JP2000135081A - Substrate for analyzing plenty of genomic gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare the subject substrate capable of effectively measuring expressivity of each gene in plants and useful as a DNA array of plants, or the like by ligating plural genomic DNA fragments from plants and including a specific number of genes. SOLUTION: The object substrate for analyzing plenty of genomic genes in which plural genomic DNA fragments from plants and including <=1 average number of genes are ligated is prepared by cloning a long DNA fragment covering a genome region, in which the base sequence has been already determined, from a genomic DNA prepared from tissue or cells of plants such as Arabidopsis thaliana (L.) Heynh., by the fragmentation of the above fragments to prepare a fraction so as its size to include <=1 average number of genes, by applying it to a substrate such as nylon filter, a slide glass or silicon and by immobilizing it by heat treatment or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a plant-derived genome.
The present invention relates to a DNA fragment-bound support and its use.

[0002]

2. Description of the Related Art As means for analyzing the function of a gene, it is common practice to measure the amount of gene transcript (mRNA) or to examine the effect of gene disruption.

[0003] When measuring the amount of gene transcript (mRNA), a DNA fragment encoding a specific gene is labeled with an isotope, a fluorescent dye, or the like, and the amount of RNA that complementarily binds thereto is measured. (Northern method, dot hybridization method). On the other hand, recently, a large amount of the nucleotide sequence (EST sequence) of the expressed gene has been analyzed, and when it becomes public, as a method of measuring the expression level of these genes, reverse transcription of total mRNA is performed. It can be converted to cDNA by the action of an enzyme, labeled, and complementarily bound (hybridized) to a support such as a filter on which a number of target DNA fragments are immobilized. It is being done.
This method is called a DNA array method.

[0004] In eukaryotes, since a poly-A chain is added to the 3 'end of mRNA, it is possible to separate and extract only mRNA from most other RNA species by using an oligo dT column. It is. Therefore, the background at the time of hybridization is reduced, and the analysis of the gene expression level by the DNA array method is easier than in the case of prokaryotes.

In fact, in the case of yeast which is a eukaryote, there is an example in which DNA of a clone contained in a genomic DNA library prepared using lambda DNA as a vector is bound to a filter, and the gene expression level is measured. (Shalon et al.,
Genome Research 6: 639-645, 1996).

However, in this case, since one clone contains several genes, in order to know the expression level of each gene, it was necessary to take out each gene and examine it again. In the case of yeast, the gene portion can be easily predicted from the nucleotide sequence.
Research has been conducted to amplify only yeast from yeast genomic DNA by PCR and to examine almost all genes in yeast using a DNA array (DeR
isi et al., Science 278: 680-686, 1997).

[0007] However, it is difficult to predict the gene portion in higher animals and plants, and at present, the DNA array method uses EST clones selected from expressed gene products,
Analyzes are performed using genomic DNA that has already been subjected to structural analysis or a part thereof.

On the other hand, as a method for disrupting a specific gene, a method for knocking out a specific gene has been established in animals (particularly mice) and microorganisms by utilizing the principle of homologous recombination. However, knockout of a specific gene using homologous recombination in plants has been reported to date in two cases (Nature 389: 80).
2-803, 1997), but it has not yet been established as a general method. In plants, knockouts can be found among transformed plants into which genes have been randomly introduced, but it is difficult to apply them to all genes. However, it is possible to introduce a transposon, a moving gene, into plants (Smith et al., Plant J. 1).
0: 721-732, 1996) In addition, it is possible to specify the location of the introduced transposon on the chromosome by using genetics techniques (Smith et al., Plant J. 10: 721-7).
32, 1996). Transposons jump out of the chromosome with a certain probability and land again at another position on the chromosome,
It is known that the landing point is overwhelmingly near the original position. Therefore, if a large number of transposon-introduced strains are prepared, it is possible to destroy a gene at an arbitrary position using the transposon. That is, in a plant, in order to perform gene disruption using the transposon method, it is important to obtain positional information of a gene on a chromosome.

