JPH08154684A - Cotton ubiquitin-binding enzymic gene - Google Patents

Abstract

PURPOSE: To obtain the subject new gene capable of participating in a ubiquitin- dependent proteolytic pathway, coding a cotton ubiquitin-binding enzyme and useful for preparation of a cotton stress-resistant individual, creation, etc., of a male sterile cotton plant. CONSTITUTION: This new DNA has an amino acid sequence containing an amino acid sequence represented by the formula or an amino acid sequence substantially the same as that and is capable of participating in a ubiquitin-dependent proteolytic pathway and coding a ubiquitin-binding enzyme derived from cotton and useful for preparation of a stress-resistant individual in cotton and creation, etc., of a male sterile cotton plant. The DNA is obtained by freezing a cotton fibrocyte with liquid nitrogen, sufficiently grinding the frozen fibrocyte in a mortar, treating the ground cell with a protease, isolating an mRNA according to a conventional method, preparing a cDNA library using the resultant mRNA as a template, screening the cDNA library with a probe comprising a part of a cotton ubiquitin-binding enzymic gene according to a plaque hybridization method, selecting a positive clone and recovering the DNA from the clone.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel cotton gene, and more particularly, to a ubiquitin-conjugating enzyme gene involved in a ubiquitin-dependent proteolytic pathway.

[0002]

BACKGROUND OF THE INVENTION Cotton is an important plant for supplying raw materials in the textile industry, but its growth is greatly affected by environmental conditions and may be greatly damaged by the weather. In addition, the appropriate growth environment among varieties is different, and some varieties grow only in specific areas.

On the other hand, in the breeding of cotton, the production of new varieties by crossing is a big problem. Cotton is a plant that self-pollinates, and when mating is performed, it requires a very laborious work of cutting out the flower buds and removing the stamens before pollination before pollination.

[0004]

As described above, the growth of cotton is affected by stress factors such as the environment. As described above, by increasing the resistance to stress, damage caused by stress such as abnormal weather can be prevented. Can be reduced.

By the way, in the living body, a protein having an abnormal three-dimensional structure or an abnormality such as degeneration is immediately decomposed in the intracellular sol. Abnormal proteins are produced by chemical damage such as incorrect incorporation of amino acids during synthesis, oxidation of amino acid side chains, and various stresses. Such abnormal proteins are decomposed by a selective decomposition reaction. Ubiquitin, a protein whose expression level is known to be increased by various stresses, plays a major role in the selective degradation reaction of proteins.

[0006] The degrading apparatus for selectively degrading proteins is complicated, and in the ubiquitin-dependent pathway used in eukaryotic cells, ubiquitin forms covalent bonds with many target proteins. This bond is catalyzed by an enzyme (ubiquitin-conjugating enzyme) produced by the gene provided by the present invention, and the ubiquitin-protein complex is recognized and degraded by a certain protease. .

Therefore, it is presumed that by increasing the expression level of this enzyme, the binding reaction between the protein to be degraded and ubiquitin is activated and the stress resistance is increased.

On the other hand, when an antisense gene against the ubiquitin-conjugating enzyme gene is introduced into cotton to suppress the expression of the ubiquitin-conjugating enzyme gene, an abnormal protein removal mechanism is impaired and stress resistance is reduced. It is thought that this interferes with the normal growth of cells.

Therefore, it is presumed that by introducing this gene into a cotton plant by linking it to an anther (pollen) -specific promoter, growth of anther (pollen) cells is impaired and a male sterility trait can be imparted. By using such a male sterile body, the labor at the time of mating can be reduced.

Although the ubiquitin-conjugating enzyme gene has already been isolated by other organisms, it is preferable to use the original gene sequence of cotton in view of expressing it in the cotton plant, and when the antisense gene is introduced, Is preferably more completely complementary to the cotton gene sequence.

An object of the present invention is to provide a gene for the above enzyme, which is useful for the production of stress-resistant individuals in cotton and the production of male sterile cotton plants.

[0012]

[Means for Solving the Problems] The present inventor
An NA library was prepared, its clones were randomly selected, the nucleotide sequence was determined, and a homology search was performed with known gene sequences in the gene database. Usually, as a gene cloning method, there is also a method of screening a library by using a part or all of a gene sequence already isolated in another organism as a probe, but the present invention is a random method from a cDNA library. Among the genes identified as having high homology with some genes by homology search between the nucleotide sequences of the clones selected as above and the known gene sequences in the gene database, the cotton ubiquitin conjugating enzyme gene The existence was recognized and it was completed.

