piggybac like elements 棉铃虫
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Journal compilation ? 2008 The Royal Entomological Society
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Insect Molecular Biology (2008) 17
(1), 9–18
piggyBac- like elements in cotton bollworm, Helicoverpa armigera (Hübner)
Z. C. Sun*, M. Wu*, T. A. Miller? and Z. J. Han*
* Key Lab of Monitoring and Management of Plant Disease and Insects, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, People’s Republic of China; and ? Department of Entomology, University of California, Riverside, CA, USA Abstract
Two piggyBac -like elements (PLEs) were identi?ed in the cotton bollworm, Helicoverpa armigera
, and were designated as HaPLE1 and HaPLE2. HaPLE1 is ?anked by 16 bp inverted terminal repeats (ITRs) and the dupli-cated TTAA tetranucleotide, and contains an open reading frame (OR F) of 1794 bp with the presumed DDD domain, indicating that this element may be an active autonomously mobile element. HaPLE2 was found with the same ITRs, but lacks the majority of an ORF-encoding transposase. Thus, this element was thought to be a non-autonomous element. Transpos-able element displays and distribution of the two PLEs in individuals from three different H . armigera popula-tions suggest that transmobilization of HaPLE2 by the transposase of HaPLE1 may be likely, and mobilization of HaPLE1 might occur not only within the same individual,but also among different individuals. In addition, hor-izontal transfer was probably involved in the evolution of PLEs between H . armigera and Trichoplusia ni .Keywords:cotton bollworm, piggyBac -like elements,transposable element, autonomous element, non-autonomous element.Introduction
T ransposable elements (TEs) or transposons are mobile genetic units identi?ed in the genomes of nearly all eukary-
otes, in which they make up a signi?cant portion of the genome (Kazazian, 2004). In humans and mice, TE-derived sequences account for more than 40% of the genome (Lander et al ., 2001; Waterston et al ., 2002). Recent genome-wide sequence analyses of fruit ?y, Drosophila melanogaster , and the malaria mosquito, Anopheles gambiae , found more than 22 and 16% of the genome sequence, respectively, was composed of TE-derived sequences (Holt et al ., 2002; Kapitonov & Jurka, 2003),indicating the importance of transposition in evolution.Since the discovery of the ?rst transposon in maize (McClintock, 1950), transposable elements have become valuable tools for genetic analysis in many organisms.The introduction of P element-mediated transgenesis and insertional mutagenesis dramatically advanced Drosophila genetics (Rubin & Spradling, 1982). But for transposons such as P elements, host factors are involved in transposition;they are nonfunctional in nondrosophilid insects. (Handler et al ., 1993).
piggyBac , a class II transposable element, was ?rst isolated from a T richoplusia ni cell line, where it caused a mutant plaque phenotype upon insertion into a baculovirus genome (Cary et al ., 1989). The element is 2476 bp in length, having 13 bp short inverted terminal repeat (ITR)sequences and a 1.8 kb open reading frame (ORF). It inserts in the centre of the tetranucleotide TT AA, which is duplicated upon insertion (Wang & Fraser, 1993). The target insertion site can be restored, leaving no footprint upon excision (Fraser et al ., 1996). Recently, several highly con-served piggyBac -like elements (PLEs) were discovered in some insects beyond T . ni . For example, nearly identical elements were discovered in the tephritid ?y, Bactrocera dorsalis (Handler & McCombs, 2000), and highly conserved,although not identical, PLE sequences were found in some noctuid species (Wang et al ., 2006; Zimowska & Handler,2006). However, except for the elements nearly identical to T . ni piggyBac , no active PLEs have been reported as yet.The piggyBac transposon has been used for germ-line transformation of more than a dozen of different insects,such as Ceratitis capitata (Handler et al ., 1998), Drosophila melanogaster (Handler & Harrell, 1999), T ribolium castaneum (Berghammer et al ., 1999), Pectinophora gossypiella
Received 17 May 2007; accepted after revision 16 September 2007. Corre-spondence: Dr Zhaojun Han, Department of Entomology, College of Plant Protection, Nanjing Agricultural University, No. 1 Weigang, Nanjing city,Jiangsu province, 210095, Peoples Republic of China. T el./fax: +86 2584395245; e-mail: zjhan@https://www.wendangku.net/doc/d23060339.html,
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(Peloquin et al ., 2000), Bombyx mori (T amura et al ., 2000), Ba. dorsalis (Handler and McCombs, 2000),
Anastrepha suspensa (Handler & Harrell, 2001), Musca domestica (Hediger et al ., 2001), Athalia rosae (Sumitani et al ., 2003)and Cochliomyia hominivorax (Allen et al ., 2004). There-fore, it was thought of as the most useful vector for animal transformation. Actually, the piggyBac transposon is not only used as a gene-transfer vector for insects of medical and economic importance, but has been further developed as a gene tagging and enhancer trapping tool for functional insect genomics (Horn et al ., 2003; Thibault et al ., 2004).Recently, it has also been used successfully in germ-line transformation of planarians and mammals (Balu et al .,2005; Ding et al ., 2005).
