Universal primers for amplifying the complete coding sequence of cytoplasmic heat shock protein 90 ( HSP 90 ) in Lepidoptera

Using sequence alignment, a conserved domain in the 3’ untranslated region (UTR) of the cytoplasmic heat shock protein 90 (HSP90) of Lepidoptera was found. This region is highly variable in other insect groups. Furthermore, universal primers were designed to amplify the complete coding sequence (CDS) of HSP90 from total genomic DNA in Lepidoptera, avoiding the commonly used reverse transcription-polymerase chain reaction (RT-PCR) and 3’, 5’-rapid amplification of cDNA ends (RACE) methods based on cDNA. These primers amplified a fragment of about 2.25 kb in the 11 species tested, which represent seven different families of Lepidoptera, including moths and butterflies. The results suggest that the conserved domain of 3’UTR is universal in Lepidoptera and these primers successfully amplify the complete CDS of cytoplasmic HSP90 from genomic DNA. 164 ** Corresponding author; e-mail: huangdw@ioz.ac.cn * These authors contributed equally to the present study. reaction consisted of an initial denaturation step (94°C for 4 min) followed by 40 cycles of 94°C for 10 s, 50–52°C for 1 min, 68°C for 2.5 min, and a final extension step (72°C for 10 min). Targeted PCR bands were purified and cloned using the pEASY-T3 Simple Cloning Vector (TransGen). Three positive clones of each insect were sequenced using a 3730XL sequencer based on the Sanger method (BioSune, Beijing, China). Nucleotide sequences were translated into amino acids to confirm translation and all sequences identified by BLAST searches implemented in National Center for Biotechnology Information (NCBI) (http://www. ncbi.nih.gov/BLAST/). RESULTS AND DISCUSSION Primer design and evaluation A highly conserved region of aligned lepidopteran cytoplasmic hsp90 sequences occurred in the 3’ UTR (Fig. 1B). Furthermore, the following set of primers were designed: L90F1 (forward primer): 5’-AMAATGCCBGAAGRDATGC-3; L90F2 (forward primer): 5’-AMAATGCCBGAAGRDATGG-3; and L90R (reverse primer): 5’-GAACTAAATCAGTCTTTGG-3. Primer L90F1 or L90F2, located at the 5’ end (Fig. 1A), contained the start codon and primer L90R1 was located in the 3’ UTR region. Theoretically, these primers could amplify a 2.25 kb fragment that spans the complete CDS and part of the 3’UTR region of hsp90. The genomic DNA of Helicoverpa armigera was used as a reference to test for optimum PCR conditions. Because a fragment of approximately 2.25 kb was to be amplified, the cycling conditions were similar to those developed for Long-PCR (Cheng et al., 1994). The results of a range of annealing temperatures and different amounts of genomic DNA are summarized in Fig. 2A, B and the amplification condition detailed in the Material and Methods were chosen. PCR amplification of 33 genomic DNA samples of 11 species from seven families of the Lepidoptera were performed. Although some weak nonspecific amplifications occurred, the targeted fragments in 11 species were successfully amplified (Fig. 2C). A very weak band of the targeted fragment was observed in Gonepteryx amintha, probably because of poor PCR amplification efficiency. Nevertheless, the specific sequence was obtained after DNA purification, cloning, and sequencing. Identification of sequences Primers were designed and 11 sequences successfully obtained. Their characteristics (molecular weights, isoelectric points, and number of encoded amino acids) are presented in Table 1. BLAST searches were employed to confirm sequence 165 AA – the number of deduced amino acids; MW – molecular weight; pI – isoelectric point; * indicates that the former submission of the hsp90 sequence to GenBank is the same as the sequence obtained in this study, which was not submitted; ** indicates that the former submission of the hsp90 sequence to GenBank is different from the sequence obtained in this study, which was submitted. 4.68 82.56 718 L90F2/ L90R1 Wild Gonepteryx amintha Pieridae 4.76 82.45 718 L90F2/ L90R1 Wild Argynnis paphia Nymphalidae 4.70 82.37 717 L90F1/ L90R1 Laboratory rearing *Plutella xylostella Plutellidae 4.74 82.44 717 L90F1/ L90R1 Wild Papilio memnon Papilionidae 4.75 82.28 716 L90F2/ L90R1 Wild Exangerona prattiaria Geometridae 4.75 82.41 716 L90F1/ L90R1 Wild Ostrinia furnacalis Pyralidae 4.73 82.62 717 L90F1/ L90R1 Wild *Spodoptera exigua 4.73 82.60 717 L90F1/ L90R1 Wild Spodoptera litura 4.71 82.56 717 L90F1/ L90R1 Wild **Mythimna separata 4.85 82.19 717 L90F1/ L90R1 Laboratory rearing Helicoverpa assulta 4.71 82.59 717 L90F1/ L90R1 Laboratory rearing Helicoverpa armigera Noctuidae pI MW (kDa) AA Primers Species origin Species name Family name TABLE 1. Lepidopteran species used to evaluate the universal primers and related information on their hsp90s. Fig. 1. The alignments of (A) the 5’ end and (B) 3’ UTR region of the heat shock protein 90 (HSP90) gene from Lepidoptera. Symbols: “–” represents a gap in the alignments, “*” identical sites in the alignments. orthology. Both nucleotide and deduced amino acid sequences were tested. All sequences obtained with these primers were very similar to cytoplasmic hsp90 of other lepidopteran species (Table 2). Moreover, the five conserved signatures of the HSP90 family (Gupta, 1995) and the conserved pentapeptide (MEEVD) of cytoplasmic HSP90 (Terasawa et al., 2005) were found in the alignments of the amino acid sequences (Fig. 3). The above analysis confirmed that the cytoplasmic hsp90 sequences were amplified by these primers. The nucleotide sequences obtained in this study were deposited in GenBank under accession numbers GU230732–GU230740. Specificity of Lepidoptera hsp90 gene and universal primers One intron is located directly upstream from the start codon of cytoplasmic hsp90 in Diptera, Hymenoptera, and Coleoptera (Blackman & Meselson, 1986; Benedict et al., 1996; KurzikDumke et al., 1996; Konstantopoulou & Scouras, 1998). This intron regulates the basal transcription of the cytoplasmic hsp90 at normal physiological temperatures (Lange et al., 1997). However, this intron is missing in the cytoplasmic hsp90 of Lepidoptera (Landais et al., 2001; Sonoda et al., 2006). In addition, the cytoplasmic hsp90 of Lepidoptera has a conserved 3’UTR region, which is not present in at least two other 166 Shown is the BLAST search with nucleotide sequence; Shown is the BLAST search with amino acid sequence; *low E values indicate high reliability of BLAST results. 93 0 100 Bombyx mori 83 0 95 Chilo suppressalis Gonepteryx amintha 96 0 100 Spodoptera frugiperda 85 0 100 Spodoptera frugiperda Argynnis paphia 94 0 100 Spodoptera frugiperda 85 0 100 Spodoptera frugiperda Papilio memnon 94 0 100 Mamestra brassicae 84 0 100 Spodoptera frugiperda Exangerona prattiaria 97 0 100 Chilo suppressalis 90 0 94 Loxostege sticticalis Ostrinia furnacalis 99 0 100 Spodoptera frugiperda 96 0 100 Spodoptera frugiperda Spodoptera litura 98 0 100 Mamestra brassicae 97 0 95 Mythimna separata Mythimna separata 98 0 100 Helicoverpa zea 97 0 95 Helicoverpa zea Helicoverpa assulta 98 0 100 Helicoverpa zea 98 0 100 Helicoverpa zea Helicoverpa armigera Similarity(%) E value* Query coverage (%) Closest species Species TABLE 2. Results of BLAST searches of hsp90 sequences. Fig. 2. Photographs of gels showing the effects of annealing temperatures and quantity of template DNA on the efficiency with which the hsp90 sequence was amplified for 11 lepidopteran species. Analysis of the effect of (A) the different annealing temperatures and (B) the different amounts of DNA in H. armigera, and (C) the result of the amplification in the 11 species. Refer to Table 1 for generic names of species examined.

