Hai-Yan Ye 2012

¤ 2012 Zoological Society of Japan ZOOLOGICAL SCIENCE 29: 90–96 (2012)Complete Mitochondrial Genome of Locusta migratoria migratoria (Orthoptera: Oedipodidae): Three tRNA-like Sequenceson the N-strandHai-Yan Ye1, Li-Li Xiao1, Zhi-Jun Zhou2, and Yuan Huang1* 1College of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, 710062, Xi’an, China 2College of Life Sciences, Hebei University, 180 Wu-Si-Dong-Lu, 071002, Baoding, Chinamigratoria has been determined. This mitogenome contains the base compositional biases andcodon usage typical of metazoans, and the RSCU values indicate a negative correlation with theC and G contents in codon. The orientation and gene order of the L. migratoria migratoria is iden-tical to Locusta migratoria migratoiodes. An unusual feature of the L. migratoria migratoria mitog-enome is the presence of three tRN A-like structures on the N-strand: one tRN A Ile-like and twotRNA Leu(CUN)-like sequences. The tRN A-like sequences have proper folding structures and anti-codons sequences. Two repeated DN A sequences, Rpt I and Rpt II, were found in the A+T-richregion of the L. migratoria migratoria mitogenome. Both repeated sequences have various features.In the 5′ region of Rpt I, a 51 bp fragment is localized in the srRNA gene; and there are two tandemlysub-repeated DNA sequences (sub-Rpts), Rpt 1–4, within Rpt I and Rpt II. One stem-loop structureon the N-strand that may be involved in the N-strand replication initiation was found in the A+T-rich region.Key words: mitochondrial genome, Locusta migratoria migratoria, tRNA-like structure, repeated DNAThe animal mitochondrial genome (mitogenome) is gen-erally a circular DNA molecule, approximately 15–20 kb in size, usually containing 37 encoding genes for two ribo-somal RNAs (lrRNA and srRNA), 13 protein-coding genes (PCGs) and 22 transfer RNAs (Wolstenholme, 1992; Boore, 1999). Although the gene content of the insect mitogenome is highly conserved, some exceptions have been reported. For example, the mitogenome of Chrysomya putoria from the order Diptera has an extra tRNA Ile (Junqueira et al., 2004), while several species of Hemiptera contain variable numbers of tRNA genes (Shao and Barker, 2003; Thao et al., 2004). The mitogenome of Bombus ignitus, in the order of H ymenoptera, contains five extra tRNA-like structures, although their functionality in amino acid transfer remains uncertain (Cha et al., 2007).Additionally, the insect mitogenome contains one major non-coding region between srRNA and tRNA Ile, known as the A+T-rich region, which may contain signals for the replication and transcription of both strands (Clary and Wolstenholme, 1985; Wolstenholme, 1992; Boore, 1999). A number of signal sequences of the replication origin have also been reported, for example, a series of thymine nucle-otides (T-stretch), stem-loop structures, and some con-replication initiation (Zhang et al., 1995; Zhang and Hewitt, 1997; Lessinger et al., 2004; Saito et al., 2005).The A+T-rich region is one of the most variable seg-ments of mitogenome, due not only to its high rates of nucle-otide substitution and insertions-deletions, but also to the notable variation in size among closely related taxa and among individuals of the same species (Duenas et al., 2006; Fenn et al., 2007). The length polymorphism often results from the presence of a variable number of tandemly-repeated DNA sequences (Zhang and H ewitt, 1997). The structure and organization of the A+T-rich region have been extensively studied (Zhang and H ewitt, 1997; Snäll et al., 2002; Lessinger et al., 2004; Duenas et al., 2006). The tan-dem repeat unit located toward the srRNA gene end has been found in the A+T-rich region of several insect mitoge-nomes, such as Aed es aegypti (Duenas et al., 2006) and Locusta migratoria migratoiodes (Flook et al., 1995).In this article, we present the complete mitogenome sequence of the Locusta migratoria