[0009] Meanwhile, in plants, whole genome sequence analysis of Arabidopsis thaliana is being promoted under international cooperation (Nature 391: 438-439, 1998). Decoding the base sequence of the genome not only decodes the sequences of individual genes, but also provides information on their chromosomal locations. As described above, the transposon method is effective as a gene disruption method in plants, but when an EST sequence is found in the genomic base sequence being decoded, it is effective in combination with a DNA array method using EST clones. Analysis can be performed. However, for many genes not included in the EST data, an effective method for analyzing the genes has not yet been developed.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for effectively measuring the expression level of individual genes in a plant and a plant DNA array used in the method. The DNA array can detect unknown genes not included in the EST data. In a preferred embodiment, it is also used for specifying a gene region on plant genomic DNA. Therefore, it can be used in combination with the transposon tagging method.

[0011]

As described above, in the conventional measurement of gene expression level in yeast, several genes are contained in a genomic DNA fragment bound to a filter for gene expression analysis. Therefore, it was difficult to detect the expression level of each gene. Therefore, the present inventors first prepared a genomic DNA fragment containing only one gene in order to improve this point.

[0012] Here, the present inventors have reported that one gene is contained in about 5 kbp in decoding the genome sequence of Arabidopsis thaliana (Nature 391: 438-43).
9, 1998), assuming that genomic DNA fragments of 2-3 kbp contain on average one or less gene, and fragment genomic DNA prepared from Arabidopsis thaliana with restriction enzymes.
A fraction with a size of about 2-3 kbp was obtained.

In order to detect the expression of the gene and to specify the map position of the gene, the inventors of the present invention determined about 1/3 of the Arabidopsis chromosome 5 from the obtained DNA fragment of about 2-3 kbp. Select DNA fragments to cover 6 regions (approximately 1/30 of the entire Arabidopsis genome)
DNA arrays were produced by binding to nylon filters.

Next, the prepared DNA array is labeled with cDNA prepared from various tissues of Arabidopsis thaliana, variously treated tissues, mutant individuals, and the like, and subjected to hybridization to detect gene expression. Was done.

As a result, we succeeded in measuring the expression levels of a large number of genes in a wide range of chromosomal regions of Arabidopsis thaliana, and succeeded in specifying their map positions. In addition, as a result of the measurement, the expression of an unknown gene not included in the EST database was detected, and thereby, the gene region that was not predicted by the computer was successfully identified.

That is, the present invention relates to a plant DNA array used for measuring the expression level of individual gene of a plant and specifying a gene region on genomic DNA and its use, and more specifically, (1) A support to which a plurality of plant-derived genomic DNA fragments containing an average of 1 or less genes are bound, (2)
The average length of the plurality of genomic DNA fragments is 2 to 3 kbp,
(3) the support according to (1) or (2), wherein the plurality of genomic DNA fragments are continuous in a specific region on the genome;
The genome on the support according to any one of (1) to (3)
A method for detecting the expression of a plant gene, which comprises hybridizing a probe cDNA to a DNA fragment,
(5) A method for identifying a plant gene region, comprising hybridizing a probe cDNA to a genomic DNA fragment on a support according to any one of (1) to (3).

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is characterized in that a plurality of plant-derived genomic DNA fragments containing an average of 1 or less genes are used as DNA fragments to be bound to a support. When the expression of a gene is detected using a support to which a genomic DNA fragment containing a plurality of genes is bound, expression is detected as the sum of the expression levels of the plurality of genes. It is not preferable because an accurate expression level cannot be measured. In addition, when using a support to which a genomic DNA fragment containing a plurality of genes is bound as described above, it is necessary to take out each gene and analyze it again in order to accurately know the expression level of each gene. Therefore, the operation is complicated. On the other hand, a plurality of genomic DNAs containing an average of 1
The use of fragments is preferable because the expression level of each gene can be accurately measured.

Whether a genomic DNA fragment contains “an average of 1 or less genes” depends on the average chain length of a plurality of genomic DNA fragments to be bound to a support and the average length of a gene (how many bp on the genome). 1 gene is included). If the comparison shows that the former is smaller than the latter,
Genome DNA fragment "contains an average of 1 or less genes"
Is determined. In Arabidopsis thaliana, the value of the latter is considered to be about 5 kb as a result of genome analysis (Nat
ure 391: 438-439, 1998). Therefore, in Arabidopsis thaliana, the average chain length of the DNA fragment
kbp or less, preferably 2-3 kbp. In addition, plants other than Arabidopsis thaliana generally have a larger genome size than Arabidopsis thaliana, but the number of genes is considered to be about the same as that of Arabidopsis thaliana.
It is considered that one gene or less per 5 kbp of DNA.

