WO2008015975A1 - Method for amplification of dna fragment - Google Patents

Abstract

Disclosed is a method for the amplification of a DNA fragment, which is intended to be used in a transposon display method for a higher plant, which can amplify only a labeled PCR product selectively, and which shows little variations in experimental results among individuals. The method for the amplification of a DNA fragment comprises the following steps in this order: a DNA extraction step for extracting genomic DNA from a cell of a higher plant; an adapter addition step for partially digesting the genomic DNA with a restriction enzyme to produce a DNA fragment and then adding an adapter to the DNA fragment; a first PCR step for amplifying the DNA fragment by an one-sided PCR procedure which uses a DNA fragment ligated to the adapter as a template and also uses only a primer specific to the transposon; and a second PCR step for amplifying the DNA fragment by a two-sided PCR procedure which uses a DNA fragment amplified in the first PCR step as a template and also uses both a labeled primer specific to the transposon and a primer complementary to the adapter.

Description

 Specification

 Amplification method of DNA fragment

 Technical field

 [0001] The present invention relates to a method for amplifying a DNA fragment, and more particularly to a method for amplifying a DNA fragment used in a transposon display method for rapidly and objectively associating a trait and a gene associated therewith.

 Background art

 [0002] With the progress of molecular biology, in addition to animals such as humans and mice, the base sequences of the whole genome of higher plants have been analyzed. For example, in the case of rice, the decoding of the draft sequence was completed in 2002 by the International Rice Genome Sequencing Project (IRGSP).

 [0003] However, in order to create plants with tolerance to environmental stresses such as drought and cold and plants with high yields by genetic recombination, genomic DNA is not limited to just determining the entire base sequence of genomic DNA. It is also necessary to analyze the effects of each gene described in the above on the traits of plants, that is, the functions of each gene. One method of analyzing the function of the gene is the transposon display method described below.

 [0004] Here, the transposon is also called a transposable genetic element or a mobile genetic element (mobile genetic element, movable genetic element). It is a DNA fragment that has the property of randomly translocating to other sites, and is known to cause gene disruption by inserting into the translocation target gene. As for transposons that function in rice, Tosl7, karma (see Patent Literature 1), mPing (see Patent Literature 2 and Non-Patent Literature 1) have already been reported.

[0005] In addition, the transposon display method is a method for examining the function of a gene by using the above-mentioned properties of the transposon, and a gene related to a certain trait is determined based on the gene disruption caused by the transposon and a change in the trait associated therewith. It is a method to identify, specifically (Refer to Patent Document 2).

[0006] First, genomic DNA is extracted from a plurality of individuals of the same strain whose traits have changed due to transposon transposition, and a DNA fragment containing a part of the transposon is amplified. Next, the amplified DNA fragments are electrophoresed to obtain waveform data for each individual, and the differences in the waveform data for each individual, in short, the presence or absence of peaks are analyzed. Finally, a specific trait is associated with a specific gene by comparing the difference in the presence or absence of the peak of each individual with the difference in the trait.

 [0007] Here, amplification of a DNA fragment containing a part of the transposon consists of (l) DNA extraction step, (2) adapter addition step, (3) first PCR step (hereinafter referred to as first PCR step) (4) It can be roughly divided into each step of the second PCR step (hereinafter referred to as the second PCR step). Details of these will be described below with reference to FIG. 8, which is a schematic diagram.

 [0008] (l) DNA extraction process

 This is a process of extracting genomic DNA from individuals of the same strain with different traits by a simple method (see Non-Patent Document 2). Figure 8 (a) schematically shows genomic DNA extracted from an individual. Show. In the figure, T means a transposon, and IR means an inverted repeat.

 [0009] (2) Adapter addition process

 The extracted genomic DNA is partially digested with a restriction enzyme, and oligomer DNA (adapter) that can bind to the recognition site by this restriction enzyme is ligated to the partially digested DNA. Fig. 8 (b) shows the state before ligation, and Fig. 8 (c) shows the state after ligation. As shown in this figure, the partial digest of genomic DNA is a mixture of partial digest 1 that contains a part of transposon T and partial digest 2 that does not contain transposon T, and the adapter ligates to either of them. Ligation products are also a mixture of 3 with and without 4!

 [0010] (3) First PCR step

As shown in Fig. 8 (c), the first PCR step is performed on both sides using two primers: primer 1 specific for a part of the base sequence of transposon T and primer 2 complementary to the adapter. A PCR reaction step, where a PCR product as shown in Fig. 8 (d) It is a process to obtain. Here, primer product 1 and primer 2 are annealed to ligation product 3 to produce PCR product 5. In contrast, primer 1 cannot be annealed to ligation product 4, so primer 2 is annealed at both ends to produce PCR product 6. The DNA fragment 4 and the DNA fragment 6 have the same force. For convenience in illustration, different numbers are assigned.

 [0011] (4) Second time? About 0¾

 As shown in Fig. 8 (d), the second PCR step consists of PCR products 5 and 6 in a saddle shape, a labeled primer 3 specific for a part of transposon T and a primer complementary to the adapter 2 Is a step of performing a PCR reaction using The PCR product 5 containing a part of the transposon is annealed with labeled primer 3 and primer 2 to generate labeled PCR product 7. In contrast, primer 3 cannot be annealed to PCR product 6, so primer 2 is annealed at both ends to produce PCR product 8.