migratoria with informa-tion on base compositional bias, codon usage of PCGs, sec-ondary structures of tRNA molecules, and the structure orien-tation of the A+T-rich region, and also compare the Locusta migratoria migratoria mitogenome with other orthopteran insects, in particular with L. migratoria migratoiodes.MATERIALS AND METHODSSpecimen collection and DNA extractionAll samples of L. migratoria migratoria were collected at BuErJin, Xinjiang, China, in August 2005, and preserved in 100% ethanol* Corresponding author.Tel.:+86-29-85307819;Fax:+86-29-85310097;E-mail:yuanh@. doi:10.2108/zsj.29.90tRNA-like Sequences in Mitogenome 91and stored at 4°C until DNA extraction.Total genomic DNA was isolated from the leg muscle tissue of one female individual (voucher code M0633) using a routine phenol/chloroform method (Zhou et al., 2007), and was diluted to 50 ng/μl with double-distilled water, and used as a template in LA-PCR.PCR and sequencingTwo sets of primers were used for amplifying the complete mitogenome in two overlapping fragments (Liu et al., 2006). The “A” fragment amplification profile consisted of 93°C for 2 min, 40 cycles of 92°C for 10 s, 57°C for 30 s, and 68°C for 8 min in the first 20 cycles and increased 20 s per cycle in the subsequent 20 cycles. The “B” fragment amplification profile consisted of 93°C for 2 min, 40 cycles of 92°C for 30 s, 59°C for 30 s., and 68°C for 7 min. The reaction mixture contained 0.9 units LA Taq polymerase, 1× LA PCR Buffer II (Mg 2+ free), 0.33 mM dNTPs, 2.5 mM MgCl 2, 1.47 mM primers, and 150 ng of genomic DNA, in final volume 15 μL. LA-PCR products were purified with DNA Gel Purification Kit (U-Gene) after separation by electrophoresis in a 1.0% agarose gel.Twenty-eight sets of primers were used for sub-PCR amplifica-tion of the complete mitogenome using LA-PCR amplicons as tem-plates. The PCR program consisted of 94°C for 2 min, 30–35 cycles of 94°C for 10 s, 38–58°C for 30 s, 72°C for 1–2 min. The final elon-gation step was continued at 72°C for 7 min. All Sub-PCR amplifi-cations were performed on the MyCycler thermal cycler with 1.5 units TaKaRa Taq polymerase (Takara), 1× PCR Buffer (Mg 2+ free), 0.2 mM dNTPs, 2.5 mM MgCl 2, 1–2 mM primers, and 50 ng the LA-PCR products, in a final volume of 25 μL reaction mixture.All purified sub-PCR products were directly sequenced from both strands using the ABI PRISM™ 3100-Avant Genetic Analyzer. Cycle sequencing conditions were: 96°C for 1 min, 35 cycles of 96°C for 10 s, 38–50°C for 1 min, and 60°C for 4 min. Sequence primers used were the same as those for Sub-PCRs.Sequence assembly, annotation and analysisSequence assembly and annotation analysis was performed using the Staden software package (Staden et al., 2000). The PCGs and rRNA coding genes were identified by comparing sequences between L. migratoria migratoiodes and Ruspolia dubia (Flook et al., 1995; Zhou et al., 2007). The tRNAs were identified by tRNAscan-SE search server v.1.21 (Lowe and Eddy, 1997). Putative tRNA genes that failed to be identified by tRNAscan-SE were further analyzed by sequence alignment, and then confirmed by respective anticodon sequences. The alignments of complete mitogenome sequences with excluding A+T-rich regions were cal-culated using ClustalX (Thompson et al., 1997) and BioEdit 7.0.0 (Hall, 1999). Positions of obviously misaligned were manually cor-rected. The aligned data were further analyzed by MEGA version 3.0 (Kumar et al., 2004) and the Staden sequence analysis package (Staden et al., 2000).The annotated complete mitogenome sequence of L. migratoria migratoria has been submitted to GenBank (EU287446).RESULTS AND DISCUSSIONGenomic structure, organization, and compositionThe L. migratoria migratoria mitogenome is 16053 bp inlength, and 331 bp larger than that of the L. migratoria migratoiodes (Flook et al., 1995). The primary size variation was found in the A+T-rich region. The sequence analysis results revealed that the mitogenome of L. migratoria migratoria contains a typical gene content in metazoan mitogenomes: 13 protein-coding genes, 22 tRNA genes, 2 rRNA (lrRNA and srRNA) subunits, and A+T-rich region (Wolstenholme, 1992; Boore, 1999). Additionally, three tRNA-like structure sequences were found in the L. migratoria migratoria mitogenome (Fig. 1; Table 1).The orientation and gene order of the L. migratoriaFig. 