KR101677045B1 - Genotyping method for various plants using a new plant cell lysis buffer - Google Patents

Abstract

The present invention relates to a method for rapidly and simply performing a single copy insertion gene test in a variety of plants by using a new plant lysis buffer for Direct PCR, If a primer set based on an exogenous gene is used for the primary screening of transformants by semi-quentitative direct-PCR analysis, the number of Southern Hybridization analysis samples at the time of mass screening can be greatly reduced And can be used to greatly reduce costs.

Description

[0001] The present invention relates to a method for analyzing genotypes in various plants using a novel plant lysis buffer,

The present invention relates to a method for rapidly and simply screening a single copy insertion gene in various plants using a novel plant lysis buffer for direct PCR.

Polymerase chain reaction (PCR) is very useful as a simple amplification technique for nucleic acids and is widely used in various biological studies and diagnostics (Florencia et al. 2006). In particular, PCR is the most widely applied in the field of plant molecular biology, PCR is the most widely applied in the field of plant molecular biology, including breeding and ecology including gene type analysis, gene mapping, gene isolation, It is also used for the proof of the conversion body and the development of the marker.

Traditional PCR techniques require isolation and purification of template DNA. This process involves two steps. The first step is to bombard tissues and cells to easily dissolve and separate DNA in plant cells. The physical method of putting plant cell tissue in liquid nitrogen and then freezing is followed by a chemical method of dissolving protein lysis enzymes (proteinase K) in plant cells and using detergents. The second step is to isolate DNA only by removing chemical molecules including enzymes that separate the DNA molecules or dissolve the plant cells and then dissolve the enzymes that interfere with the PCR reaction from the components of the separated cells (Yang et al., 2007) . Since the separation and purification processes of these DNA templates require a lot of labor, time and cost, these processes show complexity in terms of various plant types and tissues when processing a large number of samples.

Since the 1990s, a variety of methods have been developed to rapidly and rapidly extract plant DNA for PCR. Genomic DNA in plant cells has been isolated from a variety of organisms including alkaline buffers (Wang et al., 1993; Xinet et al., 2003), PVPP (John et al., 1997; Kim et al. As various plant cell lysis buffers such as CTAB (Cetyltrimethyammonium bromide) (Hwang et al. 2000) are used, they can be easily separated from secondary metabolites, including phenol compounds bound by DNA in cells after cells and other materials have dissolved there was.

On the other hand, Direct-PCR techniques have been developed to directly PCR an animal, microorganism or plant tissue without a DNA separation purification process (Pierre and Christian 1991; Victor et al. Direct-PCR technology is an efficient, fast, and convenient way to save time and money over the same time period over traditional PCR methods. However, sometimes this is a daunting task to get accurate results in plant biotechnology. Because various components such as secondary metabolites, enzymes and phenolic compounds in plant cells delay PCR reaction.

HelixAmp direct PCR (3G), Nanohelix Co. Commercial products such as the Korea and Phire Direct PCR kit, Thermo Fisher Scientific Co., USA are being developed and are being marketed for Direct-PCR. However, these are mainly provided by animals and microorganisms and have problems in applying them to various plants. Recently, the miraculous demand for Direct-PCR has skyrocketed in botany to demonstrate the function of genes in GMO crops or in various plants. However, these technological developments lag behind recent trends.

Therefore, the present inventors have developed a novel plant lysis buffer modified from the existing cell dissolution buffer (alkaline polyethylene glycol, Piotr and Michal 2006). Direct-PCR was performed on various plants using a new buffer, The inventors of the present invention have completed the present invention.

1. A Simple and Non-destructive Method of Direct-PCR for Plant System (J.plant biol / (2012) 55: 114-122) 2. Direct PCR method for detection of Xanthomonas axonopodis pv.glycines in soybean seeds (Plant Disease Res.Plant Dis 15 (2): 83-87 (2009))

It is an object of the present invention to provide a method for analyzing a genotype in a plant.