For molecular studies of HSPs90 it is necessary to identify long stretches of DNA sequences as complete coding sequence (CDS) to draw conclusions.The complete CDS of HSP90 is commonly obtained using reverse transcription-polymerase chain reaction (RT-PCR) and 3', 5'-rapid amplification of cDNA ends (RACE) from cDNA templates (Sonoda et al., 2006;Li et al., 2009;Feng et al., 2010).Both these methods are difficult and costly.
Lepidopteran cytoplasmic hsp90 is a single-copy gene without introns (Landais et al., 2001;Sonoda et al., 2006).After aligning existing sequences of cytoplasmic HSP90, a highly conserved region was located in the 3' untranslated region (UTR) in Lepidoptera, which is not present in the alignments of Diptera and Hymenoptera (data not shown).
Furthermore, a set of universal primers were designed for amplifying the gene that spanned the complete CDS and partial 3'UTR region.The primers successfully amplified specific cytoplasmic hsp90 sequences from genomic DNA templates in all of the 11 species, representing seven lepidopteran families, tested.

DNA extraction and primer evaluation
Three individuals of each of 11 species from seven families of Lepidoptera, including both moths and butterflies, were used to evaluate the primers (Table 1).Genomic DNA was extracted from each individual using EasyPure Genomic DNA Extraction Kit (TransGen, Beijing, China), which purifies DNA using proteinase K digestion and silica-membrane technology.

