JPH06233685A - Peptide gene of flower bud-inducing substance - Google Patents

Abstract

PURPOSE:To obtain the subject peptide by a genetic engineering means comprising obtaining a DNA fragment containing a gene coding a peptide exhibiting a flower bud-inducing activity and utilizing the obtained DNA fragment. CONSTITUTION:The objective peptide is produced by extracting a DNA fragment containing a gene coding the flower bud-inducing substance peptide from Lemna paucicostata Hegelm, etc., determining the DNA base sequence of the DNA fragment, transforming a procaryotic cell with the DNA fragment, culturing the obtained transformant, and subsequently recovering the flower bud-inducing substance peptide from the cells or culture medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA fragment containing a gene encoding a flower bud inducer peptide which controls flowering of plants, a recombinant DNA having this DNA fragment, and the recombinant D.
The present invention relates to a transformant having NA introduced therein and a method for producing the flower bud inducer peptide.

[0002]

2. Description of the Related Art Many types of plants change their growth mode from vegetative growth to reproductive growth in response to environmental conditions such as photoperiod and temperature, form flower buds in meristems, and eventually flower.

From the viewpoint of agriculture, it is economically important to control the flowering and fruiting of plants. For example, in cereals, fruits and vegetables, a crop is formed after flowering, and the flowering time determines the harvesting / shipping time. Furthermore, in leafy vegetables, reproductive growth is suppressed by suppressing flowering, which leads to increased yields.

Conventionally, as a method of controlling the flowering time of plants, a method of artificially controlling environmental conditions such as day length and temperature in a specific facility has been widely used. However, these methods have a problem that the cost for cultivating and producing a plant becomes high.

Therefore, it is considered that instead of controlling the environment of a target plant, a substance that directly induces a flower bud formation reaction is found and its action regulates flower bud formation. Plants form flower buds and bloom under various environmental conditions, but substances that induce a flower bud formation reaction within the plant are basically considered to be common to all plants. 50
Years ago Chailakhyan suggested the presence of flower bud-inducing hormones that were synthesized in the leaves as such substances and transported through phloem sap. After that, further experiments using grafts have revealed that the flower bud inducer hormone is present in many plant species and has a broad spectrum of action. After that, isolation of a flower bud inducer was attempted, and Cleland first isolated salicylic acid as a flower bud inducer from Phalaenopsis phloem sap using C. duckweed as the assay system (C. F. Cleland and
A. Ajami, Plant Pyisiol., 54 , 904, 1974). Furthermore, to date, other researchers have isolated phenolic compounds such as benzoic acid and pipecolic acid as substances that induce flower buds in duckweeds. However, the unknown amount of the above compound was not present in the plant body, which suggested other unknown substances.

On the other hand, Takeba et al. Isolated and identified a polypeptide showing a flower bud-inducing activity in a trace amount from a water extract of duckweed by the same assay method (JP-A-4-117397). Furthermore, the existence of similar substances with similar activity was suggested in other plants such as morning glory and Japanese radish.

At present, the above-mentioned polypeptide is produced by extraction and purification from duckweed, but production using genetic engineering techniques is indispensable for quantitative production increase and industrialization. Therefore, it is necessary to obtain the gene of this peptide and establish a mass production system using a host such as a microorganism.

On the other hand, in recent years, the development of plant genetic engineering has made it possible to introduce a target gene into a plant and express it. Thereby, for example, a certain gene can be expressed in a large amount in a plant, or can be suppressed by the expression of its antisense gene. In order to use such a method, it is necessary to isolate a gene of a peptide involved in flower bud induction in controlling flowering of plants.

[0009]

The problem to be solved by the present invention is to obtain and provide a DNA fragment containing a gene encoding a peptide exhibiting flower bud-inducing activity, and further by utilizing it, genetic engineering. To provide the peptide by a specific method. By the above-mentioned genetic engineering means, more efficient acquisition than the acquisition of the peptide by conventional extraction and purification methods can be achieved.

