JP2981549B1 - promoter - Google Patents

Abstract

Abstract: PROBLEM TO BE SOLVED: To provide a bacterial promoter trap vector and a bacterial promoter using the vector. A sequence having a restriction enzyme site, a bacterial ribosome binding site sequence, and a sequence encoding GFP are arranged in this order. Create a vector with The bacterial chromosome is inserted into this restriction enzyme site, and the expression of GFP is detected by UV. The promoter of Erwinia ananas strain is obtained with this vector. An efficient biological control agent is produced using the promoter of this Erwinia ananas strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a promoter trap vector for searching for a bacterial promoter, and a method for searching for a bacterial promoter using the vector. The present invention also relates to a promoter derived from a bacterium belonging to the genus Erwinia.

[0002]

2. Description of the Related Art Protecting the above-ground disease of a plant by bacteria living on the leaf surface of the plant (leaf bacteria), that is, biological control (biocontrol) has been a subject that has long been of interest. Many research results have been published so far. However, although effects are observed in laboratory experiments, the expected effects are not obtained at the field level in many cases, and few have reached the stage of practical application (Sato et al., Microorganisms useful for agricultural use, p123-135, Yokendo) (Tokyo) (199
0)).

[0003] In recent years, attempts have been made to develop a more effective biocontrol microbial agent by incorporating a foreign gene into leaf bacterium using genetic recombination technology (Isumi Iyo)
(Iyosumi) et al., Ann. Phytopathol. Soc. Jpn., 62: 559-
565 (1996)).

[0004] By the way, in the case of obtaining recombinant microorganisms using leaf bacteria as a host, promoter sequences, which are switches for exogenous gene expression, have not been considered so far, and mainly promoter sequences derived from Escherichia coli have been used. For this reason, it has been pointed out that in the recombinant leaf bacteria, the promoter for expressing the foreign gene does not function sufficiently, and as a result, the expression level of the foreign gene may be insufficient.

[0005] Therefore, in order to produce a more sophisticated and effective recombinant biocontrol microbial agent, it is desired to search for a promoter derived from leaf bacteria as a host.

[0006] Methods for searching for a promoter can be roughly classified into a method using a transposon and a method using a trap vector (Van Overbeek et al., Modern Soil Microbiology, p.
441-477, Marcel Dekker (New York) (1997)).

A method for searching for a promoter using a transposon is as follows. First, a reporter gene lacking a promoter is inserted into a transposon. Next, when a transposon having a reporter gene lacking this promoter is introduced into a target host bacterium via a suicide plasmid, this transposon is inserted into the genome of the host bacterium by transposon mutation. When the transposon is inserted downstream of the promoter region of the host genome, the reporter gene is expressed by the action of the promoter (endogenous promoter) of the host genome. Clones into which the reporter gene has been inserted.
Then, this promoter gene can be isolated.

However, in the search method using this transposon, in order to isolate the promoter gene from the clone, a clone containing the promoter must be separately selected from the whole genomic DNA library of the host bacterium using this transposon as a probe. It has to be complicated.

[0009] On the other hand, in the trap vector method, first, a vector into which a reporter gene lacking a promoter is incorporated is prepared, and a host whole genomic DNA fragment is shotgun cloned upstream of the reporter gene of this vector, followed by recombination. This is a method of obtaining a plasmid, transforming the obtained recombinant plasmid into a host bacterium, and analyzing the expression of a reporter gene in the host bacterium. Since the reporter is expressed if the DNA fragment of the host genome has a promoter activity, a clone having the promoter activity can be easily selected, and the promoter sequence can be easily isolated.

[0010] As described above, the reporter gene is used in both the method using a transposon and the method using a trap vector. As reporter genes, besides antibiotic resistance genes such as tetracycline and chloramphenicol,
Glucuronidase (GUS), β-galactosidase (lac
Z), luciferase (lux) and other chromogenic / luminescent genes have been used. However, since these are all enzymes, there is a disadvantage that it is necessary to separately add a substrate for the enzyme reaction. In addition, when the target host microorganism originally has antibiotic resistance or similar enzyme activity, there is a disadvantage that these reporter genes cannot be used.