On the other hand, if the chain length of the genomic DNA fragment to be bound to the support is too short, there is a problem in the specificity of hybridization when detecting gene expression, which is not preferable. Therefore, the length of the genomic DNA fragment to be bound to the support is usually 50 bp or more.

The genomic DN used in the present invention
It goes without saying that the plant species from which the A fragment is derived is not particularly limited due to the nature of the present invention.

In a preferred embodiment of the present invention, a plurality of continuous genomic DNA fragments in a specific region on the genome are used as the genomic DNA fragments to be bound to the support. This makes it possible to specify the map position of the gene together with the detection of the gene expression. Here, the “specific region on the genome” may be, for example, at the chromosome level, or at the genome size level (about 80 to 200 kbp) inserted into the BAC vector or the genome size level (about 80 to 200 kbp). About 80 kbp), the genome size level inserted into the TAC vector (about 60 to 80 kbp), and the genome size level inserted into the cosmid vector (about 10 to 25 kbp). Preferably, in the plurality of continuous genomic DNA fragments, the genomic DNA fragments have mutually overlapping regions.

In the present invention, a genome to be bound to a support
The DNA fragment can be prepared, for example, as follows. Long DNA fragment clones (eg, YAC, cosmid, BAC, P1 clones) that cover the genomic region whose nucleotide sequence has been determined from genomic DNA prepared from plant tissues and cells
Is prepared and fragmented using a restriction enzyme or the like to obtain a fraction (for example, about 2-3 kbp in Arabidopsis thaliana) having a size that contains an average of 1 or less genes. This is subcloned into a commercially available Escherichia coli vector, the nucleotide sequence is determined from both ends thereof, and the sequence is compared with a known genomic sequence to determine the position on the original clone (long DNA fragment clone). Then, small fragment clones that cover the original clones with minimal overlap are selected and amplified by PCR. As a result, a genomic DNA fragment covering a certain region on the genome can be obtained. Genomic DNA fragments can also be obtained by random cleavage of DNA by sonication or random amplification with random primers. The genomic DNA fragment thus obtained can be blunt-ended at its ends,
Alternatively, it can be inserted into a vector by adding a recognition sequence for a restriction enzyme to the 5 'side of the random primer. In subcloning, if a TA cloning vector is used, the PCR product can be directly cloned.

In the present invention, the support to which the genomic DNA fragment is bound is not particularly limited as long as it can fix the genomic DNA fragment.
A slide glass, silicon, or the like is used. Genome DN
Various methods can be used for binding the A fragment to the support (Protein, Nucleic Acid and Enzyme, Vol. 43, No. 13, p. 52).
-59 (1998)), for example, when a nylon filter or the like is used as a support, a genomic DNA fragment can be applied to the filter and immobilized by heat treatment or the like.

As the probe cDNA to be hybridized to the genomic DNA fragment on the support, various types can be used depending on the purpose. For example, cDNAs prepared from various tissues and cells of Arabidopsis thaliana, tissues subjected to various treatments, and mutant individuals are used. By comparing gene expression levels using probes prepared from plant tissues or cells in different states, it is possible to detect changes in the expression states of individual genes. Therefore, the detection of gene expression in the present invention can also be used as an application of the cDNA subtraction method (Biochemical Experiment Method 41, Introduction to Plant Cell Engineering, edited by Atsushi Komamine and Minato Nomura, Gakkai Shuppan Center, 1998). is there. CDNA used for probe
Can be prepared by extracting mRNA from plant tissues or cells, synthesizing cDNA using reverse transcriptase, and labeling it with, for example, an isotope or a fluorescent dye.

The expression level of the gene can be measured by hybridizing the probe to a genomic filter and counting the amount of the bound probe with a measuring instrument using the label bound to the probe as an index.
If the obtained signal intensities are compared with a signal of a housekeeping gene expressed at a constant level irrespective of cells as a control, quantitative comparison between samples is possible.

