Abstract:
The present invention provides a chitinase that can be used as a disease control agent for plants, as well as a gene encoding the chitinase. A family 19 chitinase isolated from yam and a gene encoding the chitinase are disclosed.

Description:
BACKGROUND OF THE INVENTION 1. Technical Field  
         [0001]    The present invention relates to a novel chitinase from yam and a gene encoding the chitinase. This chitinase has a strong lytic activity and, thus, is useful as an agent for controlling plant pathogens.  
           [0002]    2. Prior Art  
           [0003]    A great number of edible or ornamental plants are cultured at present. Generally, such cultivars are weaker against pathogenic fungi and bacteria than wild-type species. Thus, it is necessary to apply large quantities of agricultural chemicals (agents for controlling plant pathogenic fungi or bacteria) for obtaining sufficient yields. As agents for controlling plant pathogenic fungi or bacteria, chemically synthesized agents of heterocyclic aromatic compound type or organic phosphate ester type have been mainly used to date. However, these chemicals not only manifest their effect on pathogenic fungi or bacteria, but they also have an adverse effect on the human body and cause the problem of residual agricultural chemicals.  
           [0004]    Chitinase is an enzyme that hydrolyzes chitin. There are known chitinases belonging to family 18 and those belonging to family 19. It is known that chitinases are involved in the plant defense mechanism against pathogenic fungi and bacteria. Plants infected by pathogenic fungi or bacteria protect themselves by producing chitinases and degrading the pathogen with the chitinase. It is expected that, when such chitinases are applied to soils or plant bodies, they would manifest the same effect as that of the chitinases produced in the plant bodies and thus could protect the plants from infection with pathogenic fungi and/or bacteria. Since chitinases are substances produced by organisms, it can be considered that agents for controlling plant pathogens utilizing chitinases are highly safe against the human body and environments.  
           [0005]    Several reports have already been made on the use of plant-derived chitinases as agents for controlling plant pathogens. For example, the present inventors have isolated a chitinase belonging to family 18 from yam and revealed that the chitinase exhibits control effect on pathogens such as  Pyricularia oryzae  (fungus that causes rice blast) (Japanese Unexamined Patent Publication No. 2000-109405).  
           [0006]    The finding of a novel plant chitinase will lead to the development of novel agents for controlling plant pathogens. Besides, for efficient production of agents for controlling plant pathogens using the chitinase, it is necessary to isolate the gene encoding the chitinase.  
         SUMMARY OF THE INVENTION  
         [0007]    The present invention has been made under these circumstances for the purpose of providing a novel gene encoding a plant chitinase.  
           [0008]    As a result of intensive and extensive researches toward the solution of the above problem, the present inventors have found in yam a novel chitinase belonging to family 19 that is different from the previously found chitinase belonging to family 18. The present invention has been achieved based on this finding.  
           [0009]    The present invention relates to a yam chitinase gene encoding the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence substantially identical thereto.  
           [0010]    The present invention also relates to a yam chitinase represented by the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence substantially identical thereto. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0011]    [0011]FIG. 1 is a diagram showing the amino acid sequence of the chitinase of the invention including the signal sequence, chitin binding domain and catalytic domain of the chitinase. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0012]    Hereinbelow, the present invention will be described in detail.  
         [0013]    The chitinase of the invention is represented by the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence substantially identical thereto; and the chitinase gene of the invention encodes the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence substantially identical thereto. The term “amino acid sequence substantially identical thereto” used herein means the amino acid sequence of SEQ ID NO: 2 having a mutation(s) (such as deletion, replacement or addition of one or more amino acids) that does/do not allow the represented protein to lose its function as a chitinase.  