 [0012] Here, as shown in FIG. 8 (e), the amplified DNA fragments are labeled PCR product 7 and unlabeled PCR product.

 8 mixture. In addition, both ligation products 3 and 4 must have adapters at both ends, and primer 2 that is complementary to this adapter is used in both the first PCR step and the second PCR reaction. Therefore, the ratio of labeled PCR product 7 to unlabeled PCR product 8 in the PCR product is almost the same as the ratio of DNA fragment 1 containing a part of transposon T and DNA fragment 2 not containing this.

 [0013] As described above, the DNA fragment thus obtained is used for electrophoresis, analysis of waveform data, and correlation between a trait and a gene. Conventionally, analysis of waveform data obtained by electrophoresis has been performed manually based on the experience and intuition of the experimenter. Therefore, it took a lot of time and labor, and the analysis results were not objective because there were individual differences. In order to easily and objectively compare the waveform data obtained by different electrophoresis, the inventors have developed the “Rice Transposon Analysis Support System” through the “Wakayama Prefectural Community Research Project”. Create and use! /, Ru (see Patent Document 3).

[0014] In this system, the size marker and the PCR product are mixed and electrophoresed to convert the migration time constituting the waveform data into the size (number of bases) of the amplified product, and the fluorescence intensity of the waveform data. Can be quantified. Therefore, waveform data based on different electrophoresis It is possible to easily and objectively compare the waveform data by correcting the force as applied by the same electrophoresis.

[0015] As described above, the transposon display method is a useful method for associating a certain trait with a gene associated therewith, and its usefulness has been further improved by using the above-described system in particular.

 [0016] However, since the intensity of each peak constituting the waveform data is weak and the attenuation is intense, the portion of the obtained waveform data longer than 300 bp is subjected to the peak and force S due to electrical noise and electrophoresis. There was a problem that they could not be distinguished and could not be used for analysis. In addition, since the intensity of each peak is weak and the detection is unstable, there is a problem that the experimenter makes a difference in the results, and a considerable amount of skill is required before stable results can be obtained. There was also.

 Patent Document 1: Japanese Patent Application No. 2002-369691

 Patent Document 2: U.S. Pat.No. 6,420,117

 Patent Document 3: Japanese Patent Application No. 2005-237438

 Non-Patent Document 1: Nakazaki et al. (Nakazaki, T.), Movement of transposon in the rice genome (Nature;, 421 (2003), p.170-172) Non-Patent Document 2: Junya Yoshida, Kuniko Shio, "Simple rice extraction method from rice grain and discrimination of sake rice varieties by RAPD", [online], Chinese Agricultural Experiment Station, 1998 research result information, [May 10, 2006 Search], Internet <URL: http: 〃 www.affrc.go.jp/seika/data#cgk/hlO / seibuti cgkgSOCM.html>

 Disclosure of the invention

 [0017] Therefore, the present invention is a method for amplifying a DNA fragment used in the transposon display method, wherein the waveform data obtained by electrophoresis of the DNA fragment has a waveform pattern that has a strong peak intensity and is difficult to attenuate. It is an object of the present invention to provide a method for amplifying a DNA fragment that can be used for analysis of a longer part and that has little individual difference in experimental results.

[0018] The inventor has examined the reason why the intensity of the peak constituting the waveform data is weak and the attenuation is intense. As a result, the inventor amplified the same amount of unlabeled PCR product 8 as well as labeled PCR product 7 in the first PCR step and second PCR step. This is because primers and nucleic acid monomers (d) that are used at the same time are wasted for amplification of the unlabeled PCR product 8, and the amplification of the labeled PCR product 7 is not performed in proportion to the number of cycles. I thought.

 [0019] Therefore, the inventor solves the above problem by amplifying only the labeled PCR product, that is, by improving the ratio of the labeled PCR product 7 in the PCR product (hereinafter, S / N ratio). Was examined. As a result, when amplifying the DNA used for transposon display, instead of performing the normal double-sided PCR reaction using two primers twice, the first PCR reaction should be performed using only primers specific to the base sequence of the transposon. It was noticed that the S / N ratio was greatly improved by replacing the one-side PCR method used.

 [0020] That is, the method for amplifying a DNA fragment of the present invention comprises (1) a DNA extraction step for extracting genomic DNA from cells of a higher plant, and (2) partial digestion of genomic DNA with a restriction enzyme to obtain a DNA fragment. An adapter addition step of ligating an adapter capable of binding to the restriction enzyme recognition site to the DNA fragment, and (3) using the primer specific to the transposon by ligating the adapter into a vertical DNA fragment. A first PCR step for amplifying a DNA fragment by a one-side PCR method, and (4) a DNA fragment amplified by the first PCR step in a saddle shape, and a labeled primer specific for the transposon and the This includes a second PCR step in this order that amplifies the DNA fragment by a two-sided PCR method using primers complementary to the adapter.