[0010]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventor has synthesized cDNA from mRNA prepared from duckweed, and obtained a full-length flower bud inducer peptide from this cDNA library. A cDNA clone encoding the same was isolated, and this was incorporated into an expression vector to successfully obtain a transformant that produces a flower bud inducer peptide, resulting in the present invention.

That is, the present invention is based on the sequence listing, SEQ ID NO: 1
, A flower bud inducer peptide having an amino acid sequence of amino acid numbers 81 to 263, a DNA fragment containing a gene encoding the flower bud inducer peptide, and the aforementioned flower bud induction downstream of a promoter that can be expressed in prokaryotic cells A DNA fragment containing a gene encoding a substance peptide is ligated to provide recombinant DNA capable of replicating in prokaryotic cells. The present invention further includes the above-mentioned rearrangement D
A method for producing a flower bud inducer peptide, which comprises culturing a prokaryotic transformant having NA, the transformant described above, and recovering the flower bud inducer peptide from the cells or the culture medium, and the above description. It also provides a plant characterized in that it retains a DNA fragment containing a gene encoding a flower bud inducer peptide in addition to the original cells.

The present invention will be described in detail below. 1. D containing a gene encoding a flower bud inducer peptide
The type of plant from which the NA fragment flower bud inducer is extracted is not particularly limited, but plants such as duckweed and bindweed family are preferable. The plants of the duckweed family are Lemna, Spirodela, Wolffia and Wolffie.
There are genera such as lla, and you can use any genera,
For example, duckweed ( Lemna paucicostata ) and duckweed ( Lemna gibba ) are preferable. The plant of the family Convolvulaceae is preferably, for example, morning glory ( Pharbitis nil chois).

The DNA fragment containing the gene encoding the flower-inducing substance peptide of the present invention is a chromosome library or cDNA derived from the above-mentioned plant body, for example, duckweed.
It can be obtained by preparing a library and screening from this library using an oligonucleoside probe or an antibody (J. Sambrook et al, Mol.
ecular Cloning, 1989, Tadashi Watanabe et al., Plant Biotechnology Experiment Manual, Cloning and Sequence, 1989).

When screening is performed using oligonucleotides, a cDNA library consisting of plaques or colonies is transferred onto a membrane filter, and DNA is released by alkaline denaturation and neutralization treatment. After immobilizing this on a filter, using a radioisotope-labeled or chemically-labeled oligonucleotide probe, a cDNA having a base sequence complementary thereto
The clone having A may be detected.

In the case of screening using an antibody, a cDNA library prepared using an expression vector is used, a replica is taken on a filter as described above, and an antigen-antibody reaction is carried out on the replica to obtain the desired cD.
Clones producing the gene product from NA can be detected.

Alternatively, a DNA containing a target gene can be obtained by PCR using an oligonucleotide as a primer (HA Erlich, PCR Technology, 1).
989, C. Wong et al, Nature, 330 , 1987). That is, an oligonucleotide serving as a primer is prepared based on the nucleotide sequence deduced from the amino acid sequence, and PCR is performed on the cDNA library to amplify the target gene or a part thereof. It becomes possible to easily obtain the target gene or a part thereof from among these. Furthermore, if necessary, by using the obtained DNA as a more accurate probe, the target clone can be obtained from the cDNA library more easily than the above method.

The nucleotide sequence of the cDNA encoding the flower bud inducer peptide thus obtained and the amino acid sequence predicted from this sequence are shown in SEQ ID NO: 1 in the Sequence Listing. The full-length amino acid sequence shown in SEQ ID NO: 1 is also a flower bud inducer peptide, but the peptide having the 81st to 263rd amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 is also a flower bud inducer peptide.

2. Recombinant DNA expressing a gene encoding a flower bud inducer peptide and a transformant into which the recombinant DNA has been introduced are expressed as a gene encoding a flower bud inducer peptide. It can be obtained by inserting a promoter into the vector at the 5'end side thereof. promoter,
Alternatively, the type of vector is appropriately selected according to the host used. As the host, for example, Escherichia coli, Bacillus, yeast or the like can be used.

The case where Escherichia coli is used as a host will be described below.