1. Linear arrangement of genes in the mitogenome. All genes are encoded on the N-strand are indicated by underbars. The tRNA genes are labeled by a one-letter symbol, and L1, L2, S1 and S2 denote codon tRNA Leu(CUN), tRNA Leu(UUR), tRNA Ser(AGN) and tRNA Ser(UCN), respectively.Table 1. Summary of the L. migratoria migratoria mitogenome.Gene DirectionPosition Size (bp)initiation codon termination codon IR/OR*(bp)Cover Score tRNA Ile(I)F 1–656519.36tRNA Gln(Q)R 63–13169–336.73tRNA Met(M)F 131–19868–133.46ND2F 199–12191023ATGTAAtRNA Trp F 1220–12866734.81tRNA Cys R 1279–134668–830.51tRNA Tyr R 1357–1424681031.94COIF 1431–29731543ATTA TAA6tRNA Leu(CUN)-likeI R 2968–30336636.12tRNA Leu(UUR)F 2969–303466–544.7COII F 3038–3721684ATG TAA3tRNA AspF 3720–37836420.05tRNA Lys F 3787–385771325.76ATP8F 3875–4033159ATC TAA 17ATP6F 4027–4704678ATG TAA –7COXIII F 4709–5500792ATG TAA4tRNA GlyF 5504–556865331.37tRNA Ile -like R 5504–55686531.37ND3F 5569–5922354ATT TAGtRNA Ala F 5921–59856530.97tRNA Arg F 5991–605565522.03tRNA AsnF 6056–61206513.32tRNA Ser(AGN)F 6121–618767 5.02tRNA Leu(CUN)-likeII R 6186–62546525.04tRNA Glu F 6188–62546732.58tRNA Phe R 6253–631967–229.14ND5R 6320–80331714ATT T tRNA His R 8049–8114661519.23ND4R 8116–94501335ATG TAA 1ND4L R 9444–9737294ATGTAA–7tRNA Thr F 9740–980364229.95tRNA Pro R 9804–98696625.49ND6F 9872–10393522ATG TAA 2CYTBF 10398–115371140ATG TAA 4tRNA Ser(UCN)F 11537–1160670–151.89ND1R 11628–12572945ATATAA21tRNA Leu(CUN)R 12576–126416623.54lrRNA R 12642–139581317tRNA Val R 13959–140307228.45srRNAR14031–14864834A+T-rich region14865–160531189* IR: intergenic spacer sequence (positive values), OR: genes overlap (negative values), without taking the tRNA-like sequences into account.H.-Y. Ye et al.92migratoria mitogenome are identical to that of L. migratoria migratoiodes. In comparison with the hypothesized ances-tral arthropod arrangement, the positions of tRNA Lys andtRNA Asp are translocated.Gene overlap is slightly different between the mitoge-nomes of L. migratoria migratoria and L. migratoria migratoiodes. When tRNA-like sequences are not accounted, overlapping genes in the L. migratoria migratoria mitoge-nome are 40 bp in total, distributed among eleven loci, withsize ranging from 1 to 8 base pairs (Table 1). In the mitog-enome of L. migratoria migratoiodes, ovelapping genes are42 bp in total, distributed among ten loci, with size rangingfrom 1 to 10 base pairs. Without considering the tRNA-like sequences, the intergenic spacer sequences (total 78 bp)are spread over 15 regions ranging in size from 1 to 21 bp,with the largest one (21 bp) located in the conserved posi-tion between tRNA Ser(UCN) and ND1 (Flook et al., 1995; Kimet al., 2005; Zhou et al., 2007).Pairwise alignment revealed significantly higher sequ-ence similarity between L. migratoria migratoria and L. migratoria migratoiod es mitogenomes. When excludingA+T-rich regions, 474 nucleotides, a proportion of 3.19% intotal of aligned 14864 bp, are variable sites between the mitogenome sequences of L. migratoria migratoria (14864bp) and L. migratoria migratoiodes (14847 bp).Protein-coding genesThe invertebrate mitochondrial genetic code was usedto identify open reading frames (ORFs) of the L. migratoria migratoria mitogenome, which were then matched to thoseof L. migratoria migratoiodes for identifying the PCGs. All ofthe putative PCGs in the L. migratoria migratoria containseven potential initiation codons (ATN and NTG), except forCOI, which contains tetranucleotide