The methods necessary for general gene recombination, such as DNA cutting, ligation, transformation of E. coli, determination of the base sequence of genes, and hybridization, are described in, for example, the literature (Sa
mbrook et al., Molecular Cloning A LABORATORY MANUAL / SE
COND EDITION).

[0027]

[Example 1] Analysis using Arabidopsis thaliana DNA array cDNA to be used as a hybridization probe from Arabidopsis thaliana (ecotype; Colombia) subjected to long-day treatment, short-day treatment, low-temperature stress treatment, drying and wound stress treatment, respectively Was isolated.

In the long-day treatment, Arabidopsis thaliana
The cells were grown on vermiculite for about one month in a growth chamber set to a cycle condition of 22 ° C., light 16 hours / dark 8 hours.

In the short-day treatment, Arabidopsis thaliana
After growing on vermiculite for about one month in a growth chamber set to a cycle condition of 22 ° C., light period of 8 hours / dark period of 16 hours, the plate was allowed to stand still for one day under dark conditions.

The low-temperature stress treatment was applied by leaving plants grown under long-day conditions in a low-temperature room at 4 ° C. for 5 hours.

Drying and wound stress were applied by leaving the leaves of plants grown under long-day conditions in a desiccator at a humidity of 40% for 5 hours.

Poly (A) + RNA was prepared from the above-treated plants and untreated plants, and further prepared poly (A) + R
CDNA was synthesized based on NA and labeled with an isotope. RNA preparation and poly (A) + RNA purification, reverse transcription, cDN
Labels of A are RNeasyplant mini kit (QIAGEN),
Oligotex-dT30 super (TaKaRa), AdvantageTM RT-for-PC
R kit (Clontech) and BcaBEST labeling kit (TaKaRa) were used.

On the other hand, Arabidopsis genomic clones and Arabidopsis thaliana DNA arrays were prepared. Specifically, Arabidopsis thaliana DNA array refers to Arabidopsis thaliana
Chromosome Genome Project (Kotani et al., DNA Re
s. 4, 371-378, 1997), about 2000 clones (about 4.7 Mbp) derived from P1 and TAC clones (Sato e
tal., DNA Res. 4, 215-230, 1997;
And a genomic DNA fragment having an average of 2-3 Kbp was cut out from the TAC library (FIG. 1), shotgun cloned, and the genomic DNA region inserted into the P1 or TAC library was covered with minimal overlap. Phage) were amplified by PCR using M13 and RV primers and blotted onto nylon membrane filters.

On the DNA array, PCR fragments of the above-mentioned phage of about 2000 clones are blotted two by two on adjacent grids. This region is about 1/6 of the chromosome 5 of Arabidopsis thaliana and about 1/30 of the whole genome of Arabidopsis thaliana, and it is estimated that about 2000 genes are encoded.

Hybridization and washing are performed using conventional protocols (Sambrook et al., Molecular Cloning ALA).
BORATORY MANUAL / SECOND EDITION). The final wash of the filter is 0.1% SDS
By shaking at 65 ° C. for 15 minutes in a 0.1 × SSPE solution containing The data was captured using a bioimage analyzer BAS2000 (Fujix). To measure the radioactivity of the data acquired by the image analyzer,
The box tool of BAStation software (FUJIFILM) was used.

For extracting and analyzing sequence data, the Kazusa DNA Research Institute (http://www.kazusa.or.jp/arabi)
/) And National Center for Biotechnology Information
(NCBI) (http://www.ncbi.nlm.nih.gov/) World Wide
I used information posted on the website.

As a result of comparing the signal patterns on the respective filters under the respective conditions, changes in the gene expression under various environments could be confirmed. FIG. 2 shows an image of the filter in which the signal was detected. Genes whose expression changed depending on the presence or absence of each treatment condition (main ones)
And their signal intensities are summarized in Tables 1 and 2. “Grid position” in the table indicates the position of each clone on the DNA array. "Parent clone name" indicates the name of the original P1 or TAC clone used to prepare the DNA array. “Clone position” indicates the position (in “bp”) within the parent clone of the clone used to prepare the DNA array. The "annotation number" indicates the number assigned by the Kazusa DN Laboratories to the result of searching the database using the base sequence contained in each clone. “Annotation position” indicates the position (in “bp”) of the annotated gene in the P1 or TAC clone.