         [0014]    By integrating the chitinase gene of the invention into a microorganism plasmid, it is possible to express this gene in the microorganism. Such a plasmid must have at least a replication origin functional (i.e. autonomously replicating) in the host microorganism. Further, it is extremely desirable for such a plasmid to have selection marker genes which are used as markers for selecting transformants. As selection marker genes, genes that are able to confer antibiotic resistance may be used. Specific examples of well-known selection marker genes include ampicillin resistance gene and tetracycline resistance gene. Furthermore, the above-mentioned plasmid often has a promoter sequence capable of directing the expression of a constitutive gene. Alternatively, a promoter sequence may be inserted into the plasmid together with a constitutive gene. These techniques are well known in the art. One of ordinary skill in the art can select and use appropriate techniques.  
         [0015]    Plasmids are introduced into microorganism cells and function therein. Methods for introducing plasmids into microorganism cells are well known. One of ordinary skill in the art may select and use appropriate methods from those known methods.  
         [0016]    As host microorganisms,  Bacillus subtilis, Escherichia coli  and  Saccharomyces cerevisiae  are well known and used widely. In particular,  E. coli  is used frequently for the purposes of gene amplification and selection.  
         [0017]    Specific examples of hosts such as  B. subtilis, E. coli  and yeast and specific examples of useful plasmids are described in a large number of documents. One of ordinary skill in the art may select and use appropriate ones from them.  
         [0018]    Culturing a microorganism transformed with a plasmid to thereby obtain a chitinase does not need to be a special process. Briefly, the transformed microorganism is cultured in a medium where it can grow well and under conditions that allow its good growth. Subsequently, the chitinase produced in the medium, inside of the cells or around the cell membranes is recovered. Methods for isolation/purification of polypeptides such as chitinase are also well known. One of ordinary skill in the art may combine these known methods to isolate and purify the chitinase.  
         [0019]    The thus obtained chitinase of the invention has a strong lytic activity. Therefore, this chitinase can be used as an agent for controlling plant pathogenic fungi and bacteria.  
       EXAMPLE  
       [0020]    (1) Determination of Partial cDNA Sequence  
         [0021]    Yam ( Dioscorea opposita  Thunb) callus was induced from seedlings on MS agar medium (containing 10 −4  M 2,4-dichlorophenoxyacetic acid [2,4-D], 10 −5  M kinetin and 5% sucrose) in the presence of 0.1% activated charcoal at 27° C. in the dark. The resultant callus (about 300 mg) was treated with 50μl of a suspension of  Fusarium oxysporum  macroconidia (0.5-1.5 mg). Total RNA was extracted from the  F. oxysporum -inoculated callus. mRNA was separated from the total RNA by affinity chromatography using oligo dT-cellulose and then cDNA was synthesized from the mRNA. A cDNA encoding a yam chitinase was selectively amplified by polymerase chain reaction (PCR). Primers for the PCR were synthesized based on nucleotide sequences deduced from partially known amino acid sequences of chitinase. The PCR products were subcloned and sequenced.  
         [0022]    (2) Determination of Partial Genomic DNA Sequence  
         [0023]    Genomic DNA was extracted from yam leaves, and PCR was performed with primers synthesized based on the partial cDNA sequence. The PCR products were subcloned and sequenced.  
         [0024]    (3) Preparation of Oligonucleotide Primers  
         [0025]    Gene specific primers were synthesized based on the partial genomic DNA sequence. Random primers were purchased from BEX Co., Ltd. Their melting temperatures (Tms) were calculated using the following formula:  
         69.3+0.41(% GC)−650/L  
         [0026]    (Mazars et al., 1991) where L is primer length (Table 1).  
                                                                                           TABLE 1                       Primer   Sequence   Melting temp.   SEQ ID NO                                Gene specific primer                GSP-F1   5′-ATGGAGAACTGCCAGTGCGA-3′   59.4   SEQ ID NO: 3                   GSP-F2   5′-TGCAGCTTACTTCGCCCAT-3′   56.7   SEQ ID NO: 4               GSP-F3   5′-CTACTGTCAAGAAAGCCAAC-3′   55.3   SEQ ID NO: 5               GSP-F4   5′-GTACTTCGGACGTGGACC3-3′   58.2   SEQ ID NO: 6               GSP-F5   5′-CTCATCAATTTCCAGCCACTC-3′   57.9   SEQ ID NO: 7               GSP-F6   5′-CGACTATTGTGGACCGGG-3′   58.2   SEQ ID NO: 8               GSP-R1   5′-AACCAGAGAGAAGTCTTGAK-3′   53.2   SEQ ID NO: 9               GSP-R2   5′-TGTAGAAGCTTTTACCGGGA-3′   55.3   SEQ ID NO: 10               GSP-R3   5′-CAFCACACTCTTGGCCGC-3′   58.2   SEQ ID NO: 11               GSP-R4   5′-TAGTCGAATTTAAGCCAAGTTC-3′   54.7   SEQ ID NO: 12               GSP-R5   5′-GGTCCACGTCCGAAGTAC-3′   58.2   SEQ ID NO: 13                    Random primer                A28   5′-TACCCTCAAGCT-3′   35.6   SEQ ID NO: 14                   A02   5′-GCCAGCTGTACG-3′   42.5   SEQ ID NO: 15                  
 