ATTA. At the sametime, the conventional termination codons (TAA and TAG)were used by most of the putative PCGs, except for the ND5gene with incomplete termination codon (T-tRNA Phe). Incom-plete termination codons (T-tRNA and TA-tRNA) are a com-monly feature of insect mitogenomes (Carapelli et al., 2006;Kim et al., 2006; Cha et al., 2007; Zhou et al., 2007), rangingfrom two of nine genes in Gomphiocephalus hodgsoni (Col-lembola: Nardi et al., 2003) and Ruspolia dubia (Orthoptera: Zhou et al., 2007) to eight of nine genes in Gryllotalpa pluvialis (Orthoptera: Gameron, unpublished data) and Gryllotalpa orientalis (Orthoptera: Kim et al., 2005). The common interpretation for the incomplete termination codon was that it might post-transcriptionally be completed to TAA after cleavage (Anderson et al., 1981; Bibb et al., 1981; Ojala, 1981; Okimoto et al., 1990; Lavrov et al., 2002) and may be a product of the selective pressure to reduce genome size (the race-for-replication hypothesis, Rand, 1993, 2001). The L. migratoria migratoria mitogenome shows the lowest level of incomplete termination codons usage by any insects to date, which may suggest that it is under relaxed selective pressure.The number of each codon, relative synonymous codon usage (RSCU) values and the amino acid compositions of the PCGs in the L. migratoria migratoria mitogenome (Table 2) were analyzed with MEGA 3.0 (Kumar et al., 2004). As shown in Table 2, the RSCU values are negatively corre-lated with the C and G contents in codon. The codon CGC (Arg) was not used by the PCGs of L. migratoria migratoria mitogenome. The total content of the four most frequently represented amino acids (Leu, Ser, Ile and Phe) was 43.07%; the ratio of Leu (14.05%) is the highest among these. The four most frequently used codons, TTA (leucine), ATT (isoleucine), TTT (phenylalanine), and ATA (methion-ine), are all wholly composed of A and/or T. These four codons accounted for 31.28% of L. migratoria migratoria mitogenome.The base composition of nucleotide sequences has been described as skewness (Perna and Kocher, 1995), which measures the relative number of A to T (AT skew = [A − T]/[A + T]) and Gs to Cs (GC skew = [G − C]/[G + C]). The AT skew of the PCGs encoded on the N-strand (−0.385) is markedly more negative than the whole set of PCGs (−0.113) and on the J-strand (0.065). These data indi-cate that the As occurred less frequently in the PCGs on the N-strand than that of the Ts, and the direction reversed on J-strand, with a slight dominance of Ts. In contrast, the GC skew of the whole set of PCGs is close to zero (−0.047), and Table 2. Codon usage and amino acid compositions of the PCGs in L. migratoria migratoria mitogenome a.AminoAcidCodonn/RSCU%AminoAcidCodonn/RSCU%Phe (GAA)b UUU243/1.449.10%Tyr (GUA)UAU151/1.69 4.80% UUC95/0.56UAC28/0.31Leu (UAA)UUA344/3.9510.31%Ter c UAA0/0.00UUG39.0/0.45UAG0/0.00Leu (UAG)CUU59/0.68 3.74%His (GUG)CAU51/1.46 1.88% CUC4/0.05CAC19/0.54CUA75/0.86Gln (UUG)CAA60/1.90 1.70%CUG1/0.01CAG3/0.10Ile (GAU)AUU331/1.7610.12%Asn (GUU)AAU141/1.49 5.09% AUC45/0.24AAC48/0.51Met (CAU)AUA244/1.717.67%Lys (CUU)AAA70/1.43 2.64% AUG41/0.29AAG28/0.57Val (UAC)GUU92/2.04 4.85%Asp (GUC)GAU67/1.70 2.13% GUC8/0.18GAC12/0.30GUA79/1.76Glu (UUC)GAA71/1.80 2.13%GUG1/0.02GAG8/0.20Ser (UGA)UCU122/2.68 6.87%Cys (GCA)UGU39/1.70 1.24% UCC7/0.15UGC7/0.30UCA121/2.66Trp (UCA)UGA95/1.92 2.67%UCG5/0.11UGG4/0.08Pro (UGG)CCU55/1.61 3.69%Arg (UCG)CGU21/1.50 1.51% CCC3/0.09CGC0/0.00CCA74/2.16CGA33/2.36CCG5/0.15CGG2/0.14Thr (UGU)ACU54/1.10 5.28%Ser (GCU)AGU23/0.51 2.93% ACC14/0.29AGC1/0.02ACA126/2.57AGA82/1.80ACG2/0.04AGG3/0.07Ala (UGC)GCU63/1.68 4.04%Gly(UCC)GGU92/1.77 5.60% GCC7/0.19GGC1/0.02GCA75/2.00GGA104/2.00GCG5/0.13GGG11/0.21a: A total of 3714 codons were analyzed, excluding the termination codonsof all PCGs and initiation codon of COI. b: The anticodons of the corre-sponding tRNA is shown in parentheses for each codon