That is, the present invention is a DNA containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence substantially the same as the amino acid sequence. Specific examples of this nucleotide sequence include the sequences of nucleotide numbers 80 to 523 in SEQ ID NO: 1 or sequences substantially the same as these. This DNA is a gene encoding a cotton ubiquitin-conjugating enzyme and is also referred to herein as a gene of the present invention.

The present invention will be described in detail below. The gene of the present invention was obtained for the first time by the method described below, but since the nucleotide sequence was clarified by the present invention, a gene well known to those skilled in the art based on this sequence, for example, PCR method. It can be easily obtained by amplification from chromosomal DNA or cDNA, screening from a library by a hybridization method, or the like.

An example of the method for obtaining the gene of the present invention will be described step by step.

<1> Preparation of Gene Library A gene library can be prepared from both chromosomal DNA and mRNA. In order to efficiently isolate and identify a structural gene, mRNA is used as a material. Preparation of the used cDNA library is desirable.

Since the gene of the present invention is considered to be expressed in almost all organs in the body, the material for extracting mRNA is not particularly limited to that organ. The extraction method of mRNA is not particularly limited, and it can be extracted from an ordinary plant.
An mRNA extraction method can be adopted.

The synthesis of cDNA is carried out, for example, by synthesizing complementary DNA with reverse transcriptase using a poly T sequence complementary to the poly A nucleotide present at the end of mRNA as a primer and making it double-stranded with DNA polymerase. You can

The method is, for example, Molecular Cloning (M
aniatis et al., Cold Spring Harbor Laboratory), but a large number of cDNA synthesis kits are commercially available from various companies, and thus they may be used.

The library is generally constructed by a phage vector, and a large number of commercially available ones can be used, but a vector for which sequencing plasmid can be immediately prepared without the need for recloning from the vector, For example, it is preferable to use a λZAP vector or the like.

<2> Determination of Gene Sequence A clone is randomly selected from the obtained gene library, and the nucleotide sequence of the insert in the clone is determined. The nucleotide sequence can be determined by the Maxam-Gilbert method or the dideoxy method, and among these, the dideoxy method is more convenient and preferable.

To determine the nucleotide sequence by the dideoxy method, a commercially available sequencing kit can be used, and the sequence of a large number of clones can be determined in a short time by using an auto sequencer. You can

It is not necessary to determine the sequence for the entire length of the insert, as long as it is possible to determine the length considered sufficient for homology search. For example, in the examples described below, 60
When the sequence of more than bases could be determined, the following homology search was performed.

<3> Homology Search with Gene Database A homology search is performed between the determined nucleotide sequence of each cDNA clone and the known nucleotide sequence registered in the gene database.

As the database, GenBank, EMBL, DDBJ, etc., which are published by Los Alamos National Laboratory, European Molecular Biology Laboratory, National Institute of Genetics, etc. can be used as a homology search program. Can purchase and use commercially available DNA analysis software, DNASIS (Hitachi Software Engineering), GENETYX (SDC Software Development Co., Ltd.) and the like. Also,
There is also a method of connecting a large-scale computer of the National Institute of Genetics and a terminal to perform high-speed homology search.

The homology search is performed by the following algorithm, for example. Homology is compared while shifting the sequence to be examined from the gene sequence in the database by one base at a time, and when the bases are consistent for 6 or more consecutive bases, a homology score table (eg M.
Dayhoff, Atlas of Protein Sequence and Structure,
vol.5 (1978, etc.)) and calculate the homology score. It is advisable to set a score having a certain value or more to be taken as a candidate having homology, and to make a gap in the sequence to be examined or the gene sequence in the database to optimize the score.

The gene of the present invention was obtained as one of a large number of genes having homology with known genes, as will be shown in the Examples below, and includes tomato, pea, arabidopsis, rice, yeast and the like. The homology with the known ubiquitin-conjugating enzyme gene was confirmed. Among them, the gene with the highest homology was the tomato gene, and the lengths of the bases in the coding region were both 447 bp including the stop codon, and 364 of them matched.
The homology was 81.4%. In addition, when the amino acid sequences deduced from the nucleotide sequences were compared, 136 amino acids of 148 amino acids were in agreement, and the homology was 91.9%. 14 when considering amino acids with similar properties
Since 6 amino acids are considered to be substantially identical and have high homology of 96.8%, the gene obtained by the present invention was identified as a gene encoding a ubiquitin-conjugating enzyme in cotton. .