Since the cross-mobilization of elements from the hA T family was determined, it is possible that homologous or related endogenous elements will affect the stability of transgenic insects mediated by a related transposable element. (Sundararajan et al ., 1999; Wimmer, 2003). There-fore, it is necessary to determine whether the transposon used as a vector will interact with related sequences that exist in potential hosts and a range of other species.
Here, we used degenerate PCR to explore PLEs in cotton bollworm (CBW), Helicoverpa armigera (Hübner) (Lepi-doptera: Noctuidae), an important pest in a wide range of agricultural and commercial crops in many parts of the world. In this paper, we report the presence of two PLEs,HaPLE1 and HaPLE2, in the H. armigera genome, which will contribute to our understanding of the distribution and characteristic of the piggyBac family and application of piggyBac in a wide range of organisms.Results
piggyBac -like elements HaPLE1 and HaPLE2 in Helicoverpa armigera
With degenerate primers designed from conserved regions of the piggyBac gene family, PCR produced a single DNA band about 458 bp long. Cloning and sequencing results revealed that this fragment was similar to known piggyBac genes when compared with published sequences in G EN- B ANK . Subsequent inverse PCR yielded a partially overlapping clone revealing ITRs and 5 ′ - and 3 ′ -?anking https://www.wendangku.net/doc/d23060339.html,ing the primers located on the ?anking region, a full-length HaPLE copy, designated as HaPLE1 (G EN B ANK accession number EF593176) was eventually isolated. HaPLE1
appears intact and is 2500 bp in length with an ORF of 1794 bp encoding a transposase, and perfect 16 bp ITRs. Similar to other PLEs, HaPLE1 contains three C/G residues at the extreme end of both the ITRs (Elick et al .,1997) and the tetranucleotide target-site duplication, TT AA (Fig. 1A).
Another copy of full-length HaPLE, designated as HaPLE2 (G EN B ANK accession number EF593175), was
also obtained by using PCR with a pair of primers designed on the ?anking sequences recovered in the vectorette PCR. Interestingly, HaPLE2 is 982 bp with the same ITRs as HaPLE1 just linked to the TT AA tetranucleotide at both ends, but most of its ORF-encoding transposase has been deleted (Fig. 1B).
T ransposase and phylogenetic analysis
Amino acid sequence alignment of PLEs from several insects showed that the conceptual translation of HaPLE1transposase shares 63 and 78% amino acid identity and similarity with T . niIFP2 (Cary et al ., 1989), 55 and 67% with Ba. dorsalis-white eye piggyBac (Handler & McCombs, 2000),30 and 48% with Heliothis virescens HvPLE1.1 (Wang et al .,2006) and 32 and 49% with Bo. mori yabusame-1 (G EN- B ANK
accession number BAD11135), respectively (Fig. 2).The alignment also showed that the N-terminal and C-terminal regions are both highly variable, indicating that these regions are not highly conserved and might be conserved only in the sequence of the DNA-binding domain (Xu et al., 2006). The conserved core region of the T. ni piggyBac transposase, from positions 130 to 522,contains about 12 highly conserved blocks of amino acids which might be the catalytic domain of this transposase and derived proteins. The DDE domain was found in many DNA transposases, and consists of two absolutely conserved aspartic acids and a highly conserved glutamic acid. Differ-ent from the widespread DDE domain, a highly divergent DDD domain has been speculated upon to be present in the transposase of the piggyBac families (Sarkar et al .,2003). In the T . ni piggyBac transposase, the presumed highly conserved aspartic acids are D268 and D346, and D447 is the third best candidate residue (Sarkar et al .,2003). However, Xu et al . (2006) consider that D450 is a better candidate for the third residue than D447 in the trans-posase sequences of the Bo. mori PLE. In the HaPLE1transposase sequence obtained in our experiments, D268,D346, D447 and D450 were all observed (Fig. 2A).