 [0021] The power of the present invention, the amplification method of DNA fragments, improves the S / N ratio of PCR products used in the transposon display method, and the intensity of peaks constituting the waveform data is strong, making it difficult to attenuate. It was. As a result, the longer part of the waveform pattern can be used for analysis, and individual differences in experimental results have been reduced. As a result, traits and related genes can be related more easily and objectively, and it has become possible to contribute to the development of better higher plants.

 Brief Description of Drawings

 FIG. 1 is a schematic view showing an outline of a DNA fragment amplification method according to the present invention.

 FIG. 2 is a diagram comparing changes in waveform data due to differences in the configuration of the PCR method.

FIG. 3 is a diagram comparing heading time and culm length of MLR4 and MLR8 lines. FIG. 4 is a graph showing the distribution of panicle length of MLR4 and MLR8 lines.

 FIG. 5 is a graph showing the distribution of the number of spikes in MLR4 and MLR8 lines.

 FIG. 6 is an example of a diagram comparing the differences in peaks between individuals belonging to the same strain.

 FIG. 7 shows a base sequence near the mPing insertion position.

 FIG. 8 is a schematic view showing an outline of a conventional DNA fragment amplification method.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0023] The method for amplifying a DNA fragment, which is particularly suitable for the present invention, is the same as the conventional method for amplifying a DNA fragment. (4) The second PCR steps are included in this order. Therefore, the details of each process will be described below with reference to FIG.

 [0024] (l) DNA extraction process

 This is a process of extracting genomic DNA with a small number of cleavage sites, that is, sufficiently long, from each individual of a higher plant of the same line with different traits. As the extraction method, a known method without particular limitation can be used as long as the above conditions are satisfied. Specifically, the CTAB method and the simple method (see Non-Patent Document 2) can be cited. The extracted genomic DNA is shown schematically in Fig. 1 (a).

 [0025] Here, the higher plant can be used without particular limitation as long as the mutant has been obtained to such an extent that the transposon display method can be conventionally applied. Specifically, from an academic point of view, Arabidopsis and morning glory that have already undergone genetic analysis are preferable. Also, from an economic point of view, there is already a huge amount of base sequence information obtained by the genome project, a small genome size, a transposon with few repetitive sequences, among cereals preferred. Rice is preferred because it has the advantages of applying the display method.

[0026] The base sequence of the transposon is known, and any transposon can be used in principle as long as it transposes by natural cultivation or tissue culture to destroy the gene. However, transposons with tens to thousands of copies per genome are preferred because of the maximization of the S / N ratio improvement that one-sided PCR brings. Use is the most efficient! / Specific examples include Tosl7, nDart, karma, and mPing. Among them, mPin has a relatively large number of copies in the genome (about 100 to several hundred copies), and exhibits moderate translocation activity (causes several to tens of copies per generation). Use of g is preferred.

 [0028] (2) Addition of adapter

 In this step, genomic DNA is partially digested with a restriction enzyme to form a DNA fragment, and an adapter capable of binding to the restriction enzyme recognition site is ligated to the DNA fragment.

 [0029] First, genomic DNA is partially digested into DNA fragments using restriction enzymes. Here, as a restriction enzyme, a commercially available restriction enzyme that has a recognition site in a transposon can be used without particular limitation. It is preferable to use a 4-base recognition enzyme (such as Csp6I) that produces a small fragment of In addition, the restriction enzyme reaction may be carried out according to the reaction conditions required by the restriction enzyme to be used, such as buffer solution and temperature.

 [0030] Next, the partially digested DNA fragment and the adapter are mixed and ligated to the ends of the DNA fragment. The adapter is a double-stranded DNA of about 10 to 20 bases having a sequence corresponding to the recognition site of the restriction enzyme, which is synthesized by a commercially available DNA synthesizer or the like. The ligation may be performed in accordance with the reaction conditions required by the ligase used. Incidentally, partial digestion and ligation may be performed separately.

 [0031] Fig. 1 (b) shows a state before ligation, and Fig. 1 (c) shows a state after ligation. Here, the partial digest of genomic DNA is a mixture of one containing part of transposon T and one not containing 2 as shown in FIG. 1 (b). The adapter will ligate to either. Therefore, the ligation product is a mixture of ligation product 3 containing a part of transposon T and ligation product 4 not including this as shown in FIG. 1 (c).

 [0032] (3) First PCR step

This is a one-sided PCR reaction using ligation products 3 and 4 in a bowl shape and using only a primer that specifically anneals to transposon T. [0033] As shown in Fig. 1 (c), the ligation product 3 has a base sequence that specifically rings on transposon T. Therefore, in the first PCR step, the ligation product 3 is amplified by the number of cycles of the PCR reaction by PCR reaction starting from the ligation product 3 in the vertical shape and using the primer 1 as a starting point, and the PCR product 5 is generated. On the other hand, the ligation product 4 does not have a base sequence that specifically anneals to transposon T, and therefore the primer cannot be annealed in the first PCR step, and the DNA fragment 4 is not amplified. That is, the ratio of the PCR product 5 containing transposon T is greatly improved by the first PCR step. DNA fragment 4 and DNA fragment 6 are the same force. For convenience in illustration, different numbers are assigned.