Recently, GFP (Green Fluorescent Protein) has attracted attention and has been used as a new reporter gene to compensate for these drawbacks (Charhuy (C
halfie) et al., Science, 263: 802 (1994)). GFP is a luminescent protein of a luminescent jellyfish, and when this protein accumulates, it develops a green color under ultraviolet irradiation.

A promoter trap vector for animal cells using the GFP gene as a reporter gene is commercially available (for example, pEGFP-1 available from CLONTECH). However, since these vectors are for animals, they cannot be used to trap bacterial promoters.

As described above, a vector for efficiently searching for a bacterial promoter has not been known yet, and how to search for and use a bacterial promoter is hardly known. Is the actual situation.
Therefore, it has been desired to provide a vector for efficiently searching for a bacterial promoter and a bacterial promoter.

[0014]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and provides a promoter trap vector for bacteria and uses the trap vector to obtain a promoter from Erwinia ananas. The present invention provides a novel promoter sequence, which can be used to create a more sophisticated and effective recombinant biocontrol microorganism.

[0015]

SUMMARY OF THE INVENTION The present invention provides a sequence having a restriction enzyme site, a bacterial ribosome binding site sequence, and
The present invention relates to a bacterial promoter trap vector having a sequence encoding GFP in this order, wherein the bacterial GFP is expressed when a bacterial promoter sequence is inserted into the restriction enzyme site.

The present invention also provides a step of inserting a gene into a restriction enzyme site of the above-described bacterial promoter trap vector to prepare a recombinant vector, introducing the recombinant vector into a host bacterium, transforming the bacterium, The present invention relates to a method for searching for a bacterial promoter, comprising a step of obtaining a transformant and a step of detecting whether the transformant expresses GFP.

The present invention also relates to a sequence comprising the 101st to 171st sequence of SEQ ID NO: 1 or a sequence having 70% or more homology with said sequence, and having the same or a similar sequence as SEQ ID NO: 1. It relates to a promoter having a further promoter activity.

In a preferred embodiment, the promoter has a sequence as set forth in SEQ ID NO: 1 and Erwinia
It is expressed in microorganisms belonging to the genus.

Furthermore, the present invention relates to a sequence comprising the sequence from the 168th to the 227th of SEQ ID NO: 2 or a sequence having 90% or more homology with the sequence, and having the same or a similar sequence to the sequence of SEQ ID NO: The present invention relates to a promoter having the above promoter activity.

[0020] In a preferred embodiment, the promoter has the sequence of SEQ ID NO: 2 and
It is expressed in microorganisms belonging to the genus.

The present invention further relates to the sequence represented by SEQ ID NO: 3, or 1 or 2
Having the insertion, deletion, or substitution of the above base, and
The present invention relates to a promoter having a promoter activity equal to or higher than that of the sequence of SEQ ID NO: 3.

In a preferred embodiment, the promoter has the sequence shown in SEQ ID NO: 3 and Erwini
It is expressed in microorganisms belonging to the genus a.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The bacterial promoter trap vector of the present invention has a sequence having a restriction enzyme site, a bacterial ribosome binding site sequence, and a sequence encoding GFP in this order. GFP is expressed when a bacterial promoter sequence is inserted into this restriction enzyme site.

The sequence having a restriction enzyme site is preferably a sequence having a plurality of restriction enzyme cleavage sites, that is, a sequence having a multiple cloning site. Such sequences are commercially available. If the desired sequence is not commercially available, one skilled in the art can easily design and synthesize it.

The bacterial ribosome binding sequence is a purine-rich sequence known as a so-called Shine-Dalgarno sequence (SD sequence), and refers to a sequence that binds to the 3'-terminal sequence CCUCCUUA of 16S rRNA.

The sequence encoding GFP (Green Fluorescent Protein) is known (Salfy et al., Supra). Also,
Improved GFP can also be used. Therefore, the term "GFP" in the present invention includes improved GFP. In addition, the improved GFP means GFP in which the efficiency of fluorescent coloring is increased, for example, the 65th amino acid serine (Ser) of GFP is replaced with threonine (Thr),
EGFP (Enhanced green fluo)
rescent protein) (Tslen et al., NATURE vol. 73, 1995).