designation. c: Termination codons. n = The number of each codon. RSCU = Relative syn-onymous codon usage. % = percentages of amino acids in compositions of protein-coding genes of the L. migratoria migratoria mitogenome. For the amino acids Leu and Ser, separate subtotals the percentage reflecting the use of each of the two codon families.tRNA-like Sequences in Mitogenome93the PCGs encoded on J-strand (−0.168) and N-strand(0.185) are in reversed direction.Transfer RNA (tRNA) genes and tRNA-like sequencesTwenty tRNA genes and three tRNA-like sequenceswith high COVE scores (over 15) were predicted using thetRNAscan-SE program (Lowe and Eddy, 1997). The COVEscores are shown in Table 1. The COVE scores of tRNA Asnand tRNA Ser(AGN) were 13.32 and 5.02. Combined with theresults from analysis using the Staden package (Staden etal., 2000), all 22 tRNA genes and three tRNA-likesequences were found in the L. migratoria migratoria mito-genome. The relative locations were shown in Table 1 andFig. 1.In the L. migratoria migratoria mitogenome, all tRNAgenes and tRNA-like sequences can be folded into typicalcloverleaf secondary structures (Fig. 2), except fortRNA Ser(AGN) according to the Steinberg Cedergren tertiarystructure (Steinberg et al., 1997; Zagryadskaya et al., 2004).The Steinberg Cedergren tertiary structure of tRNA Ser(AGN)was more similar to normal tRNA in the L-shape of three-dimensional modeling (Fig. 3). The tRNA Ser(AGN) sequencecould not form a stable stem loop structure in the DHU armsimilar to those found in some other insects (e.g., Kim et al.,2005, Cameron and Whiting, 2007; Wolstenholme, 1992;Zhao et al., 2010) and bilaterian animals (e.g., Kim et al.,2005, 2006). Garey and Wolstenholme (1989) proposed thatthe missing D-stem in tRNA Ser(AGN) evolved very early in theevolution of Metazoa. Despite lacking this stem, this tRNAis normally considered to be functional (Steinberg andCedergren, 1994; Stewart and Beckenbach, 2003).One unusual feature of the L. migratoria migratoria mito-genome is the presence of three tRNA-like sequences: twotRNA Leu(CUN) and one tRNA Ile structure, as shown in Fig. 4.These tRNA-like sequences is encoded on the N-strand,and is located at the rough position of tRNA Leu(UUR), tRNA Glyand tRNA Glu, which is encoded on the J-strand. Those threetRNA-like sequences can also be folded into clover-leaf sec-ondary structure with the proper anticodons. H owever, thesequences on other opposite strand position of the tRNAgenes cannot be folded into the clover-leaf secondary struc-ture as any forced folding will result in mismatch or incorrectanticodons. The uniform tRNA-like sequences can also befound in the L. migratoria migratoiod es, although a fewnucleotides and structures were modified on the tRNA Ile-likeand tRNA Leu(CUN)-likeII, respectively (Fig. 4). Similar tRNA-like sequences (tRNA Leu(UUR), tRNA Ser(AGN) and tRNA Phe)have been reported in the B. ignitus mitogenome (Cha etal., 2007).The tRNA-like sequence may have originated by partialduplication of mtDNA caused by erroneous initiation or ter-mination in replication (Macey et al., 1997) or strand slip-migratoria migratoria mitogenome. The tRNAs are labeled with theabbreviations of their corresponding amino acids. Dashes (–) indi-cate Watson-Crick base-pairing and plus signs (+) indicate G/Ubase-pairing.H.-Y. Ye et al.94 page and mispairing (Madsen et al., 1993), and have been involved in the processes of translation regulation (Fechter et al., 2001). The three tRNA-like sequences have anti-codons and clover-leaf structures, which make us reluctantly to designate them as pseudogenes. Mimicry at the molecu-lar level is considered to have important functional implica-tions, not only as a curiosity of nature (Romby and Springer, 2003). In consideration of the content of amino acid compo-sitions, Leu at 14.05% and Ile at 10.12% (Table 2), the tRNA-like sequences found in the L. migratoria migra-toiodes