         [0027]    (4) Cloning of the 5′ Region of the Chitinase Gene  
         [0028]    The primary PCR was carried out in a 50 μl solution containing 50 ng of genomic DNA, 0.4 μM gene specific primer (GSP-R1), 0.4,μM random primer (A28), 200 μM each of dNTPs, 1 U of Ex Taq polymerase (TAKARA BIO INC.) and l×Ex Taq™ buffer. Thermal cycling conditions were set as shown below.  
                               TABLE 2                                       Denaturation   94° C. × 2 min    1 cycle           Denaturation   94° C. × 1 min   35 cycles           Annealing   50° C. × 2 min           Extension   72° C. × 3 min           Extension   72° C. × 7 min    1 cycle                      
 
         [0029]    The PCR was performed with Astec Program Temp Control System PC-800.  
         [0030]    The PCR products were purified with QIA Quick PCR Purification kit (Qiagen) and eluted with 50 μl of an elution buffer consisting of 10 mM Tris-HCI (pH 8.5).  
         [0031]    The secondary PCR was performed in three ways using (i) a combination of 0.4 μM GSP-R2 (this primer is located at a nested position) and 0.4 μM random primer A28 (the same primer used in the primary PCR); (ii) GSP-R2 alone; or (iii) A28 alone. The reaction composition and the thermal cycling conditions were the same as in the primary PCR except that 1 μl of the primary PCR product was used as a template and that 35 cycles were reduced to 25 cycles. The PCR products were separated by ⅕% agarose gel electrophoresis. The DNA band obtained from the PCR using the primer combination of GSP-R2 and A28 was cut out from the agarose gel and purified with Geneclean II kit (BIO 101, Inc.). The purified DNA fragment was subcloned into TOPO vector, which was introduced into  E. coli  using TOPO™ TA Cloning kit (Invitrogen). Positive clones were selected by colony PCR as described below. Briefly, a colony was picked up with a sterile toothpick and swilled in 40 μl of sterile water. The colony in sterile water was transferred into a heat block pre-heated to 95° C., boiled for 10 min, placed on ice immediately and used as a template. With this template, PCR was performed in a 50 μl solution containing 0.4 μM each of GSP-F1 and GSP-R2. Other components of the reaction solution were the same as in the primary PCR. The thermal cycling conditions were set as shown below.  
                               TABLE 3                                       Denaturation   94° C. × 2 min    1 cycle           Denaturation   94° C. × 1 min   25 cycles           Annealing   56° C. × 1 min           Extension   72° C. × 1 min           Extension   72° C. × 7 min    1 cycle                      
 
         [0032]    Plasmid DNA was prepared from each of the positive clones using QIAprep Spin Miniprep kit (Qiagen) and then sequenced.  
         [0033]    (5) Cloning of the 3′ Region of the Chitinase Gene  
         [0034]    The primary PCR was performed using 0.4 μM GSP-F2 which was used both as a gene specific primer and as a random primer. The reaction composition, the thermal cycling conditions and the purification of PCR products were the same as in the primary PCR for cloning the 5′ region. The secondary PCR was performed in three ways using (i) a combination of 0.4 μM GSP-F3 (this primer is located at a nested position) and 0.4 μM random primer A02; (ii) GSP-F3 alone; or (iii) A02 alone. The reaction composition and the thermal cycling conditions were the same as in the primary PCR except that 1 μl of the primary PCR product was used as a template. The PCR products were separated by 1.5% agarose gel electrophoresis. The DNA band obtained from the PCR using the primer combination of GSP-F3 and A02 was cut out from the agarose gel and purified with Geneclean II kit (BIO 101, Inc.). The purified DNA fragment was subcloned in the same manner as described in the cloning of the 5′ region. Then, positive clones were selected by the colony PCR method described in the cloning of the 5′ region. This PCR was performed using 0.4 μM each of GSP-F4 and GSP-R3. The reaction composition was the same as in the primary PCR. The thermal cycling conditions were set as shown below.  
                               TABLE 4                                       Denaturation   94° C. × 2 min    1 cycle           Denaturation   94° C. × 30 sec   25 cycles           Annealing   60° C. × 30 sec           Extension   72° C. × 30 sec                      
 