As the promoter, a promoter usually used for heterologous protein production in Escherichia coli can be used. For example, T7 promoter, t
Examples include rp promoter, lac promoter, tac promoter, PL promoter and the like.

The vector is preferably a so-called multi-copy type, and is a plasmid having a replication origin derived from Col E1, such as pUC type plasmid, pBR322 or its derivative, and a transformant is selected. Therefore, those having a marker of ampicillin resistance gene are more preferably used.

As such a plasmid, an expression vector having a strong promoter is commercially available (pGE
MEX-1 (manufactured by Promega Corp.), pUC system (Takara Shuzo)
Others), pPROK system (manufactured by Clontech), pKK23
3-2 (manufactured by Clontech) and the like) can be used in the present invention.

In order to increase the production amount, a terminator which is a transcription termination sequence is preferably linked downstream of the gene encoding the flower bud inducer peptide. For example,
T7 terminator, fd phage terminator, T
4 terminator, trpA terminator and the like.

3. Plant transformant carrying a DNA fragment containing a gene encoding a flower bud inducer peptide To express a gene encoding a flower bud inducer peptide in a plant cell, the gene functions at the 5'end of the gene in the plant cell. It is necessary to connect a promoter.

For example, cauliflower mosaic virus
A constitutively expressed promoter such as 35S promoter, NOS promoter, OCS promoter can be used. Alternatively, phenylalanine ammonia lyase gene promoter (salicylic acid, glutathione, injury, etc.), PR promoter (salicylic acid-induced), rab, lea, em gene promoter (abscisic acid-induced), etc. A promoter or the like that can induce expression by artificial means can also be used. Also preferably, a terminator which is a transcription termination sequence downstream of the DNA, for example, 35S terminator, NO
Connect an S terminator, etc.

The introduction of a DNA fragment containing a gene encoding a flower bud inducer peptide into a plant can be carried out by a known method. For example, the electroporation method (ME Fromm et al., Nature, 319 , 1986) and the polyethylene glycol method (J. Paszkowski et al., E).
DN to protoplasts using MBO J., 3 , 1984)
A can be introduced directly. The particle gun method (DE McCabe et al., Bio / Technology,
6 , 1988) and can be directly introduced into plant tissues. Alternatively, infection with Agrobacterium can be used to integrate the gene of interest into the plant chromosome (SB Gelvin et al., Plant Morological Biology).
Manual, 1988).

4. Method for Producing Flower Bud Inducer Peptide By culturing a transformant with a recombinant DNA that expresses a gene encoding the flower bud inducer peptide, the flower bud inducer peptide is produced in the cells or the culture medium.
It is preferable that the culture is performed under the condition that the promoter is induced in the main culture after the preculture.

For example, when the lac promoter is used, the transformant is grown in liquid culture, and then IPTG is added to the medium so that the final concentration is 1 mM, for example. After a few hours, the gene product will accumulate.

[0029]

【Example】

[Example 1] Acquisition of cDNA encoding flower bud inducer peptide 30 g of Lemna paucositata 441 was added to a small amount of GIT buffer (4 M guanidine isothiocyanate,
After adding 5 mM sodium citrate (pH 7.0), 0.1 M 2-mercaptoethanol, 0.5% sodium N-lauroylsarcosine) and crushing with a homogenizer, 6000 rpm
It was centrifuged for 15 minutes. GIT buffer was added to the obtained supernatant to make the total volume 60 ml, and 24 g of cesium chloride was dissolved to obtain an extract. 2 ml cesium chloride solution (5.7M
10 ml of the extract was put into a centrifuge tube containing 0.1 M EDTA containing cesium chloride, and centrifuged (Beckman SW41) at 35,000 rpm for 16 hours at 20 ° C. After centrifugation, the resulting RNA precipitate was treated with 300 μl of TE-SDS solution (10 mM Tris-HCl buffer, 5 mM EDTA, 0.1% SDS, pH).
7.4) and then dissolved in chloroform-butanol (4:
1) The solution was extracted several times. 0.1 volume of 3M sodium acetate (pH5.2) and 2.2 volume of 100% ethanol were added and ethanol precipitation treatment was carried out according to a conventional method to obtain a total RNA precipitate.