<4> Isolation of full-length clone of cotton gene having homology with gene in gene database The clone obtained as described above does not always contain the entire nucleotide sequence of the gene.
In that case, the clone containing the full-length gene can be obtained from the library by using the clone as a probe and performing screening by plaque hybridization. The specific method is Molecu
lar Cloning 2nd Edition (Maniatis et al., Cold Spring Harbor
Laboratory) 12.30-40.

In the examples, randomly selected c
Since it was possible to obtain a full-length clone from the DNA clone, it was not necessary to perform rescreening. However, for example, an appropriate 5 ′ non-coding region (nucleotide sequence 1 to 79) shown in the sequence listing was selected. By selecting an oligonucleotide sequence and using it as a probe, clones having a full length can be easily screened from the library. Alternatively, a plurality of clones having partial sequences may be ligated to obtain a full-length DNA fragment.

The nucleotide sequence of the gene of the present invention obtained as described above and the amino acid sequence predicted from this sequence are shown in SEQ ID NOS: 1 and 2 of the Sequence Listing. This amino acid sequence is a novel sequence, and all genes having a nucleotide sequence encoding this amino acid sequence are included in the present invention.
Further, in the above amino acid sequence, as long as it does not substantially affect the activity of the ubiquitin conjugating enzyme, it may include substitution, deletion or insertion of amino acid residues. Such substitution, deletion, or insertion of amino acid residues can be obtained as an expression product of a gene DNA that is randomly or intentionally mutated and has enzymatic activity.
Further, depending on the cotton cultivar, or due to natural mutation or the like, there may be some that carry an enzyme or gene having a sequence partially different from the sequence shown in the sequence listing. Included in the gene.

It is expected that the gene of the present invention can be used for the production of stress resistant cotton and the production of male sterile cotton plants.

[0032]

EXAMPLES Examples of the present invention will be described below.

<1> Preparation of Total RNA from Cotton Cotton (Gossypium hirsutum L.) Coker 312 was used as a material, and its fiber cells were collected in liquid nitrogen. 75 g of cotton fiber cells were thoroughly ground in a mortar while freezing with liquid nitrogen. Transfer the powdered fiber to a centrifuge tube with a lid, add 375 mg of DTT as powder, and then heat the XT buffer (90 mM EDTA and 0.2 M sodium borate containing 1% SDS) heated to 90-95 ° C. After adjusting to pH 9.0, treat with diethylpyrocarbonate and autoclave.Add vanadyl ribonucleoside to this solution to 10 mM)
200 ml was added and stirred well.

To this, 100 mg of protease K was added, and the mixture was stirred again and incubated at 40 ° C. for 2 hours, and then 16 ml of 2M KCl
Was added. After stirring well again, leave on ice for 1 hour,
It was centrifuged at 12,000 g for 20 minutes (4 ° C) in a high speed cooling centrifuge.

The supernatant was filtered to remove the suspended matter, transferred to a graduated cylinder to measure the volume, transferred to another centrifuge tube,
85 mg of lithium chloride was added per 1 ml of the extract to a final concentration of 2M. This solution was allowed to stand overnight at 4 ° C., and then the precipitated RNA was centrifuged at 12,000 g for 20 minutes. Obtained RNA
The precipitate was washed and precipitated twice with cold 2M lithium chloride.

The obtained RNA was treated with 10 mM Tris buffer (pH 7.
Dissolve in 5) to about 2 mg / ml and add 5M potassium acetate.
After adding 200 mM, ethanol was added to 70% and cooled at −80 ° C. for 10 minutes. After centrifugation at 4 ° C. and 15,000 rpm for 10 minutes, the RNA sample was suspended in an appropriate amount of sterilized water. As a result of quantifying this RNA sample, 2 mg of total RNA was obtained.

<2> Purification of mRNA From the total RNA obtained above, mRNA was purified as a poly (A) ⁺ RNA fraction. For purification, poly (A) ⁺ RNA purification oligo (d
T) Immobilized latex Oligotex-dT30 <Super> (purchased from Toyobo Co., Ltd.) was used.

Elution buffer (elution buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% SD) was added to a solution of 1 mg of total RNA.
S) to make the total volume 1 ml, and Oligotex-dT30 <Sup
er> 1 ml was added, the mixture was heated at 65 ° C. for 5 minutes and rapidly cooled on ice for 3 minutes. To this, 0.2 ml of 5M NaCl was added, incubated at 37 ° C for 10 minutes, and then centrifuged at 15,000 rpm for 3 minutes,
The supernatant was carefully removed.