[0027] The bacterial promoter trap vector having these sequences must further have an origin of replication (Ori) so that it can replicate in bacterial cells. It preferably has a selectable marker (eg, a drug resistance gene).

The bacterial promoter trap vector of the present invention can be prepared using a commercially available vector generally used for bacteria as a starting material. A restriction enzyme site, a sequence encoding GFP, and a ribosome binding sequence may be placed at an appropriate site in a commercially available vector according to procedures well known to those skilled in the art.

The bacterial promoter trap vector can also be prepared from an animal promoter trap vector. For example, a sequence having a restriction enzyme site, a bacterial ribosome binding site sequence, and a sequence encoding GFP are inserted in this order into an appropriate site of a vector having an origin of replication (Ori) and a drug resistance gene. Is done. The preparation method uses a recombinant DNA technique and technique well known to those skilled in the art.

Hereinafter, a commercially available plasmid pEGFP-1 (CLONTEC
An example of constructing the bacterial promoter trap vector of the present invention using (Company H) as a starting vector will be described with reference to FIG.

The plasmid pEGFP-1 is a promoter trap vector for animals and is shown at the upper left of FIG. This plasmid pEGFP-1 has a modified EGFP gene, but has no promoter sequence for expressing EGFP and no SD sequence. However, since it has a multiple cloning site and has bacterial Ori (pUC ori) and kanamycin / neomycin resistance gene, it can replicate in bacteria and transformed bacteria are screened for drug resistance Therefore, this skeleton can be used. Therefore, first, from plasmid pEGFP-1, EGFP
Prepare a skeleton from which the gene has been removed.

On the other hand, a commercially available plasmid pEGFP (CLONTECH
(Top right of Fig. 1) shows E. coli Lac promoter and SD
And a bacterial expression vector having an EGFP gene downstream thereof. An SD sequence and an EGFP gene sequence are cut out from the plasmid pEGFP, and inserted into the prepared skeleton, thereby preparing a target bacterial promoter trap vector.

One example of the obtained vector is pEGFP-V1 shown in FIG. This vector has a multi-cloning site, an SD sequence and an EGFP gene sequence in this order,
It has a drug (kanamycin / neomycin) resistance gene and Ori of pUC that functions in bacteria.

Hereinafter, a method for searching for a promoter using this vector will be described. First, whole genomic DNA is extracted from a specific bacterium by an ordinary method, and the whole genomic DNA is cut with a restriction enzyme. As the restriction enzyme, a restriction enzyme corresponding to the multiple cloning site may be used, or a restriction enzyme generating an insertable sequence may be used. Genome DNA size is about 100
It is preferable to cut with a restriction enzyme so as to be ~ 1000 bp. Further, the DNA may be synthesized.

The obtained genomic DNA is inserted into the multiple cloning site of the above promoter trap vector to prepare a recombinant plasmid. The recombinant plasmid is introduced into a bacterium to transform the bacterium. A transformant having a promoter is detected by irradiating the transformant grown on an agar medium containing kanamycin (neomycin) with UV (365 nm) and selecting a clone that emits light. By selecting a transformant having a high luminescence intensity from the detected transformants, a promoter having high activity can be obtained.

Using this promoter trap vector, the promoter of the bacterium Erwinia ananas can be searched. For example, the whole genomic DNA of Erwinia ananas is isolated and decomposed with, for example, a restriction enzyme Sau3AI to prepare a genomic DNA fragment. Then, for example, a genomic DNA fragment was cloned into the BglII site of the multiple cloning site of the plasmid pEGFP-V1, and Erwinia ananas
To obtain a transformant, obtain a kanamycin-resistant strain, and detect the expression of EGFP by UV to obtain a promoter.

The obtained DNA fragment containing the promoter is
It is isolated and sequenced by conventional methods.

According to the above exemplified method, SEQ ID NOs.
And 3 Erwinia ananas promoters are obtained.

The sequence shown in SEQ ID NO: 1 corresponds to Erwinia an
anas A promoter sequence obtained from MAFF 301714. Homology search of the promoter sequences of the SEQ ID NO: 1 was performed in D DBJ database results, of SEQ ID NO: 1
Sequences 101-171 and 52-12 of the E. colienvA promoter
The second sequence shows 67% homology, and corresponds to SEQ ID NO: 1
It is highly likely that the sequence from 01 to 171 is a promoter.

