hydroxylates E All the oligonucleotide primers (table 1) used in this study 20E, an active form of ecdysteroid. During the past decade, were commercially synthesized by Invitrogen (China). The the orthologs of these Halloween genes have been also identified cloning of each gene was performed by three steps. Firstly, a or inferred in several other insects (Christiaens et al., 2010; Iga & fragment of cDNA was amplified by PCR using degenerate Smagghe, 2010;Jiaet al., 2013;Wanet al., 2014a;Cabreraet al., primers. Then the 3′-end and 5′-end of this cDNA fragment 2015). Although many ecdysteroidogenic enzyme-encoding were obtained respectively via rapid-amplification of cDNA genes are well conserved in the genomes of insects (Niwa & ends (RACE) using SMART™ RACE cDNA amplification kit Niwa, 2014), previous studies also raise the possibility that ec- according to the manufacturer’s protocols (Clontech, CA, dysteroid biosynthesis is differentially regulated among various USA) with gene-specific primers used for 3′- and 5′-RACE. insect species (Enya et al., 2014). This situation highlights the ne- Lastly, the full-length open reading frames were amplified cessity to know the specific molecular mechanism of ecdysteroid for sequence confirmation using corresponding primers synthesis in certain insects of interest. (table 1), which were designed based on the sequence informa- From the aspect of pest control, P450 enzymes that catalyze tion obtained from 3′- and 5′-RACE ends. Products which physiologically important reactions offer insect-selective tar- amplified using the high-fidelity DNA polymerase (Pfu) gets for discovering and developing new insecticides (Niwa were gel purified (Takara, Dalian, China) and then subcloned & Niwa, 2014; Feyereisen, 2015). Several studies have showed into pEasy-T1 and transformed the Escherichia coli Trans 5α that such gene can serve as a potential target gene for strain (Transgen, Beijing, China). At least three positive clones RNA-interference-based pest management (Luan et al., 2013; were sequenced for each gene. Kong et al., 2014; Wan et al., 2014b; Wan et al., 2015). For ex- ample, knockdown of CYP314A1 in Leptinotarsa decemlineata by dietary introduction of double-stranded RNA into the Sequence identification and phylogenetic analysis secondary instar larvae caused larval lethality, delayed de- velopment and reduced pupation ratio (Kong et al., 2014). Bioinformation analysis was conducted using on line soft- Similarly, knockdown of CYP306A1 in the white-backed ware (http://web.expasy.org/protparam/). Alignment of planthopper Sogatella furcifera through dietary delivery amino acid sequence was performed by using Sequence into the 2nd instars caused lethality and slowed down ecdy- Alignment tool Clustal W program, and a neighbor-joining sis during nymphal stages (Wan et al., 2014b), and oral de- phylogenetic tree was generated by MEGA 6 (Tamura et al., livery of double-stranded RNA of Laodelphax striatellus 2013). CYP315A1 at the nymph stage caused nymphal lethality and delayed development in a dose-dependent manner Spatial and temporal expression analyses using RT–PCR (Wan et al., 2015). The cotton bollworm Helicoverpa armigera is a dreaded The expression profiles were evaluated in various tissues pest worldwide (Fitt, 1989). Although molting and metamor- and at various developmental stages of H. armigera by RT– phosis are known to be controlled by 20E in this holometabo- PCR. Total RNA from eggs (12 h old), selected tissues (head, lous insect, the enzymes in ecdysteroid synthetic pathway MG, Malpighian tubules, FB and epidermis) of the 5th-instar remain to be characterized. As the first step toward charac- larvae (1 day old), female and male pupae (4–5 days old), and terizing the enzymes involved in ecdysteroid biosynthesis, different parts from female and male adults (3–4 days old), in this study, we tried to probe H. armigera for Halloween were prepared using TRIzol according to the manufacturer’s gene orthologs, and investigated their transcriptional pat- protocol (Invitrogen, Carlsbad, CA). Ten individuals were terns. This effort may advance our knowledge about the mo- used for each preparation.
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