A phylogenetic tree was generated using amino acid sequences of 19 PLEs including the sequence of HaPLE1.As is shown in Fig. 3, these transposase sequences are distributed within three major clades (I, II, III) with higher than 50% bootstrap support. In the ?rst clade, the HaPLE1isolated from H. armigera is clustered with the original T. niIFP2. BdopiggyBac , AgaP
B , BmoY abusame-1 and HvPLE1.1 are also grouped in this clade. Although H. armigera is taxonomically the closest to Heliothis vires-cens , the relationship between HaPLE1 and HvPLE1.1 is not very close, not even in the nearest branch. The second and third clades comprise a mixture of insect and mammal PLEs. Obviously , the evolutionary pattern within the piggyBac family deviates from the phylogeny of their host species,which indicates that horizontal transfer was probably involved in the evolution of PLEs.
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Figure 1.Nucleotide sequence and conceptual
translations of putative transposase for the full-length piggyBac -like element HaPLE1 (A), nucleotide
sequence and putative translation of HaPLE2 (B) and their structures (C). The putative 16 bp inverted
terminal repeats (ITRs) are indicated by the arrows. The potential duplicated tetranucleotide targets TT AA are in bold. The regions corresponding to the degenerate primers used in this study are underlined on the HaPLE1. Structures of HaPLE1 and HaPLE2 are
depicted with ITRs (arrowheads) and transposase open reading frames (box). The indel in HaPLE2 is indicated by hash marks (#).
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Insertion sites and inverted terminal repeats
Products of vectorette PCR showed that the insertions of HaPLE usually varied with insect individuals (Fig. 4). From 12 individual CBW of NJ strain, we obtained a total of 12 5′-and 8 3′-?anking sequences of HaPLE (Fig. 5A). Sequence analysis revealed that 11 5′- and 7 3′-?anking sequences obtained belong to HaPLE2. All of the insertions occurred at a TT AA target site, which is characteristic of all members of the TT AA-speci?c family of DNA transposons (Fraser et al ., 1996). A B LAST search of the ?anking sequences of the HaPLEs found no signi?cant match to known genes in the database. The 16 bp perfect ITRs are well conserved in both HaPLE1 and HaPLE2 (Fig. 5B).
Distribution of HaPLEs in different populations of Helicoverpa armigera
A pair of primers designed to the common region of HaPLE1 and HaPLE2, HaPLE-F and HaPLE-R, were used to investigate the distribution of the HaPLEs in different populations of H. armigera . Genomic DNA of 17 individuals from each strain, NJ, GY and Oxford, was used as the PCR template. As a result, HaPLE2 was found in all the indi-viduals of the three CBW strains, but HaPLE1 was only observed in six of 17 individuals in the NJ strain, six of 17individuals in the GY strain and four of 17 individuals in the Oxford strain (Fig. 6).
Discussion
In our experiments, two types of full-length PLEs were iden-ti?ed in H. armigera by a sequential approach: degenerate PCR, inverse PCR, vectorette PCR and ?anking PCR.HaPLE1 seems potentially activated with an intact ORF encoding transposase and perfect ITRs of 16 bp linked to the canonical TT AA tetranucleotide at both ends. HaPLE2has the intact 16 bp ITRs, but has lost the majority of its ORF-encoding transposase. A similar phenomenon has been observed in D. melanogaster P elements. There are two distinct types of P elements, autonomous and non-autonomous, in the D. melanogaster genome (O’Hare &Rubin, 1983; Karess & Rubin, 1984). Autonomous P ele-ments are intact and can mobilize independently (Karess &Rubin 1984; Rio et al ., 1986). Non-autonomous P elements occur naturally through internal deletions of the auto-nomous P elements. Such elements lack the transposase gene but retain the parts of the sequence required for transposition. Mobilization of non-autonomous P elements occurs only if there is at least one autonomous P element present to supply transposase (Engels 1984, 1996). Thus,of the two HaPLEs, HaPLE1 was thought to be the active autonomous element, and HaPLE2 was thought to be the non-autonomous element.
Phylogenetic analyses of transposase sequences revealed three major clades with higher than 50%
Figure 1.Continued.