The pellet was washed with Washing Buffer (wash: buffer 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M NaCl, 0.1
% SDS) was suspended in 2.5 ml, centrifuged at 15,000 rpm for 3 minutes, and the supernatant was carefully removed. Pellet the TE Buffer
It was suspended in 1 ml and then heated at 65 ° C. for 5 minutes. This was rapidly cooled on ice for 3 minutes and then centrifuged at 15,000 rpm for 3 minutes to recover poly (A) ⁺ mRNA in the supernatant.

As described above, from 1 mg of total RNA, about 10
μg of poly (A) ⁺ mRNA was obtained. 5 μg of which is cDN
Used to make A library.

<3> Preparation of cDNA Library (1) Synthesis of cDNA Using the mRNA obtained above as a template, Stratagene λZA
CDNA synthesis was performed using the P cDNA synthesis kit.

The following solutions were mixed in a tube. 5.0 μl 10 × 1st Strand Buffer (Reverse transcription reaction buffer) 3.0 μl 10 mM 1st Strand Methyl Nucleotide Mix (A,
G, C, U mixture) 2.0 μl Linker-Primer (linker / primer) H ₂ O (adjust the total volume to 50 μl) 1.0 μl RNase Block II (RNAse inhibitor)

Each of the above components is the contents of the kit,
The sequence of nker-Primer is as shown in SEQ ID NO: 3. The methylated nucleotide is used so that the cDNA will not be cleaved by the subsequent restriction enzyme reaction.

After thoroughly stirring the above reaction solution, poly (A) ⁺ m
5.0 μg of RNA was added, and the mixture was reacted at room temperature for 10 minutes. 2.5 more
After adding μl M-MuLV RTase (reverse transcriptase) (at this time, the total volume becomes 50 μl), gently mixing, centrifuging gently, the reaction solution was dropped to the bottom of the tube, and reacted at 37 ° C. for 60 minutes.

The following solutions were then mixed into the tube in the order given. 45.0 μl Reaction solution containing cDNA primary strand 40.0 μl 10 × 2nd Strand Buffer (polymerase reaction buffer) 6.0 μl 2nd Strand Nucleotide Mixture (A, G, C, T mixture) 302.0 μl H ₂ O

Further, the following solution was added, but RNase and DNA were added.
The enzyme solution was attached to the wall of the tube so that the polymerases could work simultaneously, then quickly vortexed and centrifuged to drop the reaction solution to the bottom of the tube, 150 ° C for 16 minutes at 16 ° C.
A DNA secondary strand synthesis reaction was performed.

0.8 μl RNase H (RNA-degrading enzyme) 7.5 μl DNA polymerase I (10.0 u / μl)

To this, 400 μl of a phenol: chloroform (1: 1) mixture was added, stirred well, and then centrifuged at room temperature for 2 minutes. Add 400 μl of phenol: chloroform to the supernatant,
It was vortexed and centrifuged at room temperature for 2 minutes, and the following solution was added to the supernatant to precipitate cDNA.

33.3 μl 3M sodium acetate solution 867.0 μl 100% ethanol

This was left overnight at -20 ° C., centrifuged at room temperature for 60 minutes, gently washed with 80% ethanol, and then centrifuged for 2 minutes. The supernatant was removed, the pellet was dried and dissolved in 43.5 μl of sterile water. The following solution was added to 39.0 μl of the solution to blunt the cDNA ends.

5.0 μl 10 × T4 DNA Polymerase Buffer
(T4 Polymerase reaction buffer) 2.5 μl 2.5 mM dNTP Mix (A, G, C, T mixture) 3.5 μl T4 DNA polymerase (2.9 u / μl)

The reaction was carried out at 37 ° C. for 30 minutes, 50 μl of sterilized water was added, 100 μl of phenol: chloroform was added, and the mixture was vortexed and centrifuged for 2 minutes. 100 μl of chloroform was added to the supernatant, vortexed and centrifuged for 2 minutes, and the following solution was added to the supernatant to precipitate cDNA.

7.0 μl 3M sodium acetate solution 226 μl 100% ethanol

The solution was left on ice for 30 minutes or longer and kept at 4 ° C.
It was centrifuged for 60 minutes. The precipitate was washed with 150 μl of 80% ethanol, centrifuged for 2 minutes, and dried. This cDNA pellet was dissolved in 7.0 μl of EcoRI Adapter (EcoRI adapter: see FIG. 1) solution, and the following solution was added to add cDN.
EcoRI adapters were ligated to both ends of A.