         [0035]    The preparation of plasmid DNA from positive clones and sequencing of the DNA were carried out in the same manner as in the cloning of the 5′ region.  
         [0036]    (6) Cloning of the Full-Length Yam Chitinase Gene by High Fidelity PCR  
         [0037]    Based on the newly identified DNA sequences, gene specific primers GSP-F5, -F6, -R4 and -R5 were synthesized (Table 1). In order to isolate the full-length yam chitinase gene, high fidelity PCR was performed in a 50 μl solution containing 50 ng of genomic DNA, 0.4 μM each of GSP-F5 and GSP-R4, 200 μM each of dNTPs, 1.25 U of Pyrobest DNA polymerase (TAKARA BIO INC.) and 1×Pyrobest Buffer IH. The thermal cycling conditions were set as shown below.  
                               TABLE 5                                       Denaturation   94° C. × 2 min    1 cycle           Denaturation   94° C. × 30 sec   25 cycles           Annealing   60° C. × 1 min           Extension   72° C. × 2 min                      
 
         [0038]    PCR products were purified with QIA Quick PCR Purification kit (Qiagen) and eluted with 30 μl of an elution buffer consisting of 10 mM Tris-HCI (pH 8.5). The purified DNA fragments were subcloned in the same manner as in the cloning of the 5′ region. Colony PCR was performed using 0.4 μM each of GSP-F6 and GSP-R5. The reaction composition was the same as in the primary PCR for cloning the 5′ region. The thermal cycling conditions were set as shown below.  
                               TABLE 6                                       Denaturation   94° C. × 2 min    1 cycle           Denaturation   94° C. × 30 sec   25 cycles           Annealing   60° C. × 30 sec           Extension   72° C. × 1 min                      
 
         [0039]    The preparation of plasmid DNA from positive clones and sequencing of the DNA were carried out in the same manner as described in the cloning of the 5′ region. The nucleotide sequence of the full-length yam chitinase gene is shown in FIG. 1 and SEQ ID NO: 1. In addition, the amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO: 2.  
         [0040]    The present invention provides a yam-derived chitinase belonging to family 19 and a gene encoding the chitinase. Since this chitinase has lytic activity, it can be used as an agent for controlling plant pathogenic fungi and bacteria.  
         [0041]    The entire disclosure of Japanese Patent Application No.2002-055222 filed on Mar. 1, 2002 including specification, claims, drawings and summary is incorporated herein by reference in its entity.  
         [0042]    All publications, patents and patent applications cited herein are incorporated herein by reference in their entity.  
     
       
       
         1 
         
           
             15  
           
           
             1  
             1631  
             DNA  
             Dioscorea oppositifolia  
             
               CDS  
               (343)...(763)  
             
             
               intron  
               (764)...(887)  
             
             
               CDS  
               (888)...(1324)  
             
           
            1 

ctcatcaatt tccagccact caatttgcat ttagacatgc atgtgtgact atatcagttt     60 

cagatctttt aatttgtatt ttttttcttt ttcatttaaa ttaaattaaa ttcagctttc    120 

agctttacat gcacacatgt gtattatttt aattaatgta ctatgaaatt aagtgggacc    180 

cttcaaacca taccataatt aatataatac aaagaagata agctcaacga aagatgagtc    240 

acagaagagg acagaagtaa cattgctata aatacgctcc gttctccatt caaaaccttc    300 

acaacaaaga aaaaaaaaca agaagtacta gtaattaaga at atg cat tca ttt       354 
                                               Met His Ser Phe 
                                                1 

aga atg ata ttc ctt gaa gct ctc ctc atc gcc gga gtt ctc tcc ggt      402 
Arg Met Ile Phe Leu Glu Ala Leu Leu Ile Ala Gly Val Leu Ser Gly 
 5                   10                  15                  20 

ctc ttc tcc agc tct gcc gtg gca caa aac tgc cag tgc gac acc acc      450 
Leu Phe Ser Ser Ser Ala Val Ala Gln Asn Cys Gln Cys Asp Thr Thr 
                 25                  30                  35 