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Figure 2.T ransposase sequence alignment for piggyBac -like elements (PLEs) (A) and alignment of putative transposase for HaPLE1 and HaPLE2 (B).
The yabusame-1 element from Bombyx mori , the HvPLE1.1 from Heliothis virescens , the piggyBac from the Bactrocera dorsalis strain white eye and the IFP2 from T richoplusia ni are aligned together with HaPLE1. The presumptive DDD domain (Sarkar et al ., 2003) is also observed and indicated by asterisks. D268, D346, D447 and D450 are the positions of aspartic acids in T . ni piggyBac transposase. Black and grey boxes are for identical and similar amino acids having a 50% majority in the sequence alignment. G EN B ANK accession numbers are given in Fig. 3.
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bootstrap support. One clade is mainly clustered with insects; the other two are clustered with mammals and insects. Overall, the piggyBac phylogeny appears to devi-ate from the phylogeny of their host species, which implies
that horizontal transfer was probably involved in the evolu-tion of PLEs. Horizontal transfer of transposons such as P .mariner , Tc1-like and Minos has been reported (Matuyama & Hartl, 1991; Clark et al ., 1994; Michael, 2001; de Almeida & Carareto, 2005), and horizontal transfer has also been implied for Ba. dorsalis and Bo. mori PLEs. (Sarkar et al .,2003; Xu et al., 2006). In our analysis, the relationship between HaPLE1 and T . niIFP2 is very close, with a boot-strap value of 99% in the nearest branch and the alignment of HaPLE1 and T . niIFP2 shows 78% similarity at the amino acid level, which is further evidence for horizontal transfer of PLEs.
To survey the distribution of HaPLE1 and HaPLE2 in different CBW populations, individuals were selected ran-domly from each of the CBW strains, NJ, GY and Oxford.As a result, HaPLE2 was found to exist in all of the individuals selected from the three populations; however, HaPLE1existed in just 23–35% of the individuals (Fig. 6). These results indicate that the intact copy HaPLE1 has potential mobility. Inactive copies of a TE will be ?xed or lost in the genome of individuals in a population over time if they are neutral (Deceliere et al ., 2005). Therefore, copies should exist in almost all individuals within the population over time. In our study, HaPLE2 was observed in all individuals within the three populations. This result for HaPLE2 was interpreted as inheritance of ancestral elements before the insect host invaded the New World. Nevertheless, HaPLE1was only observed in some individuals in the three popula-tions, including the Oxford strain which has been kept in laboratories for more than 20 years. This result for HaPLE1implies that the mobilization of HaPLE1 occurs not only within the genome of the same individual but also among genomes of different individuals. However, we consider that the HaPLEs obtained in our experiments have existed in the H. armigera genome for more than 20 years because of the presence of HaPLE1 and HaPLE2 in the Oxford strain.Although germ-line transformation of insects using piggyBac -based vectors is developing rapidly, knowledge of the biological characteristics and evolutionary history of piggyBac elements is still limited. Experiments investi-gating the transposition of the piggyBac element in embryos of T . ni showed that endogenous piggyBac ele-ments in the target species may act to repress transposition of introduced piggyBac vectors (Lobo et al ., 1999). A similar phenomenon was also observed with the P element of D. melanogaster (Rio, 1990). Another example is endo-genous mariner -like elements, which affected the trans-position of the mariner -based transformation vector (Coates et al ., 1995). However, this phenomenon was not observed in Ba. dorsalis , where germ-line transformation occurred (at 2–3% per fertile G0) despite the existence of nearly identical piggyBac elements in its genome (Handler & McCombs, 2000). The two phenomena mentioned above
have not been con?rmed by compelling evidence. Regardless,
Figure 3.Phylogenetic relationships among piggyBac-like element transposase amino acid sequences. T rees were generated by the
neighbor-joining method. Numbers at the nodes are bootstrap values for 1000 replications. Abbreviations: Aga, Anopheles gambiae ; Bdo, Bactrocera dorsalis ; Dme, Drosophila melanogaster ; Dpu, Daphnia pulicaria ; Ha, Helicoverpa armigera ; Hsa, Homo sapiens ; Hv, Heliothis virescens ; Mmu, Mus musculus ; Mfa, Macaca fascicularis ; Tni, T richoplusia ni . G EN B ANK accession numbers are: AgaPBD1(XM_312615), AgaPBD2(XM_320414), AgaPBD3(XM_310729), Bdo PiggyBac(AF289123), BmoY abusame-1(BAD11135), DmeCG9839(AAL39784), DmeCG13151(AAF58496),
DmeLOOPER1M(AAM50981) DpuPokey(A Y115589), HsaPGBD1(NM_032507), HsaPGBD2(NM_170725), HsaPGBD3(NM_170753), HsaPGBD4(NM_152595), HsaPGBD5(NM_024554),
HvPLE1.1(DQ407726), MmuPGBD5(NM_171824), MfaPGBD3
(AB179012), TniIFP2(J04364), HaPLE1(EF593176).