1.0 μl 10 × Ligation Buffer (buffer for ligase reaction) 1.0 μl 10 mM ATP 1.0 μl T4 DNA ligase

The reaction solution was lightly centrifuged and left at 4 ° C. overnight or more. The sequence of each strand of the EcoRI adapter is shown in SEQ ID NOs: 4, 5 and FIG. This solution was treated at 70 ° C. for 30 minutes, then lightly centrifuged, allowed to stand at room temperature for 5 minutes, and the following solution was added to phosphorylate the 5 ′ end of the EcoRI adapter.

1.0 μl 10 × Ligation Buffer (buffer for ligase reaction) 2.0 μl 10 mM ATP 6.0 μl H ₂ O 1.0 μl T4 Polynucleotidkinase (10.0 u /
μl)

After reacting at 37 ° C. for 30 minutes and after treating at 70 ° C. for 30 minutes, it was lightly centrifuged and left at room temperature for 5 minutes. Further, the following solution was added, and the mixture was reacted at 37 ° C. for 90 minutes to cut the XhoI site introduced by Linker-Primer with XhoI, and then left at room temperature to cool.

28.0 μl XhoI Buffer 3.0 μl XhoI (45 u / μl)

5.0 μl of 10 × STE (10 mM Tris
-HCl (pH8.0), 100mM NaCl, 1mM EDTA) was added and the short column removal centrifugal column (Sephacryl Spin Col)
umn) and centrifuged at 600 g for 2 minutes to give a fraction 1. This operation was repeated 3 times to obtain fractions 2, 3 and 4, respectively.

Fractions 3 and 4 were combined, phenol: chloroform (1: 1) was added, and the mixture was stirred well and centrifuged at room temperature for 2 minutes. An equal amount of chloroform was added to the supernatant, and the mixture was stirred well, centrifuged at room temperature for 2 minutes, and twice the 100% ethanol was added to the supernatant, and the mixture was allowed to stand at -20 ° C overnight. This was centrifuged at 4 ° C. for 60 minutes and then washed with an equal volume of 80% ethanol. 4 more
After centrifugation at 60 ° C. for 60 minutes, the cDNA pellet was suspended in 10 μl of sterilized water.

(2) Preparation of cDNA Library The double-stranded cDNA obtained above was ligated to a λ phage expression vector to prepare a recombinant vector.

The following solutions were mixed in a tube, reacted at 12 ° C. overnight and left at room temperature for 2 hours to ligate cDNA to a vector. 2.5 μl cDNA solution 0.5 μl 10 × Ligation Buffer 0.5 μl 10 mM ATP 1.0 μl λZAP vector DNA (1 μg / μl) 0.5 μl T4 DNA ligase (4 Weiss u / μl)

(3) Packaging of Phage DNA into Phage Particles A phage vector into which a cDNA was inserted was prepared in vitro.
Packaging Kit (GigapackII Gold packaging ex
tract; Stratagene) was used to package the phage particles.

Freeze-thaw extract immediately after thawing (Freeze
/ Thaw extract) with recombinant phage solution, place on ice and immediately add 15 μl of sonicated extract (Sonic extra).
ct) was added and mixed by pipetting well. This was lightly centrifuged and left at room temperature (22 ° C) for 2 hours.

To the above reaction solution, 500 μl of phage dilution buffer (Phage Dilution Buffer) was added, and further 20 μl of chloroform was added and mixed. To measure the titer of the library, 2 μl of the 500 μl aqueous phase was added to 18 μl of SM.
buffer (in 1 L, NaCl 5.8g, MgSO ₄ / 7H ₂ O 2g, 1M Tris
Diluted 1:10 with HCl (pH 7.5) 50 ml, 2% gelatin 5 ml). 1 μl dilution and 1 μl phage stock solution,
The plate was plated with 200 μl of the culture solution of Escherichia coli PLK-F ′ strain that had been cultured until the OD ₆₀₀ reached 0.5. That is, E. coli
The PLK-F ′ strain and the phage solution were mixed, incubated at 37 ° C. for 15 minutes, added to 2-3 ml of top agar (48 ° C.), and immediately overlaid on an NZY agar plate warmed to 37 ° C. 37 ° C
After culturing overnight in, the number of emerging plaques was counted and the titer was calculated. As a result, the titer was 1.2 × 10 ⁶ pfu / ml.