 [0034] Further, the primer length, base sequence, PCR reaction conditions, specifically the temperature and reaction time of the thermal denaturation step, annealing step, extension step, the number of reaction cycles, etc. should be covered. The type can be adjusted while looking at the experimental results according to the primer. However, the proportion of PCR product 5 in the PCR product (S / N ratio) increases in proportion to the number of reaction cycles. The number of cycles for one-sided PCR can be determined as needed from the 20 cycles that provide a certain S / N ratio improvement effect to the 40 cycles that are the upper limit of DNA synthase normally used. I'll do it.

 [0035] (4) Second PCR step

 As shown in Fig. 1 (d), PCR products 5 and 6 are made into a saddle shape, and labeled primer 3 that is the same as or inside the primer used in the first PCR step (below the transposon) and In this step, a two-sided PCR reaction is performed using primer 2 complementary to the adapter.

 [0036] Since both the labeled primer 3 and the primer 2 are used in the second PCR step, the labeled primer 3 and the primer 2 are annealed to the PCR product 5 as shown in Fig. 1 (d). A PCR reaction is performed to produce labeled PCR product 7. In contrast, labeled primer 3 cannot be annealed to DNA fragment 6 and primer 2 is annealed at both ends, resulting in non-PCR product 8. Thus, in the second PCR step, both PCR products 5 and 6 are amplified. Therefore, between the first PCR step and the second PCR step, the ratio of the DNA fragment containing a part of the transposon and the DNA fragment not containing it is almost the same.

[0037] The labeled primer 3 is a primer labeled with a labeling substance such as a fluorescent substance. The labeling substance and the labeling method can be used without particular limitation as long as they are known. The primer length, base sequence, and various PCR reaction conditions can be adjusted while looking at the experimental results as in the first PCR step.

 [0038] As described above, since the DNA fragment amplification method of the present invention amplifies only a DNA fragment containing a part of transposon T in the first PCR step, the ratio of labeled PCR product 7 in the PCR product is as follows. The (S / N ratio) is improved, the intensity of the peaks that make up the waveform data is strong, making it difficult to attenuate, and the longer part of the waveform pattern can be used for analysis. Therefore, the association between the presence / absence of a DNA fragment and the difference in trait, that is, the association between a certain trait and its related gene can be performed more easily and objectively than in the conventional method.

 It should be noted that the present invention is not limited to the above-described embodiments, and various modifications can be made within the technical scope described in the claims. For example, other steps may be added as necessary. Specifically, before a PCR reaction using a labeled primer, you can add a one-sided PCR reaction or a PCR reaction set consisting of a one-sided PCR reaction and a two-sided PCR reaction! /,. In this way, the S / N ratio can be further improved by repeating the one-side PCR reaction a plurality of times.

 [0040] Also, during each of the above steps, the enzyme such as restriction enzyme, ligase, and DNA polymerase coexisting with the DNA fragment or PCR product is inactivated by removing, for example, phenol 'chloroform extraction. And a step of purifying these DNAs by ethanol precipitation or the like.

 [0041] Hereinafter, the present invention will be described in more detail based on examples. The scope of claims of the present invention is not limited in any way.

 Example 1

 [0042] In the transposon display method, the effect of improving the amplification efficiency of a DNA fragment containing a part of transposon T by one-side PCR reaction was investigated. Specifically, (a) When only the first PCR step is a one-sided PCR reaction, (b) When only the second PCR step is a one-sided PCR reaction, (c) When one-sided PCR reaction is performed only once (D) The comparison with the result by the conventional method became fi.

[0043] (1) Plant material and DNA extraction DNA extracted from the leaves of five rice fine lines M294 planted in the field in 2004 by a simple method (see Non-Patent Document 2) was used as a sample.

 [0044] (2) Adapter addition process

 First, an adapter was prepared by associating a synthetic oligo DNA, Csp6I_Al and Csp6I_A2. Table 1 shows the base sequences of the oligo DNA of adapters such as Csp6I_Al and Csp6 to A2, and primers described later. Next, 10 ng of genomic DNA of each individual was placed in an Eppendorf tube, partially digested with the restriction enzyme Csp6I, and an adapter addition reaction was performed using T4 ligase (Ligation high, TOYO BO). After completion of the addition reaction, Csp6I and T4 ligase were inactivated, and the reaction product was purified and concentrated by ethanol precipitation and dissolved in 40 1 TE (pH 8.0) buffer.

 [0045] [Table 1] Oligo DNA nucleotide sequence

 Csp6 I -A 1 TAGTCAGGACTGAT (Major IJ number 1)

 Csp6 I -A2 GACGATCAGTCCTGAC (SEQ ID NO: 2)

 Srt-Pl TAGTCTTGGAAACGTCAA (SEQ ID NO: 3)

 Srt-P3-D4 TCATTTGAGAGAAGATGGTA (SEQ ID NO: 4)

 Csp6 I -AP GACGATCAGTCCTGACTAC (SEQ ID NO: 5)

[0046] (3) First PCR step

 A part of the reaction product (1 H 1, DNA 250 pg) was collected for each individual, and the first PCR step was performed with the primer combinations shown in Table 2 (concentration: 25 0101/1). In Table 2, Srt-PI is an mPing-specific primer, Srt-P3_D4 is labeled with a fluorescent dye (D4, BEC MAN COU LTER, USA), and is a primer specific for mPing. Is a primer complementary to the adapter. Table 3 shows the composition of the solution when the PCR reaction was performed, and Table 4 shows the PCR cycle of the PCR reaction.