Accordingly, the 101st to the 171st of SEQ ID NO: 1
A promoter having a sequence having 70% or more homology with the second sequence or the sequence and having a promoter activity equivalent to or greater than that of the sequence of SEQ ID NO: 1;
Unlike the envA promoter of li, it is a promoter expressed in microorganisms of the genus Erwinia. Such an arrangement is performed by means well known to those skilled in the art. For example, substitution, deletion, or insertion of one or more bases is performed by site-directed mutagenesis, or 70% or more of the 101st to 171st sequences of SEQ ID NO: 1 by DNA synthesis. Are synthesized, and a sequence containing the same is also synthesized. Then, the obtained sequence is cloned into a promoter trap vector, and the promoter activity of the obtained sequence may be examined based on the expression level of GFP or EGFP.

The sequence shown in SEQ ID NO: 2 corresponds to Erwinia an
anas A promoter sequence obtained from MAFF 301714. Homology search of the promoter sequences of the SEQ ID NO: 2 was performed in D DBJ database results, of SEQ ID NO: 2
Sequences 168-227 and 137-1 of E. colifabB promoter
It shows 83% homology with the 96th sequence.
It is highly possible that the sequence at positions 168 to 227 and its vicinity are promoters.

Accordingly, it has a sequence at positions 168 to 227 of SEQ ID NO: 2 or a sequence having 90% or more homology with the sequence, and has a promoter activity equivalent to or higher than that of the sequence of SEQ ID NO: 2. The promoter has E. coli fa
Unlike the bB promoter, it is a promoter expressed in microorganisms of the genus Erwinia. Such promoters
It can be obtained using a method similar to that described in SEQ ID NO: 1 and well known to those skilled in the art.

The sequence shown in SEQ ID NO: 3 corresponds to Erwinia an
anas A promoter sequence obtained from MAFF 301714. As a result of homology search for promoter sequences of the SEQ ID NO: 3 in D DBJ database, not able to be found out sequences showing high homology are likely to be novel promoter. And, in the sequence of SEQ ID NO: 3, has an insertion, deletion, or substitution of one or more bases,
In addition, a promoter having a promoter activity equal to or greater than that of the sequence of SEQ ID NO: 3 is
It can be obtained by using a method well known to those skilled in the art in the same manner as for producing a promoter having a high homology with.

[0044]

Hereinafter, the present invention will be described with reference to examples.
The present invention is not limited only to the examples.

Example 1 Promoter Capture Vector pE
Construction of GFP-V1) FIG. 1 shows a commercially available plasmid of CLONTECH.
Promoter capture vector pEGFP based on pEGFP, pEGFP1
It is a schematic diagram which constructs -V1. pEGFP was digested with the restriction enzymes SmaI and NotI, and EG by agarose electrophoresis.
The vector portion not containing the FP gene was recovered and further dephosphorylated with alkaline phosphatase to prevent self-ligation. Next, pEGFP1 was replaced with the restriction enzyme Bs.
Digest with rBI and NotI, containing about 8 RBS (ribosome binding site, SD sequence) and EGFP necessary for initiation of bacterial translation.
A 00 bp DNA fragment was recovered. Ligation of this DNA fragment with the vector portion not containing the EGFP gene resulted in pE
GFP-V1 was constructed. The structure of pEGFP-V1 is shown in FIG.

In addition, pEGFP was used as a control vector.
PEGFP-V1 plac was constructed by linking a lac promoter to the Eco47III site upstream of RBS (see FIG. 1, lower right).

This pEGFP-V1 was used as a promoter trap vector for searching for a promoter of Erwinia ananas.

Example 2 Isolation of Promoter of Bacterium of the Genus Erwinia Bacterium Erwinia an isolated from the leaf surface of rice
The promoter of anas MAFF 301714 strain was searched and isolated.
Erwinia ananas has the ability to survive and colonize the leaves of plants, and is a strain that has recently attracted attention as a biocontrol agent (biocontrol agent) for controlling plant diseases. This strain is kept at the National Institute for Biological Resources, Ministry of Agriculture, Forestry and Fisheries.