may have important role in protein biosynthesis or transcription regulation.Ribosomal RNA (rRNA) genes and A+T-rich regionAs in most insect mitogenomes, the two rRNA genes in L. migratoria migratoria mitogenome are located in the con-served position between tRNA Leu(CUN) and tRNA Val , and between tRNA Val and the A+T-rich region, respectively (Table 1). The length of the lrRNA and srRNA are 1317 bp and 834 bp, respectively. The A+T content of the lrRNA and srRNA are 78.50% and 76.26%.The A+T-rich region is well known for the initiation of replication and transcription in animals, and the length ranges from tens to several thousands bp (Lewis et al., 1995; Shao et al., 2001; Zhou et al., 2007). The A+T-rich region of the L. migratoria migratoria mitogenome locates between the srRNA and tRNA Ile gene. This region is 1189 bp in size and contains 84.86% A+T. It is larger than in L. migratoria migratoiodes (875 bp, 86.0%) in size, though their base composition is very similar.Sequence analysis using Dot-Plot (Sonnhammer and Durbin, 1995) showed that there are two repeat DNA sequences (Rpt I and Rpt II, with an interval of 31 bp between them) in the A+T-richregion of the L. migratoria migrato-ria mitogenome. In Rpt I, a 51 bp sequence in the 5′ region is shared with the srRNA gene. But the tan-dem repeat DNA sequences loca-tion toward the srRNA gene end have been found in the A+T-rich region of several insect mitogenomes , such as L. migratoria migratoiod es (Flook et al., 1995) and Aedes aegypti (Duenas et al., 2006). An unusual feature of the L. migratoria migratoria mitogenome is that two tandemly sub-Rpt (Rpt 1–4, 123 bp in size, only Rpt4 showed one deleted base) were found in the interior of Rpt I and Rpt II, but thishas not been found in L. migratoria migratoiodes . The structural organi-zation and relative positions of these Rpts are shown in Fig. 5.Previous studies found that the replication origin (O R ) of four Drosophila species, Bombyx mori and Triborium casta-neum (Saito et al., 2005), which are holometabolous insects,Fig. 3. The L-shape modeling of tRNA Ser(AGN) three-dimensional (A) and the secondary structure of tRNA Ser(AGN) (B) in the L. migra-toria migratoriamitogenome. Fig. 4. Changes of predicted secondary structure of the tRNA-like sequence between L. migra-toria migratoria and L. migratoria migratoiodes mitogenomes. In comparison with L. migratoriamigratoria , the modified parts in the L. migratoria migratoiodes are highlighted in boxes.Fig. 5. The structural organization and relatively position of the Repeated DNA sequence (Rpt) in the A+T-rich region of L. migratoria migratoria (A) and L. migratoria migratoiodes (B) mitoge-nome. The number indicted the relatively positions of Rpts in the A+T-rich region (on) and wholemitogenome (down), respectively.tRNA-like Sequences in Mitogenome 95located immediately downstream of a series of thymine nucleotides (T-stretch). For L. migratoria migratoiod es (hemimetabolous insect), in which none of the long T-stretch was found in the upstream portion of the O R , one possible stem-loop structure formed immediately on the upstream of the O R on the N-strand (Saito et al., 2005), and this possible stem-loop structure was also located at a similar position in the desert locust Schistocerca gregaria and the meadow grasshopper Chorthippus parallelus (Zhang et al., 1995). This stem-loop structure can also be found in the A+T-rich region (N-strand) of the L. migratoria migratoria mitogenome (Fig. 6).ACKNOWLEDGMENTSWe thank Dr. Lu Hui-meng for his assistance in primer design and discussion during in the experiments.This study was supported by the National Natural Science Foundation of China (Grant No. 30970346) and Natural Science Foundation of Shaanxi Province (Grant No. 2007C101).REFERENCESAnderson S, Bankier AT, Barrell BG, de Bruijin MHL, Droujn ARJ,Eperon IC, et al. 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