[0047] [Table 2] Test area used

 Primer a b c d

 Srt-Pl 0.06 a 1 0.06 / i 1 0.06 a 1 First PCR Srt- P3-D4

 Csp6 I-AP 0.06μ 1 0.06μ 1

 Srt- P3-D4 0.06 a 1 0.06μ 1 0.06 a 1 Second PCR

 Csp6 I -AP 0.06μ 1 0.06μ 1

 [0048] [Table 3] First PCR Second PCR

 10 X PCR buffer 1.00μ 1 1.00 μ 1

 2 mM dNTPs 1.00 μ 1 1.00 μ 1

2omM MgS0 ₄ 0.40μ 1 0.40 1 Primer (25 pmol//i 1) 0.06 /; 1 or 0.12 / i 1 0.06 1 or 0. \ 2, ι \

KOD-Plus (1U / _W 1) 0.20 oμ 1 0.20 _W 1

 H O 6.84 μ 1 or 6.78 // 1 6.84 a 1 or 6.78 / x 1 Vertical UNA 0.50μ 1 (250pg) 0.50μ 1

 Total amount

 [0049] [Table 4] o

PCR cycle

 94 ° C 2 minutes o

94 ° C 15 seconds

 58 ° C 1 minute> 30 cyclore

 68 ° C 1 minute

 Re

 68 ° C 1 minute

 4 ° C storage

 [0050] (4) Second PCR step

 The PCR product of the first PCR step was diluted to 2.5 culture medium with ΤΕ (ρΗ8 · 0) buffer, and this was used as a cage to perform the second PCR step using the primers shown in Table 2. The PCR product was purified by ethanol precipitation. The solution composition and reaction cycle when performing the PCR reaction were the same as in the first PCR step.

[0051] (5) Electrophoresis

The PCR product thus obtained was dissolved in a size marker (CEQ DNA Size Standard it-600 (BEC MAN COULTER, USA), labeled with Dl) 0.375 1 Solution (CEQ Sample Loading Solution (BEC MAN COULTER, USA) 30 1) and then applied to the sample plate of the first type DNA sequencer (CEQ 2000 Fragment Analysis System, BEC MAN COULTER, USA) The waveform data obtained by electrophoresis was converted to text data format using the computer software that came with the DNA sequencer, and the waveform data here refers to “fluorescence” at a specific “electrophoresis time”. Strength ".

 [0052] (6) Waveform data analysis

 This text data is input to the “Rice Transposon Analysis Support System”, and the waveform data obtained by (a) to (c) and the waveform obtained by the conventional method by the “variation comparison” of the system. Comparison with the data was performed. The results are shown in Fig. 2 (a) to (c). In the figure, solid lines indicate waveform data from (a) to (c), and dotted lines indicate (d) waveform data by the conventional method.

 [0053] (7) Experimental results

 (a) When only the first PCR step is a one-sided PCR method, the maximum value of the fluorescence intensity is about 20,000, as shown in Fig. 2 (a), over a wide range from 80 bp to 600 bp. I was able to observe the peak. In addition, the fluorescence intensity decayed more slowly than the PCR product obtained by the conventional method, and a larger amplified product tended to be obtained. Unlike the conventional method, this method made it easy to learn experimental techniques with little difference in the success rate by the experimenter. Specifically, even beginners completed the operation within a few days, and the results were stable even after 5 iterations. In the case of the conventional method, eight people worked on it, and only one person obtained a result above a certain level. On the other hand, with this method, 7 people worked and 7 people were able to obtain satisfactory results.

[0054] (b) When only the second PCR step is a one-sided PCR method, the fluorescence intensity decreases over the entire range as shown in Fig. 2 (b), particularly at 200 bp or more. did. This is thought to be due to the significant consumption of primers and monomers during the second PCR step as a result of the amplification of many DNA fragments that did not contain transposons during the first PCR step! available. In the first PCR step, the ratio of target fragment 3 and unwanted fragment 4 in Fig. 1 (c) does not change, so target fragment 3 is used for exponential amplification of PCR products in the second PCR step. It is thought that there is no priority. [0055] (c) When the one-side PCR reaction was performed only once, as shown in FIG. 2 (c), the apparent attenuation was smaller than that of the PCR product obtained by the conventional method. One-sided PCR reactions are shown to be effective in amplifying large fragments. However, since this method does not use a single mPing-specific primer, it may be possible to narrow down the target sequence. In fact, this is why the number of amplified fragments in (c) is larger than the number of amplified fragments in (a).