(1) Whole genome of E. ananas MAFF strain 301714
Preparation of DNA First, the whole genome DN of E. ananas MAFF 301714
After extracting and purifying A, it is completely digested with the restriction enzyme Sau3AI that recognizes 4 bases, and subjected to agarose electrophoresis.
DNA fragments of 0 to 1000 bp were separated and recovered (FIG. 3, upper left).

(2) Preparation and Transformation of Recombinant Plasmid On the other hand, pEGFP-V1 was digested with BglII and dephosphorylated to prevent self-ligation (FIG. 3, upper right).
The vector cut with BglII and the above separated about 100
A DNA fragment of 10001000 bp was ligated and introduced into the Erwinia ananas MAFF 301714 strain by electroporation (FIG. 3, middle). After culturing at 30 ° C. for 3 days in an LB plate medium containing 40 ppm of kanamycin, transformed colonies of about 2000 clones were obtained (FIG. 3, bottom). Observation of the activity of GFP under irradiation of ultraviolet rays (UV 365 nm) revealed that 27 clones emitted light under irradiation of ultraviolet rays. These 27 clones were isolated as positive clones.

Example 3 Analysis of Positive Clones and Determination of Sequences (1) Analysis of Positive Clones Of the 27 clones, 10 clones (PCF1, 3, 5, 6, 9 10, 13, 15, 26, 27), the plasmid was recovered, and the restriction enzymes Eco47III and EcoR
Double digested with I, agarose electrophoresed, insert D
The presence and size of the NA fragment were confirmed. As a result, inserts of different sizes (250 bp to 1700 bp) were confirmed in the DNA recovered from the positive clone (see FIG. 4).

(2) Measurement of GFP Activity by Spectrofluorometer The luminescence obtained by the spectrofluorometer was quantitatively analyzed. 10 positive clones, 50pp kanamycin
Incubate with shaking at 30 ° C in LB liquid medium containing m, sample over time, collect cells, resuspend in sterile water,
After adjusting to = 1, the emission intensity of GFP was measured with a spectrofluorometer. In addition, pEGF was used as a positive control.
E. ananas into which P-V1plac was introduced was used, and the original strain Erwinia ananas MAFF 301714 was used as a negative control.

In all of the 10 clones tested, a difference in luminescence intensity was already observed 14 hours after the start of culture. Thereafter, the luminescence intensity increased with time, but the relative relationship of each clone did not change (see FIG. 5). From these, 3 clones (PCF1, 9, 15) were selected as clones having the same or higher emission intensity than the positive clones.

(3) Determination of base sequence of DNA fragment showing promoter activity DNA was purified from the obtained three clones by a conventional method and sequenced. Fluorescent primers upstream and downstream of the BglII site of pEGFP-V1 were synthesized, and Auto Read Sequencing K
After the dideoxy reaction using it, the sequence was determined by DNA Sequencer.

PCF1 was 411 bp in total length (SEQ ID NO: 1). When we performed a homology search on that sequence, we found that 101
~ 171 sequence and 52-122 of E. coli envA promoter
The 67th sequence showed 67% homology (FIG. 6), indicating that the 101st to 171st sequences of SEQ ID NO: 1 are likely to be promoters.

PCF9 had a total length of 458 bp (SEQ ID NO: 2).
When a homology search of the sequence was performed, SEQ ID NO: 2
The 137-196-th sequence of the promoter of 168-227 in SEQ E. coli fatty acid substrate transfer protein synthetic gene (fabB) of, shows a 83% homology (Fig. 7), SEQ ID NO: 2
It was shown that the sequence at positions 168 to 227 is likely to be a promoter.

The Escherichia coli promoter showing these homology is a promoter of a gene (housekeeping gene) essential for the survival of bacteria. It is considered an expression type promoter.

PCF15 had a total length of 214 bp (SEQ ID NO: 3).
However, none shows homology with a known gene, and is a novel promoter sequence. However, it was unclear whether the promoter was a constant expression type or a condition inducible type.

[0059]

EFFECT OF THE INVENTION Using the promoter trap vector of the present invention, bacterial promoter screening can be performed efficiently. In particular, promoters of bacteria belonging to the genus Erwinia, a leaf bacterium, can be searched. By using the obtained promoter of the bacterium of the genus Erwinia, a foreign gene, for example, a chitinase gene or the like can be efficiently expressed, and a sophisticated and effective recombinant biocontrol microorganism can be produced.