(4) Amplification of library To a centrifuge tube, add a packaging solution containing about 50,000 recombinant bacteriophages, and ₆₀₀ μl of a culture solution of Escherichia coli PLK-F 'strain cultured to an OD ₆₀₀ of 0.5, and add at 37 ° C In 1
Cultured for 5 minutes. To this culture medium, 6.5 ml of top agar kept at 48 ° C after thawing was added and warmed to about 37 ° C1.
The cells were overlaid on a 50 mm NZY plate and incubated at 37 ° C for 5 to 8 hours. 10 ml of SM Buffer was added to each plate, and the plate was incubated overnight at 4 ° C with slow shaking.

Collect SM Buffer in each plate into a sterilized polypropylene tube and add 2 ml of SM to each plate.
Rinse with buffer and also collect in the same tube. Chloroform, which is 5% of the total amount, is added and mixed at room temperature for 15
The cells were allowed to stand for 4 minutes and centrifuged at 4,000 g for 5 minutes to remove bacterial cells. Chloroform (0.3% of the total amount) was added to the supernatant, and the mixture was mixed at 4 ° C.
Saved in.

The titer of the library thus amplified was measured in the same manner as above, and the result was 2.3 × 10 ⁹ pfu / ml.

(5) Excision of Plasmid from Phage DNA In vitro excision of the plasmid portion was performed from the recombinant phage DNA. The following were mixed in a 50 ml conical tube and infected at 37 ° C for 15 minutes.

E. coli XL1-Blue culture solution (OD ₆₀₀ = 0.1) 200 μ
l Phage solution after amplification 200 μl (> 1 × 10 ⁵ phage particles) Helper phage R408 1 μl (> 1 × 10 ⁶ pfu / ml)

5 ml of 2 × YT medium was added to the above mixture, and 37
After shaking culture at ℃ for 3 hours and heat treatment at 70 ℃ for 20 minutes,
It was centrifuged at 00 g for 5 minutes. The supernatant was decanted and transferred to a sterile tube. Centrifuge this, and dilute the supernatant 100-fold.
200 μl of a culture solution of Escherichia coli XL1-Blue, which had been cultivated until the OD ₆₀₀ reached 1.0, was mixed and infected at 37 ° C. for 15 minutes.

1 to 100 μl of the culture solution was plated on an LB plate containing ampicillin, and then cultured at 37 ° C. overnight. Colonies that appeared were randomly selected, glycerol was added, and the mixture was stored at -80 ° C.

(6) Preparation of plasmid A plasmid was prepared using Magic mini-prep kit manufactured by Promega. A culture solution of Escherichia coli carrying the plasmid stored at -80 ° C was inoculated into 5 ml of 2XYT medium and cultured at 37 ° C overnight. Centrifuge for 5 minutes (4,000 rpm,
The supernatant was removed by decanting, and 1 ml of TE buffer was added to the bacterial cell pellet and vortexed. Transfer the cell suspension to an Eppendorf tube and centrifuge for 5 minutes (5,00
The supernatant was removed by decanting.

300 μl of Cell Resuspensio was added to the cell pellet.
n Solution (solution for cell suspension) was added, well suspended, and transferred to an Eppendorf tube. This was stirred with a mixer for 2 minutes, 300 μl of Cell Lysis Solution (cell lysis solution) was added, and the mixture was stirred until it became transparent. Further 300 μl Ne
After adding utralization Solution (solution for neutralization), the mixture was shaken by hand and stirred, and then centrifuged (15,000 rpm) for 10 minutes.

The supernatant alone was transferred to a new Eppendorf tube (1.5 ml). Connect a cock, a mini column, and a syringe (syringe) in this order to the suction tube, and then attach 1 resin to the syringe.
I added ml. The above supernatant was poured into this syringe, stirred well, and then aspirated. Add 2 ml of Column Wash Solution to wash by aspirating and
I kept sucking for 2 minutes. Remove the mini column,
It was set in a new Eppendorf tube (1.5 ml). Inject 100 μl of sterilized water warmed to 65-70 ° C into the mini column and centrifuge together with the Eppendorf tube for 1 minute (5,000 rp
m) did.