 [0056] From the above results, it was found that (a) when only the first PCR step is a one-sided PCR, a PCR product containing a part of the transposon Τ can be obtained at a higher rate than the conventional method. . Example 2

 [0057] By the transposon display method using the DNA amplification method of the present invention, an attempt was made to identify a DNA fragment containing a gene related to economic properties. Specifically, we attempted to identify DNA fragments associated with (5a) leaf presence, (5b) number of spikes, (5c) spike length, and (5d) pod length.

 [0058] (1) Cultivation test and DNA extraction

 Individuals with non-fine-grained gold-hull traits were obtained from ML fine-grained lines grown in the field in 2004. Such individuals do not occur in the breeding of fine-grained and normal-grained individuals! /, Thus it can be deduced that they are mutants induced by mPing metastasis.

 [0059] Next-generation individual lines (MLR lines, all 9 lines) of these individuals were transplanted to the field. The heading date was investigated for all plants planted, and the culm length, head length, number of ears, etc. were investigated approximately one week after heading.

 [0060] After all the traits were examined, the leaf immediately below the leaf was manually collected for each individual, and genomic DNA was extracted by the simple method described in Example 1 for each individual.

 [0061] (2) Transposon display and waveform data analysis

 Using the obtained genomic DNA of each individual, the transposon display method and waveform data analysis were performed in the same manner as in Example 1. Only the PCR cycle of the PCR reaction was changed to that shown in Table 5. We then examined the association between the found mutation and the mPing insertion polymorphism and detected mPing copies associated with the mutated trait.

[0062] [Table 5] PCR cycle

 94 ° C 2 minutes

94 ° C for 15 seconds,

 58 ° C 30 seconds 30 cycles

 68 ° C 1 minute

68 ° C 1 minute

 4 ° C storage

 [0063] (3) Results of cultivation test

 First, when the traits of all individuals were investigated in all 9 MLR lines, mutations were observed in traits such as heading length and cocoon length in all lines. For example, FIG. 3 to FIG. 5 show variations in heading date, culm length, head length, and number of heads of MLR4 and MLR8 belonging to the MLR line. Figure 3 shows a comparison of the heading date and culm length of these two lines. As shown in this figure, mutations of 30 days or more were observed on the heading date and 30 cm or more on the culm length. Figures 4 and 5 show the distribution of spike length and spike number, respectively.As shown in these figures, mutations of 8 to 22 cm were observed in the spike length, 1 to 16 mutations were observed. From the above results, it was clarified that genetic variation exists in these two strains. Peaks indicating amplification of PCR products were confirmed in all individuals, and differences in peaks generated from individuals belonging to the same strain were also confirmed. For example, in the case of MLR8, all 79 individuals obtained, in the case of MLR4, the gold-hull trait, which is an indicator of illegal crossing, is missing! / ヽ 8 individuals and 4 individuals for whom data were not obtained. By using the excluding 70 individuals, it was possible to confirm the peak indicating amplification of the PCR product in the range from 80 bp to 600 bp in any strain, and to confirm the difference in peak between each individual.

FIG. 6 is an example of a diagram comparing the differences in peaks between individuals belonging to the same strain. Specifically, the data (partial) of MLR4-2 individuals (indicated by dotted lines) that were the reference individuals in the MLR4 strain and MLR4-79 individuals (hereinafter referred to as comparative individuals, indicated by solid lines) different from that were compared. It is a result. In this figure, the vertical axis indicates the fluorescence intensity (relative value) proportional to the amount of DNA fragment, and the horizontal axis indicates the size (bp) of the DNA fragment. In this figure, the reference individual MLR4-2 has a peak and the comparative individual MLR4-79 has no peak. The location is indicated by (-), and the location where the reference individual has no peak and the comparative individual has a peak is indicated by (+).

 [0066] In other words, (1) indicates that mPing was inserted in the reference individual and the PCR product was amplified, or mPing was excised in the comparative individual and the PCR product was not amplified. On the other hand, (+) indicates that mPing was excised in the reference individual and PCR product was not amplified, or that mPing was inserted in the comparative individual and PCR product was amplified. That is, (1) and (+) show the insertion polymorphism of mPing between the reference individual and the comparative individual.

 [0067] Thus, as a result of transposon display, it was possible to summarize the increase / decrease in the peak between the reference individual and the comparison individual, that is, the mPing insertion polymorphism. Table 6 shows a part of the summary of ML R4. MLR4-2 was used as the reference individual.

 [0068] [Table 6]

 [0069] (5) Integration of trait data and insertion polymorphism

Comparison of trait data obtained from field surveys and mPing insertion polymorphism We investigated the insertion polymorphism of mPing. As a result, (5a) presence or absence of leaf hair, (5b) number of spikes, (5c) spike length, and (5d) spike length, mPing, which has a significant correlation at 5% level with the presence or absence of mutation Found a type.

 [0070] (5a) Presence or absence of leaf hair

DNA fragments related to the presence or absence of leaf hair were searched based on whether or not the presence or absence of the DNA fragment had a significant difference in the presence or absence of leaf hair. Specifically, a null hypothesis is established that there is no association between the presence or absence of leaf hair and a specific DNA fragment, the significance probability p is calculated, and the significance probability is 5% or less. determining the presence or absence of related by the X ² test to determine (relationship. between the two) to reject the null hypothesis in. Table 7 shows the results that were found to be relevant. The p value in this table means the significance probability p.

