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 
     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. 
     2. Prior Art 
     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. 
     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. 
     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). 
     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 
     The present invention has been made under these circumstances for the purpose of providing a novel gene encoding a plant chitinase. 
     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. 
     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. 
     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 
         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 
     Hereinbelow, the present invention will be described in detail. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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 
     (1) Determination of Partial cDNA Sequence 
     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 50 μl 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. 
     (2) Determination of Partial Genomic DNA Sequence 
     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. 
     (3) Preparation of Oligonucleotide Primers 
     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
 
(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′-GTACTTCGGACGTGGACC-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′-AACCAGAGAGAAGTCTTGAA-3′   53.2   SEQ ID NO: 9       GSP-R2   5′-TGTAGAAGCTTTTACCGGGA-3′   55.3   SEQ ID NO: 10       GSP-R3   5′-CATCACACTCTTGGCCGC-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                    
(4) Cloning of the 5′ Region of the Chitinase Gene
 
     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                        
The PCR was performed with Astec Program Temp Control System PC-800.
 
     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). 
     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 
               
               
                   
                   
               
             
          
         
       
     
     Plasmid DNA was prepared from each of the positive clones using QIAprep Spin Miniprep kit (Qiagen) and then sequenced. 
     (5) Cloning of the 3′ Region of the Chitinase Gene 
     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 
               
               
                   
                   
               
             
          
         
       
     
     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. 
     (6) Cloning of the Full-Length Yam Chitinase Gene by High Fidelity PCR 
     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 II. 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 
               
               
                   
                   
               
             
          
         
       
     
     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 
               
               
                   
                   
               
             
          
         
       
     
     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. 
     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. 
     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. 
     All publications, patents and patent applications cited herein are incorporated herein by reference in their entity.