Figure 4.The 5′ and 3′ transposable element (TE) displays (upper and lower panel, respectively) by using vectorette PCR for individual cotton bollworm (CBW) of the NJ strain. Individual CBW gDNA used for the TE displays were digested with either Hind III (lanes 1–4), Mlu I (lanes 5–8) or Sal I (lanes 9–12) and ligated to the anchoring bubble linker oligos.
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Figure 5.Nucleotide sequences of the inverted terminal repeats (ITRs) and the ?anking genomic sequences in HaPLE1 and HaPLE2 (A) and the ITR sequences for HaPLE1, HaPLE2, Heliothis virescens HvPLE1.1, T richoplusia ni piggyBac and Bombyx mori yabusame-1 (B). Underlining shows the ITRs, and the duplicated tetranucleotide targets are in bold.
Figure 6.PCR ampli?cation of HaPLE1 (1931 bp) and HaPLE2 (420 bp) from individuals in three Helicoverpa armigera populations; the NJ, GY and Oxford strains.
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as noted previously (Zimowska & Handler, 2006), it is important to be aware of potential interactions between piggyBac vectors introduced into species containing endogenous piggyBac elements.
Thus far, many piggyBac -like sequences have been found in the genomes of phylogenetically diverse range of organisms including fungi, plants, insects, crustaceans,urochordates, amphibians, ?sh and mammals, but most of them appear to be defective and are presumably now inactive (Sarkar et al ., 2003). Here, the identi?cation and analysis of PLEs in the H. armigera genome provide useful information for further study. The intact PLE (HaPLE1)obtained in our experiments is currently being tested for function and the feasibility of its being reconstructed as a transformation vector with high ef?ciency.Experimental procedure Insect strains
The H. armigera strain NJ was collected from cotton plants in Nanjing, Jiangsu Province, China in July 2004, and routinely reared on an arti?cial diet in an insectary. T wo laboratory strains,GY and Oxford were kindly provided by Professor Yidong Wu (Nanjing Agricultural University, Nanjing, China). The GY strain was originally collected from cotton plants in Gaoyang County,Hebei Province, China in August 2001, and reared on an arti?cial diet in the laboratory. The Oxford strain originated from Africa and has been kept in laboratories for more than 20 years. The samples used in degenerate PCR, inverse PCR and vectorette PCR in our study were randomly chosen individuals from the NJ strain.
Genomic DNA isolation, cloning and sequencing
Genomic DNA for PCR was extracted from single larval or adult insects by a phenol–chloroform extraction method (Wang et al .,2005). The PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced. The DNA sequences were compared with nonredundant databases by using the NCBI server with tblastx and tblastn (https://www.wendangku.net/doc/d23060339.html,/cgibin/BLAST). Sequence alignments were performed with CLUSTAL × 1.8 (Thompson et al ., 1997). The aligned sequences were used for construction of phylogenetic tree in MEGA version 3.1(Kumar et al ., 2004).
PCR strategies
A pair of degenerate primers, PLEF: 5′-TTYTTYACNGAYGARA T -HA T -3′ (Fig. 1A: 415–434) and PLER: 5′-GGYTTRTTNGGDA TRT -ACA T -3′ (Fig. 1A: 853–872), were designed in the highly conserved regions of the piggyBac gene family. In general, PCR was performed in a 50 μl reaction volume containing 50–100 ng genomic DNA, 0.8 μM of each degenerate primer, 0.2 mM of each dNTP , 2 mM of MgCl 2, and 2.5 U T akara LA -T aq DNA polymerase .A touchdown PCR protocol for the degenerate PCR consisted of denaturing genomic DNA at 94 °C for 3 min, then 20 cycles at 94 °C for 30 s, 55–45.5 °C (decreasing by ?0.5 °C per cycle) for 30 s, 72 °C for 1 min; 15 cycles at 94 °C for 30 s, 45 °C for 30 s,72 °C for 1 min and a ?nal extension at 72°C for 10 min.