Transfer the eluate to an Eppendorf tube,
Add 5 μl of 3M sodium acetate solution, and add cold ethanol.
250 μl of (99.5%) was added. Centrifuge this (15000 rpm, 25
Minutes), the supernatant was discarded, 1 ml of ethanol (70%) was added to the precipitate, and the mixture was centrifuged again (15000 rpm, 3 minutes). The ethanol was completely removed and the tube was vacuum dried in a desiccator. Dissolve the precipitate in 20 μl of sterile water,
Stored at ° C. 1 μl of this solution was taken and subjected to electrophoresis together with a volume marker to quantify the plasmid DNA.

<4> Determination of nucleotide sequence of cDNA and homology search with gene database (1) Determination of nucleotide sequence of cDNA For analysis of nucleotide sequence of cDNA, DNA Auto Sequencer 373A manufactured by Applied Biosystems (ABI) was used. went. Sequence reaction is Dye Primer Cycle Sequenci manufactured by the same company
ng kit using T3 primer according to the attached instructions. The nucleotide sequence of about 750 clones randomly selected was determined.

(2) Homology search with gene database The determined nucleotide sequence was subjected to the homology search function of DNA analysis software DNASIS (Hitachi Software Engineering). We chose GenBank as the database. The homology search algorithm compares the sequence to be searched while shifting it by 1 base from the sequence in the database, and calculates the homology score according to the homology score table when 6 or more consecutive bases match. It is to do. The homology score of 160 points or more was set as a candidate, and the gap was further optimized to optimize the score.

Among the sequences of about 750 clones searched, 282 clones had homology with some sequences in the database, and among them, clones having homology with plant ubiquitin-conjugating enzyme gene were found. The gene with the highest homology score was the same gene isolated from tomato, but by comparing the clones obtained and the tomato gene with each other, it was found at this point that the above clone contained the start codon. did.

(3) Determination of the full-length nucleotide sequence of the cotton ubiquitin conjugating enzyme gene To determine the full-length sequence of the clone which was found to contain the initiation codon and encoded the cotton ubiquitin conjugating enzyme, 3 ' In order to determine the sequence from the terminal side, the nucleotide sequence was determined by an auto sequencer using the T7 primer. By using both T3 and T7 primers, all the base sequences were revealed as shown in the sequence listing.

(4) Comparison of the gene of the present invention with the tomato ubiquitin conjugating enzyme gene The homology search with the database was performed again using the entire sequence of the revealed gene of the present invention. as a result,
It was found that there is homology with the ubiquitin-conjugating enzyme genes of tomato, pea, arabidopsis, rice, yeast and the like. Among them, the tomato-derived gene had the highest homology with the gene of the present invention.

The lengths of the bases in the coding region were both 447 bp including the stop codon, of which 364 bases were in agreement, and the homology was 81.4% (FIG. 2). Also,
When the amino acid deduced from the nucleotide sequence of the gene was compared, all consisted of 148 amino acids, of which 136 amino acids were in agreement and had a homology of 91.9% (Fig. 3). 14 when considering amino acids with similar properties
The clones obtained above were identified as the cDNA of the gene encoding the ubiquitin conjugating enzyme in cotton, because 6 amino acids were considered to be in agreement and had high homology of 98.6%.

[0084]

Industrial Applicability According to the present invention, a cotton-derived ubiquitin-conjugating enzyme gene was obtained for the first time. It is expected that this gene can be used for the production of stress resistant cotton and the production of male sterile cotton plants.