The total RNA precipitate was dissolved in 10 mM Tris-hydrochloric acid buffer (pH 7.5) 0.5 M sodium chloride, 1 ml of 0.5% SDS aqueous solution, and the following oligotex-dt30 (Oligote) was used.
x-dt30, Nippon Roche Co., Ltd.)
NA was collected and used as the mRNA fraction. Furthermore, phenol / chloroform extraction and ethanol precipitation were performed,
The mRNA was purified.

The mRNA solution is cDN manufactured by Pharmacia.
It was subjected to A synthesis kit to prepare cDNA. Created c
After adding an Eco RI / Not I adapter to the DNA,
Incorporated into gt11 vector, Stratagen
e) Gigapack II Gold (GIGAPACK
II GOLD) was used to construct a cDNA library.

The obtained cDNA library is about 33 million
Had a recombinant. from here ³²Synthesis labeled with P
DNA probe (5 'TGITCIAAIA CIGC
ITCIGC ITCIAAITTI GGIGCIG
Use TIT TICCIAC 3'Sequence Listing SEQ ID NO: 2)
Was screened. Placement of synthetic DNA probe
The row is amino acid number 72 of the flower bud inducer determined by Takeba et al.
To 87 amino acid sequences. In addition,
Inosine was used for unknown bases in the don.
Finally screened in 3xSSC buffer at 42 ℃ for 30 minutes
Of the filter for about 1.0 kb
A positive clone containing the insert was obtained. Positive clone
Was about 1 in 10,000.

From the cDNA clone thus obtained, the insert fragment was cut out with NotI, and the vector Bluescript + KS (Stratagene) was added.
Sub-cloning into a recombinant DNA. The recombinant DNA was named pLK1. When the nucleotide sequence of the cDNA inserted into the recombinant DNA was determined by the dideoxy method according to a conventional method, there was a nucleotide sequence complementary to the used DNA probe, and the amino acid sequence revealed as a flower bud inducer was determined. Since the encoding nucleotide sequence exists, it was confirmed to be the target clone. The DNA base sequence thus obtained is shown in SEQ ID NO: 1 in the sequence listing.

Escherichia coli harboring pLK1 (Escher
ichia coli AJ12819) has been deposited with the Institute of Microtechnology,
The deposit number is FERM P-13383.

Example 2 Expression of cDNA Encoding Flower Bud Inducer Peptide in Escherichia coli cDNA Encoding Flower Bud Inducer Peptide Obtained Above
To express the desired peptide using the clone,
It is necessary to perform the following operations to align the reading frames for codons. First, the plasmid pLKl was cleaved with Apa I and Xho I, and then exonuclease III / Mung bean nuclease isolation method (Exonuclease III / Mung B
Various plasmids were prepared by partially deleting the 5'end of cDNA by the ean Nuclease Deletion method).

E. coli (MV
1184) was transformed.

Clones producing the desired peptide were screened by the following method. Transformed E. coli in LB liquid medium containing 3 ml of ampicillin at 37 ° C
It was pre-cultured overnight in. 0.3 ml of pre-culture liquid to 3 m of new liquid medium
After inoculating to 1 l and culturing for 4 hours, IPTG was added to a final concentration of 1 mM.
Was added. Furthermore, the cells were cultured overnight and collected by centrifugation. 200 μl of bacterial cells, SDS sample buffer (8M urea, 0.125M Tris-HCl buffer (pH 6.
3) 4% SDS, 5% 2-mercaptoethanol)
The resulting suspension was boiled for 10 minutes and centrifuged at 15000 rpm for 5 minutes. The supernatant was subjected to SDS-page (S
DS-PAGE) was carried out, and after CBB staining, one producing a protein having the highest molecular weight among those producing a new protein was selected. This fungus is LK1-10,
The plasmid retained by this bacterium was designated as pLK1-10.