 [0071] [Table 7]

DNA fragment With or without leaf hair

 Strain Number P Number

 (bp) Yes No

 MLR4 Yes 50 38 12

 120 0. 0000

 20 19

 Yes 62 31 31

 103 0. 0117

 Yes 61 37 24

 227 0. 0302

 MLR8 Yes 65 56

 120 0. 0000

 13 13

 Yes 64 49 15

 142 0. 0218

 4ε 15

 Yes 54 42 11

 238 0. 0348

 4ε 24 13 12

 [0072] As can be seen from this table, three DNA fragment candidate forces S, MLR4 and 3 MLR8 were detected, which are related to the presence or absence of leaf hair. Of these, the 120 bp fragment was detectable in both strains, and the p-value was as low as 0%. From this, it can be considered that hairlessness was expressed by excising mPing from this locus.

 [0073] (5b) Number of ears

Specific DNA fragments associated with the number of spikes were sought by t-test based on whether the presence or absence of the DNA fragment significantly affected the number of spikes. Specifically, first, the number of spikes and a specific DN A null hypothesis that there is no association with the A fragment was calculated, and the average number of ears and the unbiased variance were calculated for the pair with and without the specific DNA fragment, respectively, and the index t was calculated based on these values. Calculated. Next, the significance probability p was obtained from the index t and the degree of freedom. When the significance probability was 5% or less, the null hypothesis was rejected (there is a relationship between the two). As a result, as shown in Table 8, four DNA fragments and three MLR8 were detected as candidates for DNA fragments related to the presence or absence of the number of spikes. The p value in this table means the significance probability p.

 [Table 8]

UNA fragments

 Strain Number of individuals 1

 (bp) Mean Unbiased variance

 MLR4 Yes 8. 25 17. 93

 297 0. 0087

 62 5. 68 5. 1 1

 Yes 10 4. 30 5. 57

 604 0. 0300

 None 60 6. 25 6. 80

 Yes 10 7. 60 14. 04

 98 0. 0347

 None 60 5. 70 5. 54

 Yes 64 6. 17 6. 94

 346 0. 0377

 None 6 3. 38 3. 37

 MI.R8 Yes 71 6. 90 4. 26

 550

 None 8 8. 88 4. 41

 Yes 57 7. 40 4. 60

 155 0. 0425

 None 11 6. 32 3. 85

 Yes 21 7. 90 3. 19

 195 0. 0439

 No 58 6. 81 4. 82

 [0075] (5c) Hocho

 As for the panicle length, the relevant DNA fragment was searched for by t-test as in (5b). As a result, as shown in Table 9, 6 DNA fragments and 3 MLR8 were detected as candidates for DNA fragments related to mutations in panicle length.

[0076] [Table 9] ^DNA fragment Number of individuals

 (bp) Mean Unbiased variance

 MLR 47 17. 37 5.bo

 243 0. 0013

 23 15. 21 8. 06

 Yes 13 18. 65 2. 94

 280 0.

 67 16. 21 Ί. 'Άό'

 Yes 47 丄 7, 13 7. 57

 122 0. 0198

 None 2.2 丄 5. 51 5. 25

 Yes 7 18. 61 5. 52

 251 0. 0441

 No 63 16. 44 7. 20

 Yes 55 17. 00 7. 03

 368 0. 0450

 None 15 15. 42 7. 14

 ΪΗ 24 17.5 (i. 78

 330 0. 0451

 No 46 16. 19 6. 17

 MLK Yes 18. ϋ'ό

 1 16. 64 10. 82

 Yes 23 16. 8 7. 91

 394 0. 0318

 No 5β 18. 07 4. 59

 Yes G7 17. 47 6. 06

 95 0. 0474

 No 66 19. 29 3. 36

 [0077] (5d) Chief

 As for the culm length, similar to (5b), we searched for specific DNA fragments related by t-test. As a result, as shown in Table 10, 5 DNA fragments and 4 MLR8 were detected as candidates for DNA fragments related to ear length mutation.

[0078] [Table 10]

DNA fragment

 System Number of enclosures P value

 (bp) Mean Unbiased variance

 MLRd Yes 51 57. 43 50. 93

 406 0. 0064

 nt 19 62. 53 29. 71

 Yes 58 57. 88 50. 11

 109 0. 0138

 VI 63. 33 ^ ϋ. 24

 Yes 61 59. 49 46. 25

 177 0. 0356

 9 54. 22 55. 94

 Yes 24 56. 42 58. 08

 330 U. Ού

 46 60. 07 42. 11

 29 60. 83 37. 86

 290 0. 0440

 41 54. 49

 MLR8 Yes 74 61. 66 49. 68

 326 U. UUo

 lot 5 71. 20 82. 70

 Yes 60 63. 35 51. 11

 167 0. 0214

 19 58. 84 59. 70

 Yes 13 66. 38 57. 42

 346 0. 0293

 66 61. 45 52. 77

 Yes 61 48. 38

 406 0. 0345

 18 59. 00 72. 47

 [0079] Thus, since the S / N ratio of the PCR product is improved by the DNA amplification method of the present invention, a wider range of waveform patterns can be used for analysis. In addition, since the variation in the experiment results by the experimenter has decreased, it has become easier and more objective to correlate traits and genes by the transposon display method.