[0085]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 733 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Cotton (Gossypium hirsutum L.) Sequence characteristics Symbol: CDS Location: 80..526 Method of determining the characteristic: S sequence CAACTCCACA ACGCCAAATT CAAGTTTACC AACCTTATTG TTTATTTGTT CGAGAATTAG 60 AGAGTTTTTT TGAATACCC ATG GCT TCG AAG CGG ATT AAT AAG GAG TTG AAA 112 Met Ala Seruu Ly Lyn Arle Lyn Arle Lys 1 5 10 GAT CTG CAG AAG GAC CCT CCT GCA TCT TGC AGT GCT GGC CCT GTT GGT 160 Asp Leu Gln Lys Asp Pro Pro Ala Ser Cys Ser Ala Gly Pro Val Gly 15 20 25 GAT GAT ATG TTC CAC TGG CAA GCA ACA ATT ATG GGC CCA GCA GAC AGT 208 Asp Asp Met Phe His Trp Gln Ala Thr Ile Met Gly Pro Ala Asp Ser 30 35 40 CCT TTT GCT GGA GGA CTA TTT CTT GTT TCC ATT CAC TTC CCC CCA GAC 256 Pro Phe Ala Gly Gly Leu Phe Leu Val Ser Ile His Phe Pro Pro Asp 45 50 55 TAT CCA TTC AAG CCT CCC AAG GTT TCT TTC CGA ACA AAG GTT TTC CAT 304 Tyr Pro Phe Ly s Pro Pro Lys Val Ser Phe Arg Thr Lys Val Phe His 60 65 70 75 CCC AAC ATC AAC AGC AAT GGA AGC ATC TGT CTT GAC ATT CTC AAG GAG 352 Pro Asn Ile Asn Ser Asn Gly Ser Ile Cys Leu Asp Ile Leu Lys Glu 80 85 90 CAG TGG AGC CCT GCC CTT ACC ATA TCC AAG GTG CTG CTT TCA ATA TGC 400 Gln Trp Ser Pro Ala Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cys 95 100 105 TCA CTT CTC ACA GAA CCA AAT CCT GAT GAC CCT CTG GTG CCT GAG ATT 448 Ser Leu Leu Thr Glu Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Ile 110 115 120 GCT CAC ATG TAT AAG ACT GAT AGA GCC AAA TAT GAG AGC ACT GCT CGC 496 Ala His Met Tyr Lys Thr Asp Arg Ala Lys Tyr Glu Ser Thr Ala Arg 125 130 135 TCA TGG ACT CAG AAA CAT GCA ATG AAT TAAGGGTCGG TCAGGCTTAG 543 Ser Trp Thr Gln Lys His Ala Met Asn 140 145 GATGCTATGT TTGATATCTT TTACTAAATA ATAGTAGTAA TAACTTTTGT CGTTTGGATCAA TTACTATTAT AAATATATTC GACAGTTCTT AAAAAAAAAA 723 AAAAAAAAAA 733

SEQ ID NO: 2 Sequence length: 148 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Ala Ser Lys Arg Ile Asn Lys Glu Leu Lys Asp Leu Gln Lys Asp 1 5 10 15 Pro Pro Ala Ser Cys Ser Ala Gly Pro Val Gly Asp Asp Met Phe His 20 25 30 Trp Gln Ala Thr Ile Met Gly Pro Ala Asp Ser Pro Phe Ala Gly Gly 35 40 45 Leu Phe Leu Val Ser Ile His Phe Pro Pro Asp Tyr Pro Phe Lys Pro 50 55 60 Pro Lys Val Ser Phe Arg Thr Lys Val Phe His Pro Asn Ile Asn Ser 65 70 75 80 Asn Gly Ser Ile Cys Leu Asp Ile Leu Lys Glu Gln Trp Ser Pro Ala 85 90 95 Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cys Ser Leu Leu Thr Glu 100 105 110 Pro Asn Pro Asp Asp Pro Leu Val Pro Glu Ile Ala His Met Tyr Lys 115 120 125 Thr Asp Arg Ala Lys Tyr Glu Ser Thr Ala Arg Ser Trp Thr Gln Lys 130 135 140 His Ala Met Asn 145

SEQ ID NO: 3 Sequence GAGAGAGAGA GAGAGAGAGA ACTAGTCTCG AGTTTTTTTT TTTTTTTTTT 54

SEQ ID NO: 4 Sequence AATTCGGCAC GAG 13

SEQ ID NO: 5 Sequence CTCGTGCCG 9

[Brief description of drawings]

FIG. 1 is a diagram showing the structure of an EcoRI adaptor.

FIG. 2 is a diagram showing a comparison of the nucleotide sequences of ubiquitin-conjugating enzyme genes of cotton and tomato. * Indicates a base that is the same in both genes.

FIG. 3 is a view showing a comparison of amino acid sequences of ubiquitin-conjugating enzymes of cotton and tomato. * Indicates the amino acid residues that are the same in both enzymes. Symbols represent amino acid residues with similar properties.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Soichiro Takenishi 1-18-1 Nishiaraicho, Adachi-ku, Tokyo Nisshinbo Ltd. Tokyo Research Center (72) Inventor Hirofumi Uchimiya 1315 Kizuki, Nakahara-ku, Kawasaki-shi, Kanagawa Kizuki Housing 1-403

Claims (2)

[Claims] 1. A DNA containing a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence substantially identical thereto. 2. The DNA according to claim 1, wherein the nucleotide sequence is the sequence of nucleotide numbers 80 to 523 in SEQ ID NO: 1 or a sequence substantially identical thereto.