atc tac tgc tgc agc cag cat ggc tac tgc ggc aac agc tac gac tat      498 
Ile Tyr Cys Cys Ser Gln His Gly Tyr Cys Gly Asn Ser Tyr Asp Tyr 
             40                  45                  50 

tgt gga ccg gga tgc caa gcc ggt cct tgt ttg gtt cct tgc gaa gga      546 
Cys Gly Pro Gly Cys Gln Ala Gly Pro Cys Leu Val Pro Cys Glu Gly 
         55                  60                  65 

aac ggc acc tta aca gtt agt gat att gta aca cag gac ttt tgg gac      594 
Asn Gly Thr Leu Thr Val Ser Asp Ile Val Thr Gln Asp Phe Trp Asp 
     70                  75                  80 

gga att gca tca caa gcc gct gcc aac tgt tcc ggt aaa ggc ttc tac      642 
Gly Ile Ala Ser Gln Ala Ala Ala Asn Cys Ser Gly Lys Gly Phe Tyr 
 85                  90                  95                 100 

acc ctg tct gcc ttc tta gaa gcc gtt tcg gct tac cct ggc ttt ggc      690 
Thr Leu Ser Ala Phe Leu Glu Ala Val Ser Ala Tyr Pro Gly Phe Gly 
                105                 110                 115 

acc aaa tgc acc gac gaa gac aga aag aga gag att gca gct tac ttc      738 
Thr Lys Cys Thr Asp Glu Asp Arg Lys Arg Glu Ile Ala Ala Tyr Phe 
            120                 125                 130 

gcc cat gtc acc cat gaa act gga c gtacgtacat ttattcattc              783 
Ala His Val Thr His Glu Thr Gly 
        135                 140 

attcatgcat gcatctcaat tatatatata tagttcatga gatatataat ataatatgag    843 

agatgaaatg ctaaagaatt gtttggcttt gttccggtta atag at tta tgt tac      898 
                                                His Leu Cys Tyr 

att gaa gaa aga gat gga cac gct aat aac tac tgt cta gaa agc caa      946 
Ile Glu Glu Arg Asp Gly His Ala Asn Asn Tyr Cys Leu Glu Ser Gln 
145                 150                 155                 160 

cag tat cca tgc aat cct aac aag gag tac ttc gga cgt gga cct atg      994 
Gln Tyr Pro Cys Asn Pro Asn Lys Glu Tyr Phe Gly Arg Gly Pro Met 
                165                 170                 175 

cag ctc tca tgg aac tac aac tac atc gac gcc ggc aag gag ctc aac     1042 
Gln Leu Ser Trp Asn Tyr Asn Tyr Ile Asp Ala Gly Lys Glu Leu Asn 
            180                 185                 190 

ttc gac ggc ttg aat gat ccg gac ata gtc ggc cgt gac ccc atc ctc     1090 
Phe Asp Gly Leu Asn Asp Pro Asp Ile Val Gly Arg Asp Pro Ile Leu 
        195                 200                 205 

tcc ttc aag act tct ctc tgg tat tgg ata agg aaa ggg gtg caa tac     1138 
Ser Phe Lys Thr Ser Leu Trp Tyr Trp Ile Arg Lys Gly Val Gln Tyr 
    210                 215                 220 

gtc ata ctt gat ccg gac cag ggc ttc gga gcc agc atc aga atc atc     1186 
Val Ile Leu Asp Pro Asp Gln Gly Phe Gly Ala Ser Ile Arg Ile Ile 
225                 230                 235                 240 

aac ggc ggc caa gag tgt gat ggc aag aac acc gcc cag atg atg gcg     1234 
Asn Gly Gly Gln Glu Cys Asp Gly Lys Asn Thr Ala Gln Met Met Ala 
                245                 250                 255 

cgt gtg gga tac tac gag caa tat tgt gcc cag ctt ggt gtc tct cct     1282 
Arg Val Gly Tyr Tyr Glu Gln Tyr Cys Ala Gln Leu Gly Val Ser Pro 
            260                 265                 270 

ggc aat gat ctc act tgt gtc act agt aac ctg gct gtt agt             1324 
Gly Asn Asp Leu Thr Cys Val Thr Ser Asn Leu Ala Val Ser 
        275                 280                 285 

tagtaagtgc atgcatgcac aagtacgtat gttactaaat cagcggctat tgagatgcag   1384 

cactgtgtgt tgtgtttccc taaataaatg ctgatgatga ataacaatgt tattcatggt   1444 

gaataaattt atctttaatt aatggctccg tctccataaa taatctttgt ttttatcgca   1504 

gaaacggttc tgaatatttg gatttttaaa aatataataa attaaatatg ataattttaa   1564 

ttctatacat atttagtcga gcaaatcaat ttggttaaag tttatgaact tggcttaaat   1624 

tcgacta                                                             1631 

 
           