 Example 3

 [0080] A 167 bp DNA fragment (see Table 10) related to panicle length and culm length shown in Example 2 was isolated by the transposon display method, and its nucleotide sequence was examined to search for related genes. Specifically, the procedure was as follows.

Use Srt-P3_D4 and a selection primer with one base added to ATG and C at the 3 'end of ply Csp6I_AP,

 Therefore, a 167 bp DNA fragment was amplified. The reason for using the selection ply instead of the ply Csp6I_AP is that there are DNA fragments of different sizes near the 167 bp amplified DNA fragment, and it is difficult to amplify only the target DNA fragment.

[0082] The results were confirmed by capillary electrophoresis. As a result, amplification of the objective 167 bp DNA fragment could be confirmed only when the selection ply to which A was added was used. on the other hand, DNA fragments of different sizes that existed around 167 bp were not amplified in this PCR reaction. Thus, by using the selection primer, only a 167 bp DNA fragment could be amplified.

 [0083] (2) Purification and subcloning of amplified DNA fragments

 The amplified DNA fragment was subjected to acrylamide gel electrophoresis, and a 167 bp amplified DNA fragment was excised from the gel to purify the DNA contained in the gel. Next, a PCR reaction was carried out using the Csp 6I-AP and Srt_P3 as primers, and the presence of the target DNA amplification fragment was confirmed. Furthermore, in order to clarify the base sequence of the amplified DNA fragment, the amplified DNA fragment was cloned into a sequencing vector (pGEM-T Easy).

 [0084] (3) Determination of the base sequence of the amplified DNA fragment and the base sequence adjacent to the mPing insertion position

 Using primers designed to sandwich the cloning vector cloning site (T7 PROM and SP6PROM, see Table 11), the nucleotide sequence of the target fragment inserted into the cloning site was determined. Further, the sequencing vector sequence and mPing sequence were removed from the obtained base sequence! / And the sequence adjacent to the position where mPing was inserted was examined.

 [0085] [Table 11] Oligo DNA nucleotide sequence

 T7PR0M TAATACGACTCACTATAGGG (SEQ ID NO: 6)

 SP6PR0M ATTTAGGTGACACTATAGAA (SEQ ID NO: 7)

As a result, a 92 bp base sequence (SEQ ID NO: 8) was obtained as shown in FIG. In FIG. 7, the boxed line portion indicates the primer-derived arrangement! J, and the underlined portion indicates the mPing sequence (part).

 [0087] (4) Search for the causative gene of the mutation

When the above nucleotide sequence was searched for in the chromosomal salt ti 歹¹ Jatahose (http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp), mPing was It is inserted near the long end (29430773bp) of chromosome 7 (AP008213, total length 29644043bp), and immediately after 170bp is a gene (Os07g0693000) encoding a protein with unknown protein kinase domain (BAF22640). It became clear that there was a transcription start point. A gene encoding ubiquitin activating enzyme E1 (BAF22639) is located 752 bp upstream of the mPing insertion position. It also became clear that it exists.

[0088] From the above results, it is inferred that the variation of culm length and panicle length was caused by insertion of mPing at the above position and change in the transcription amount of a gene of unknown function downstream thereof. On the other hand, for the upstream ubiquitin activating enzyme E1 gene, mPing is inserted downstream from the transcription end position of mRNA, so it is considered that the relationship between the cocoon length and the mutation related to panicle length is low. .

 [0089] Thus, by the transposon display method using the DNA amplification method of the present invention, the mutant trait and the mPing insertion mutant gene are linked, and the gene of unknown function (Os07g0693000) is used to change the head length. It was revealed that it was a dominant gene in a short period of time and low effort compared to the conventional method.

Claims

Claims [1] A method for amplifying a DNA fragment used in a transposon display method for higher plants, (1) a DNA extraction process for extracting genomic DNA from cells of higher plants;  (2) An adapter addition step of ligating an adapter capable of binding to the restriction enzyme recognition site into a DNA fragment by partially digesting genomic DNA with a restriction enzyme;  (3) a first PCR step of amplifying the DNA fragment by a one-side PCR method using a DNA fragment ligated with the adapter as a saddle and using only a primer specific for the transposon;  (4) The second DNA fragment is amplified by the double-sided PCR method using the DNA fragment amplified in the first PCR step as a saddle and using a labeled primer specific for the transposon and a primer complementary to the adapter. PCR process of  A method for amplifying a DNA fragment comprising: [2] The method for amplifying a DNA fragment according to claim 1, wherein the PCR cycle power in the first PCR step is S, 20 to 40 cycles. [3] The method for amplifying a DNA fragment according to [1], wherein the higher plant is rice. [4] The method for amplifying a DNA fragment according to claim 1, wherein the transposon has a copy number S of 100 to 1000 copies in a higher plant genome. 5. The method for amplifying a DNA fragment according to claim 4, wherein the transposon is mPing.