When the base sequence of pLK1-10 was examined, it was found that the downstream region from base number 241 of SEQ ID NO: 1 in the sequence listing was inserted into the vector. Therefore, p
LK1-10 is a part of the β-galactosidase of the vector and a part of the flower bud inducer peptide, that is, SEQ ID NO: in the Sequence Listing.
It encodes a fusion protein with the peptide of amino acid numbers 81 to 263 of 1.

Escherichia coli harboring pLK1-10 (Es
cherichia coli AJ12820) has been deposited with the Institute of Microtechnology, and the deposit number is FERM P-13384.

LKl-10 was cultured in the same manner as above, and IPT
After adding G, the culture was continued for 4 hours. 10 ml of 50 mM phosphate buffer (pH 7.0) was added to the centrifuged bacterial cell precipitate,
After suspending by adding a 15 M sodium chloride solution, 200 μl of 10 N sodium hydroxide was added to lyse the cells. After neutralization with 6N hydrochloric acid, the method of Takeba et al. (G. Takeba et al., Plant
Physiol., 94 , 1677, 1990) to examine the flower bud inducing activity.

That is, Lemna paucosit
Ata ) 151 was used as an index for the flower bud induction. Substances to be assayed for flower bud inducing activity were added to 1/10 M medium (see Table 1 for M medium) at various concentrations, and 3 frond duckweed 151 fronds were planted and cultured under continuous light at 25 ° C. did.
The flower buds that differentiated after 1 week were observed with a stereoscopic microscope, and the flower bud-inducing activity was indicated by FL (%), which represents the number of flower buds per flond as a percentage.

[0042]

[Table 1]

As a result, the activity was confirmed (Table 2).

[0044]

[Table 2]

1 liter of the culture broth induced by IPTG was collected by centrifugation and frozen and thawed with liquid nitrogen to 2 times.
I went there. 10 ml, 50 mM Tris-HCl buffer (pH 8.0),
Add 25% sucrose and 1 mM EDTA solution to suspend, and add 5
400 μl of 0 mg / ml lithosome solution was added and treated on ice for 30 minutes.

It was frozen and thawed twice in liquid nitrogen again, and magnesium chloride, manganese chloride and DNase I (DNase I) were used.
I) was added so that the final concentrations were 10 mM, 1 mM, and 10 g / ml, and the mixture was treated on ice for 30 minutes. 10 ml, 0.2 M sodium chloride, 1% deoxycholic acid, 1% nonidet P-40
(Nonidet P-40), 20 mM Tris-HCl buffer (pH 7.
5) A 2 mM EDTA solution was added, the mixture was treated on ice for 15 minutes, and then centrifuged at 5000 rpm for 10 minutes. The obtained precipitate was 80 ml, 1% Triton X-100 (Triton X-100), 1%.
% EDTA, and the mixture was centrifuged at 5000 rpm for 10 minutes. After carrying out this operation 5 times, the precipitate was dissolved in SDS and subjected to SDS-PAGE (SDS-PAGE) to cut out the desired band. After extracting the target protein from this gel using a protein extractor (manufactured by Nippon Eido Co., Ltd.), 50 mM phosphate buffer (pH 8.0), 0.15M
The sample was dialyzed against a sodium chloride solution to obtain a standard product.

10 mg of this standard protein, 100 μg of trypsin
Prepare a 10 mM piperazine solution (pH 8.0) containing
And reacted overnight. Further, 100 μg of trypsin was added and the reaction was carried out for 6 hours, which was then subjected to reverse phase chromatography using PepPRC HR5 / 5 (Pharmacia). The elution conditions were as follows. Elution rate: 0.5 ml / min Dissolution solution: A ... Aqueous solution containing 0.1% TFA B ... Acetonitrile solution containing 0.1% TFA 0 to 10 minutes A 100% A 10% to 40 minutes A 100% → 40% linear gradient

Fractions showing activity (shaded area in FIG. 1) were taken as Cosmo
It was subjected to reverse phase chromatography using sil C18-AR (manufactured by Nacalai Tesque) to isolate a peak fraction showing activity. The elution conditions were as follows. Elution rate: 1 ml / min Dissolution liquid: A ... water B ... acetonitrile 0 to 40 minutes B 10% → 80% linear gradient

When the fraction showing activity (FIG. 2A) was applied to a gas phase protein sequencer, SEQ ID NO: 133 in the Sequence Listing was found.
To 141 sequences were determined.