             2  
             286  
             PRT  
             Dioscorea oppositifolia  
           
            2 

Met His Ser Phe Arg Met Ile Phe Leu Glu Ala Leu Leu Ile Ala Gly 
 1               5                  10                  15 

Val Leu Ser Gly Leu Phe Ser Ser Ser Ala Val Ala Gln Asn Cys Gln 
            20                  25                  30 

Cys Asp Thr Thr Ile Tyr Cys Cys Ser Gln His Gly Tyr Cys Gly Asn 
        35                  40                  45 

Ser Tyr Asp Tyr Cys Gly Pro Gly Cys Gln Ala Gly Pro Cys Leu Val 
    50                  55                  60 

Pro Cys Glu Gly Asn Gly Thr Leu Thr Val Ser Asp Ile Val Thr Gln 
65                  70                  75                  80 

Asp Phe Trp Asp Gly Ile Ala Ser Gln Ala Ala Ala Asn Cys Ser Gly 
                85                  90                  95 

Lys Gly Phe Tyr Thr Leu Ser Ala Phe Leu Glu Ala Val Ser Ala Tyr 
            100                 105                 110 

Pro Gly Phe Gly Thr Lys Cys Thr Asp Glu Asp Arg Lys Arg Glu Ile 
        115                 120                 125 

Ala Ala Tyr Phe Ala His Val Thr His Glu Thr Gly His Leu Cys Tyr 
    130                 135                 140 

Ile Glu Glu Arg Asp Gly His Ala Asn Asn Tyr Cys Leu Glu Ser Gln 
145                 150                 155                 160 

Gln Tyr Pro Cys Asn Pro Asn Lys Glu Tyr Phe Gly Arg Gly Pro Met 
                165                 170                 175 

Gln Leu Ser Trp Asn Tyr Asn Tyr Ile Asp Ala Gly Lys Glu Leu Asn 
            180                 185                 190 

Phe Asp Gly Leu Asn Asp Pro Asp Ile Val Gly Arg Asp Pro Ile Leu 
        195                 200                 205 

Ser Phe Lys Thr Ser Leu Trp Tyr Trp Ile Arg Lys Gly Val Gln Tyr 
    210                 215                 220 

Val Ile Leu Asp Pro Asp Gln Gly Phe Gly Ala Ser Ile Arg Ile Ile 
225                 230                 235                 240 

Asn Gly Gly Gln Glu Cys Asp Gly Lys Asn Thr Ala Gln Met Met Ala 
                245                 250                 255 

Arg Val Gly Tyr Tyr Glu Gln Tyr Cys Ala Gln Leu Gly Val Ser Pro 
            260                 265                 270 

Gly Asn Asp Leu Thr Cys Val Thr Ser Asn Leu Ala Val Ser 
        275                 280                 285 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            3 

atggagaact gccagtgcga                                                 20 

 
           
             4  
             19  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            4 

tgcagcttac ttcgcccat                                                  19 

 
           
             5  
             20  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            5 

ctactgtcaa gaaagccaac                                                 20 

 
           
             6  
             18  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            6 

gtacttcgga cgtggacc                                                   18 

 
           
             7  
             21  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            7 

ctcatcaatt tccagccact c                                               21 

 
           
             8  
             18  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            8 

cgactattgt ggaccggg                                                   18 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            9 

aaccagagag aagtcttgaa                                                 20 

 
           
             10  
             20  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            10 

tgtagaagct tttaccggga                                                 20 

 
           
             11  
             18  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            11 

catcacactc ttggccgc                                                   18 

 
           
             12  
             22  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            12 

tagtcgaatt taagccaagt tc                                              22 

 
           
             13  
             18  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            13 

ggtccacgtc cgaagtac                                                   18 

 
           
             14  
             12  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            14 

taccctcaag ct                                                         12 

 
           
             15  
             12  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            15 

gccagctgta cg                                                         12