PCR amplifications, sequencing and sequence confirmation
PCR reagents (25 µl) contained 300 ng of template DNA, 0.5 µl of each primer (10 mM), 3 µl of dNTP mixture containing 2.5 mM of each dNTP, 2.5 µl of 10 × reaction buffer and 1 U of High Fidelity Expand Taq polymerase (TransGen).The PCR reaction consisted of an initial denaturation step (94°C for 4 min) followed by 40 cycles of 94°C for 10 s, 50-52°C for 1 min, 68°C for 2.5 min, and a final extension step (72°C for 10 min).Targeted PCR bands were purified and cloned using the pEASY-T3 Simple Cloning Vector (TransGen).Three positive clones of each insect were sequenced using a 3730XL sequencer based on the Sanger method (BioSune, Beijing, China).Nucleotide sequences were translated into amino acids to confirm translation and all sequences identified by BLAST searches implemented in National Center for Biotechnology Information (NCBI) (http://www.ncbi.nih.gov/BLAST/).
The genomic DNA of Helicoverpa armigera was used as a reference to test for optimum PCR conditions.Because a fragment of approximately 2.25 kb was to be amplified, the cycling conditions were similar to those developed for Long-PCR (Cheng et al., 1994).The results of a range of annealing temperatures and different amounts of genomic DNA are summarized in Fig. 2A, B and the amplification condition detailed in the Material and Methods were chosen.PCR amplification of 33 genomic DNA samples of 11 species from seven families of the Lepidoptera were performed.Although some weak nonspecific amplifications occurred, the targeted fragments in 11 species were successfully amplified (Fig. 2C).A very weak band of the targeted fragment was observed in Gonepteryx amintha, probably because of poor PCR amplification efficiency.Nevertheless, the specific sequence was obtained after DNA purification, cloning, and sequencing.

Identification of sequences
Primers were designed and 11 sequences successfully obtained.Their characteristics (molecular weights, isoelectric points, and number of encoded amino acids) are presented in Table 1.BLAST searches were employed to confirm sequence 165 AA -the number of deduced amino acids; MW -molecular weight; pI -isoelectric point; * indicates that the former submission of the hsp90 sequence to GenBank is the same as the sequence obtained in this study, which was not submitted; ** indicates that the former submission of the hsp90 sequence to GenBank is different from the sequence obtained in this study, which was submitted.orthology.Both nucleotide and deduced amino acid sequences were tested.All sequences obtained with these primers were very similar to cytoplasmic hsp90 of other lepidopteran species (Table 2).Moreover, the five conserved signatures of the HSP90 family (Gupta, 1995) and the conserved pentapeptide (MEEVD) of cytoplasmic HSP90 (Terasawa et al., 2005) were found in the alignments of the amino acid sequences (Fig. 3).The above analysis confirmed that the cytoplasmic hsp90 sequences were amplified by these primers.The nucleotide sequences obtained in this study were deposited in GenBank under accession numbers GU230732-GU230740.
In addition, the cytoplasmic hsp90 of Lepidoptera has a conserved 3'UTR region, which is not present in at least two other   1 for generic names of species examined.insect orders.However, due to the limited information on the occurrence of cytoplasmic hsp90 sequences in insects, it is not possible to confirm that this conserved 3'UTR region only occurs in Lepidoptera.Given this difference, this important functional marker could greatly facilitate research in Lepidoptera.The universal primers designed here amplified the complete CDS from genomic DNA, and, consequently, circumvented the use of costly RACE methods.
The amplification yielded some nonspecific bands, which might have resulted from the nonspecific binding of the primers.Nevertheless, identification and purification of the expected fragment for cloning and sequencing proved to be easy.Most of the products were amplified with primers L90F1/L90R and yielded the most consistent results during initial trials.
In conclusion, the results suggest that the cytoplasmic hsp90 of Lepidoptera has a universally conserved 3'UTR region.The primers described here proved to be very promising for the amplification of the cytoplasmic hsp90 fragment containing the complete CDS from Lepidoptera.

Fig. 2 .
Fig. 2. Photographs of gels showing the effects of annealing temperatures and quantity of template DNA on the efficiency with which the hsp90 sequence was amplified for 11 lepidopteran species.Analysis of the effect of (A) the different annealing temperatures and (B) the different amounts of DNA in H. armigera, and (C) the result of the amplification in the 11 species.Refer toTable 1 for generic names of species examined.

Fig. 3 .
Fig. 3. Partial alignments of the amino acid sequences obtained in this study.The five conserved signatures of HSP90 family and the conserved pentapeptide of cytoplasmic HSP90 are shaded.Symbol "*" indicates the stop codon.GenBank accession numbers are given in left column.

TABLE 1 .
Lepidopteran species used to evaluate the universal primers and related information on their hsp90s.

TABLE 2 .
Results of BLAST searches of hsp90 sequences.