[0038]

[Table 1]

[0039]

[Table 2]

As shown in the table, clones always expressed strongly, clones induced under long-day conditions, clones induced under short-day conditions, clones induced under short-day and drought conditions, and clones induced at low temperatures , Several clones induced by drying and low temperature and drying were detected.

Further, as shown as "no annotation", the presence of the gene was detected at a position on the genome where the presence of the gene has not been confirmed so far.

In this example, higher eukaryotes were used on a genome scale (approximately 1/10 of the entire genome sequence of Arabidopsis thaliana).
This is the first example of analyzing the expression of a gene.

[0043]

As described above, a method for detecting gene expression using a DNA array in which a cDNA is bound to a support has been used until now. However, even in recent EST databases, cDNA sequences of only a part of all genes have been reported. No (about half of all genes in Arabidopsis). The EST database accumulates genes that are frequently expressed in plants, for example, tissue-specific genes, environmentally responsive genes,
Little is known about things that are specific to the stage of growth.

The method using the genomic DNA fragment of the present invention can detect the expression of all genes including sequences not in the EST database. further,
You can know the promoter sequence and map position of all genes. In higher plants, it is difficult to accurately predict the protein coding region due to the complexity of the gene structure.However, the method of the present invention can be used to analyze the structure of genes as effectively as the ORF prediction by computer. It can be.

Up to now, gene tagging projects using T-DNA or transposons have been conducted worldwide, but according to the method of the present invention, the expression of genes in specific genomic regions is intensively investigated. Therefore, it can be used in combination with transposon tagging or T-DNA tagging. In addition, analysis using a number of mutants,
It is very important for analyzing the biological function of the whole genome, and will be very useful for understanding various signaling pathways and networks.

[Brief description of the drawings]

FIG. 1 shows genomic DNA aligned with a P1 or TAC clone identified on chromosome 5 of Arabidopsis thaliana.
The relationship with the clone is shown. Clones starting with M indicate P1 clones, and clones starting with K indicate TAC clones. In this figure, the K18P6 clone (74589 bp) is used as an example
It shows a state where the cells were sorted as A clones.

[Fig. 2] Fig. 2 shows Arabidopsis thaliana mRNA grown on a support by fixing Arabidopsis genomic DNA to a support.
Shows the results of hybridization performed using the cDNA probe prepared from Example 1. The left shows the results of the entire support, and the right shows a part of the support enlarged to show a grid for measuring the signal intensity of each clone.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shigeru Hanano 2-1-6 Sengen, Tsukuba, Ibaraki Pref. Tsukuba Research Support Center D-21 Inside the Plant Biotechnology Research Institute, Mitsui Co., Ltd. (72) Inventor Daisuke Shibata Ibaraki 2-1-6 Sengen, Tsukuba Tsukuba Research Support Center D-21 Mitsui Okayama Plant Biotechnology Research Institute, Inc. (72) Inventor Kiichi Kaneko 1532-3 Yanauchino, Kisarazu-shi, Chiba Kazusa DNA Research Institute ( 72) Inventor Tetsuyuki Tabata 1532-3 Yanauchi, Kisarazu-shi, Chiba F Kazusa DeNA Research Laboratory F-term (reference) 4B024 AA20 BA80 CA04 HA14 HA19 4B029 AA07 BB12 CC03 FA15 4B063 QA13 QQ04 QR32 QR42 QS28 QS34 QS39 Q07

Claims (5)

[Claims] 1. A support to which a plurality of plant-derived genomic DNA fragments containing an average of 1 or less genes are bound. 2. The average length of a plurality of genomic DNA fragments is 2 to 3 k.
The support according to claim 1, which is bp. 3. The support according to claim 1, wherein the plurality of genomic DNA fragments are continuous in a specific region on the genome. 4. A method for detecting the expression of a plant gene, which comprises hybridizing a probe cDNA to a genomic DNA fragment on a support according to any one of claims 1 to 3. 5. A method for identifying a gene region of a plant, comprising hybridizing a probe cDNA to a genomic DNA fragment on a support according to any one of claims 1 to 3.