Inverse PCR was followed with primers designed based on sequence information obtained from the degenerate PCR.
Genomic DNA (~1 μg) was digested with either Pst I or Hin dIII (Promega) for 10–16 h at 37 °C in a 25 μl total volume. The enzyme was inactivated at 80 °C for 20 min. The digested DNA was diluted to a ?nal concentration of 4–6 ng/μl and ligated using T4 DNA-ligase (Promega) at 4 °C for 16 h, followed by precipita-tion and resuspension in 25 μl water.
The following two primer pairs were used for the nested inverse PCR:
External primer pair:
HaPEF: 5′-GTGCCAGAGAA TGT AGCGTCAAACAA-3′ (Fig. 1A:601–626)
HaPER: 5′-ACCAACTCTTCGTAGCGATGATGCTT-3′ (Fig. 1A:705–730)
Internal primer pair:
HaPIF: 5′-CAGCCGTCAGAGTGAGTATTCCGACT-3′ (Fig. 1A:543–568)
HaPIR: 5′-TAGATGAGCAATTGTTGGGTTTTCGC-3′ (Fig. 1A:809–834)
The full-length PLE copy HaPLE1 was successfully ampli?ed from the same genomic DNA that was used for inverse PCR. The primers used for HaPLE1 were located in the 5′ and 3′ ?anking sequences:HaPF1: 5′-GGAAGACAGT ACCGAAGA TGGG-3′HaPR1: 5′-CT AGGTTCGCGCCA T AAAGCT -3′
The full-length PLE copy HaPLE2 was obtained by using com-binatorial pairs of primers designed on each ?anking sequence captured in the vectorette PCR. The pairs provided successful ampli?cation of HaPLE2 were:
HaPF2: 5′-A TGAAAACCTCTGCGAAC-3′HaPR2 : 5′-A TTTGAAGGGGTGT AGTGAA-3′
A pair of primers located in the common region of HaPLE1and HaPLE2, HaPLEF: 5′-CAGT AGCCA TTGGGACCTCG-3′ and HaPLER: 5′-TCCACAATAGCGTCGTTTCT -3′, were used to survey the distribution of HaPLE in different populations of H. armigera . The PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining.
Vectorette chain reaction for diversity of insertion sites
We employed a vectorette PCR strategy (Ko et al ., 2003; Wang et al ., 2005) to obtain the ?anking sequences of HaPLE and to examine the diversity of insertion sites. The vectorette constructs were modi?ed from Wang et al . (2005). Four anchoring bubble linker oligos were designed to make the vectorette unit for ligation to the Hind III digested genomic DNA, Mlu I digested genomic DNA and Sal I digested genomic DNA:
About 1 μg genomic DNA was digested at 37 °C overnight by using either Hind III, Mlu I or Sal I (Promega) in 25 μl total volume.After digestion, 3 pmol of anchor bubble unit, 50 nmol A TP (Sigma,St Louis, MO, USA) and six units of T4 DNA ligase (Promega) were added and the reaction was incubated at 4 °C for 16 h. Two consecutive rounds of nested PCR with two sets of primers were carried out. The primers used for the vectorette PCR were:
VPCR1: 5′-CCCTTCTCGAA TCGT AACCG-3′ (vectorette external primer)
VPCR2: 5′-CGT AACCGTTCGGTCCTCTG-3′ (vectorette internal primer)
SPR1: 5′-GAAACAAA TTCAAAAGTT ACACAAAGCCC-3′ (5′ speci?c
external primer)
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SPF1: 5′-AACGACGCT A TTGTGGAGTTTGCT -3′ (3′ speci?c external primer)
SPR2: 5′-CAAAAGTT ACACAAAGCCCAA T -3′ (5′ speci?c internal primer)
SPF2: 5′-AAAAGGCGGA TGACCAAAGCG-3′ (3′ speci?c internal primer)
Acknowledgements
We would like to thank Dr Alfred M. Handler for helpful comments on and reviewing the manuscript, and Professor Yidong Wu for providing two H. armigera strains, GY and Oxford. This work was supported by National Basic Research project (2006CB102003), NSFC project (30471144) and IAEA project (12 826/RO/RBF).References
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