[0050]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide means for easily mass-producing the flower bud inducer peptide.
By using this flower bud inducer peptide, it is possible to control the flowering and fruiting of plants and adjust the harvest time of crops. Therefore, the present invention makes it possible to bring about planning easily and cheaply in agricultural production.

[Sequence list]

SEQ ID NO: 2 Sequence Length: 47 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TGITCIAAIA CIGCITCIGC ITCIAAITTI GGIGCIGTIT TICCIAC 47

[Brief description of drawings]

FIG. 1 shows an elution curve obtained by subjecting 10 mg of a flower bud inducer peptide-labeled protein to trypsin digestion and subjecting it to reverse phase chromatography.

FIG. 2 shows an elution curve obtained by subjecting the fraction showing the activity in the reversed phase chromatography elution of FIG. 1 (shaded area) to another reverse phase chromatography.

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[Procedure amendment]

[Submission date] April 2, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

[0029]

[Example 1] Acquisition of cDNA encoding a flower bud inducer peptide Lemna paucicostat ( Lemna paucicostat)
a ) To 441 g of 441, a small amount of GIT buffer (4M guanidine isothiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M 2-mercaptoethanol, 0.5% sodium N-lauroylsarcosine) was added, and the mixture was homogenized with a homogenizer. After crushing, 15 at 6000 rpm
Centrifuge for minutes. GIT buffer was added to the obtained supernatant to make the total volume 60 ml, and 24 g of cesium chloride was dissolved to obtain an extract. 2 ml cesium chloride solution (5.
Put 10 ml of the extract into a centrifuge tube containing 0.1 M EDTA containing 7 M cesium chloride, and 35000 rpm
Centrifuge for 16 hours at 20 ° C (Beckman SW
41) was performed. After centrifugation, the RNA precipitate obtained was washed with 3
00 μl of TE-SDS solution (10 mM Tris-HCl buffer, 5 mM EDTA, 0.1% SDS, pH 7.
After dissolution with 4), chloroform-butanol (4:
1) The solution was extracted several times. 0.1 volume of 3M sodium acetate (pH 5.2) and 2.2 volume of 100% ethanol were added, and ethanol precipitation treatment was carried out according to a conventional method to obtain a total RNA precipitate.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

That is, Lemna p.
aucocosta ) 151 flower bud induction was used as an index for assay. Substances to be assayed for flower bud inducing activity were added to 1/10 M medium (see Table 1 for M medium) at various concentrations, and 37 frond duckweed 151 fronds were planted and cultured under continuous light at 25 ° C. did. The flower buds that differentiated after 1 week were observed with a stereoscopic microscope, and the flower bud-inducing activity was indicated by FL (%), which represents the number of flower buds per flond as a percentage.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction content]

[0050]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide means for easily mass-producing the flower bud inducer peptide.
By using this flower bud inducer peptide, it is possible to control the flowering and fruiting of plants and adjust the harvest time of crops. Therefore, the present invention makes it possible to bring about planning easily and cheaply in agricultural production.

[Sequence list]

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Claims (6)

[Claims] 1. A flower bud inducer peptide having an amino acid sequence from amino acid No. 81 to amino acid No. 263 of SEQ ID No. 1 in the sequence listing. 2. A DNA fragment containing a gene encoding the flower bud inducer peptide according to claim 1. 3. A recombinant DNA which is replicable in a prokaryotic cell, wherein a DNA fragment containing the gene encoding the flower bud inducer peptide according to claim 2 is connected downstream of a promoter that can be expressed in a prokaryotic cell. 4. A prokaryotic transformant carrying the recombinant DNA according to claim 3. 5. A method for producing a flower bud inducer peptide, which comprises culturing the transformant according to claim 4 and recovering the flower bud inducer peptide from the cells or the culture medium. 6. A plant characterized in that it retains a DNA fragment containing a gene encoding the flower bud inducer peptide according to claim 2 in addition to the original cell.