[0060]

[Sequence List] SEQUENCE LISTING <110> National Institute of Agrobiological Resources Ministry of Agric ulture Forestry and Fisheries <120> Promoter trap vector and method for isolating promoters of Genus Erwinia using the vector <130> P198N07015 <160> 3 <210> 1 <211> 411 <212> DNA <213> Erwinia ananas <220> <221> promoter <222> (101)… (171) <223> n (A or C or G or T or U) or (unknown or other base) <400> 1 gatcagagtg cacagtcaaa caaagagccc gattaccttg atattccggc gttttgcgca 60 agcaggccga ctaggaataa ccttataatt gggattcccg ctctttgtgc taaagtgttc 120 gcccgctggt aacgtatact ggcggtcgga aagcgatatt gcgagataat aacgatgatc 180 ccgtctacgg gtttttcata ccatcgggta taattacgca tcctcatcgg ggcgctgaag 240 atgcccagcg cagcaaggcc gtgtgaaatc cggccaatgt accgggatgt cccgctatct 300 gtgttgttct gggcttccca cngcngttca ggcgtaaagg tttcaagacg gggatcgtta 360 gcgcgctgaa acaagatttt ccctggctga tgggcaccag taactacgat c 411 <210> 2 <211> 458 <212> DNA <213> Erwinia ananas <220> <221> promoter <222> (168)… (227) <400> 2 gatcctca cg ccgtcagtct gggttaaaga aagaaggacc tccggccagt attgtgtcat 60 cgcgtgtgaa cgtttattga tccgcatcgt cagattgcga ttgctaaccc aatggcgctg 120 cccgttcgtg tctaagctga tcttatcggt cggcgtgaca tgacgcaaat ttacaaaagg 180 aaatggctga tcggacttgt tcggcgtaca agtgtaagct agagtgctcg tcaaataaca 240 gaagtggtga ggaagattga atgaaacgtg ctgtgattac tggcttaggg atcaccttgt 300 ttctgtgggc aatggcaacc tttgccacgg tccccggcct gcagatctgt ccctgattgc 360 cagaaagcag cttgccccgc cgcgtggctt ttgttgagca acatgcgtta accagcgcca 420 atatgaccgt acgtgatgtc gtaaagcttg gaaggatc 458 <210> 3 <211> 214 <212> DNA <213> Erwinia ananas <220> <221> promoter <222> (1)… (214) <400> 3 gatctgtggc gccctgagtc ttgttacggt aagtctaaaa cgtgcagggatct ggctttcttc acgctttatt acgttaagga aatcacaatg aatctgatga 120 ataattgtgc atcgcctgcc gcagatgtta tattaaaata taatattcgg aggtgctcta 180 tgtataccac tcgtctgaaa aaggttggcg gatc 214

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for constructing a promoter trap vector pEGFP-V1 and a positive control vector pEGFP-V1 plac.

FIG. 2 is a diagram showing the structure of a promoter trap vector pEGFP-V1.

FIG. 3 is a diagram showing a method for searching for a promoter by shotgun cloning.

FIG. 4 is a view showing an electrophoresis photograph for confirming a captured DNA fragment having promoter activity.

FIG. 5 is a diagram showing a change over time in the luminescence intensity of a positive clone.

FIG. 6 is an alignment showing the homology between clone PCF1 and envA.

FIG. 7 is an alignment showing the homology between clone PCF9 and fabB.

Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 GENESEQ DDBJ

Claims (5)

(57) [Claims] 1. The 101st to 1st sequence of SEQ ID NO: 1 in the sequence listing
A promoter having the 71st sequence. 2. The promoter according to claim 1, wherein the sequence is the sequence of SEQ ID NO: 1 in the sequence listing. 3. The 168th to the 2nd sequence of SEQ ID NO: 2 in the sequence listing.
Promoter having the 27th sequence. 4. The promoter according to claim 3, wherein the sequence is the sequence of SEQ ID NO: 2 in the sequence listing. 5. A promoter having the sequence of SEQ ID NO: 3 in the sequence listing.