In order to solve the above object,

The present invention

1) collecting leaf tissue using a punch;

2) adding the leaf tissue to a tube containing an alkaline polyethylene glycol lysis buffer;

3) vortexing the tube containing the leaf tissue and culturing at room temperature;

4) The primer set of SEQ ID NO: 1 and SEQ ID NO: 2, the primer set of SEQ ID NO: 3 and SEQ ID NO: 4, the primer set of SEQ ID NO: 5 and SEQ ID NO: 6, the primer set of SEQ ID NO: 7 and SEQ ID NO: The primer set of SEQ ID NO: 9 and SEQ ID NO: 10, the primer set of SEQ ID NO: 11 and SEQ ID NO: 12, the primer set of SEQ ID NO: 13 and SEQ ID NO: 14, the primer set of SEQ ID NO: The primer set of SEQ ID NO: 18, the primer set of SEQ ID NO: 19 and SEQ ID NO: 20, the primer set of SEQ ID NO: 21 and SEQ ID NO: 22, the primer set of SEQ ID NO: 23 and SEQ ID NO: (Direct PCR) reaction using any one of the primer set of the group consisting of the primers set;

5) identifying the amplified band by electrophoresis analysis of the reactant, and providing a method for analyzing a genotype in a plant.

If a primer set based on the endogenous and exogenous genes of the plant of the present invention and the plants of the present invention are used for the first screening of transformants by semi-quentitative direct-PCR analysis, Southern hybridization (Southern Hybridization) analysis can be usefully used to greatly reduce time and cost.

Figure 1 is a flow diagram of a direct-PCR method for plant genotyping.
Figure 2 shows the results of analysis of dissolved PCR products dissolved in various plant leaf tissues according to different concentrations of NaOH:
Lane 1; commercial direct-PCR kit, lane 2; 6% PEG 200 solution + 0 mM NaOH, lane 3; 6% PEG 200 solution + 1 mM NaOH, lane 4; 6% PEG 200 solution + 5 mM NaOH, lane 5; 6% PEG 200 solution + 10 mM NaOH, lane 6; 6% PEG 200 solution + 20 mM NaOH, lane 7; 6% PEG 200 solution + 50 mM NaOH, lane 8; 6% PEG 200 solution + 100 mM NaOH; A. rice, TPI (360 bp); B. Arabidopsis, Actin (491 bp); C. Potato, Actin (320 bp).
FIG. 3 is a diagram showing the results of direct-PCR amplification of endogenous genes in various plants using Alkaline PEG lysis buffer established in Example 2. Lanes 1 to 3 show that each crop is divided into three independent lines ) Loading the PCR product; A. rice TPI , 360 bp; B. Arabidopsis Actin 491bp; C. Tobacco Actin , 524bp; D. Potato Actin , 320 bp; E. Tomato Actin , 247 bp; F. Chrysanthemum Actin , 243 bp; G. Rapid Actin , 512bp.
Figure 4 shows the result of direct-PCR amplification of exogenous genes from various plants: lane 1 Wt; wild type; Lane 5 no template control PCR reaction; Lanes 2 to 4, three independent T ₀ Transgenic plant lines (A and C to F); Three independent T1 Transgenic Arabidopsis line (B); A. rice, Cry1Ac (658 bp); B. Arabidopsis Hyg (510 bp); C. Tobacco, Bar (170 bp); D. Potato, Km ( NPTII ) (249 bp); E. chrysanthemum, Hyg (510 bp); F oil, Bar (170bp).
Figure 5 shows the results of Southern Hybridization and semi-quantitative direct-PCR analysis on an independent transgenic rice line for CryIAc Copy number of the exogenous gene is a diagram showing a result of measurement (copy numbers).
Figure 6 is a graph showing the results of hybridization with Hyg (SEQ ID NO: 2) by semi-quantitative direct-PCR analysis in Southern hybridization and independent transgenic rice lines Copy number of the endogenous gene is a diagram showing a result of measurement (copy numbers).
Figure 7 shows the results of Southern hybridization and semi-quantitative direct-PCR analysis in an independent transgenic rice line to determine the CaMV 35S promoter Copy number of the exogenous gene is a diagram showing a result of measurement (copy numbers).
Figure 8 shows the results of Southern hybridization and semi-quantitative direct-PCR analysis of the CaMV 35S promoter in independent transgenic rice lines Copy number of the exogenous gene is also shown (copy numbers).

Hereinafter, the present invention will be described in detail.

The present invention

1) collecting leaf tissue using a punch;

2) adding the leaf tissue to a tube containing an alkaline polyethylene glycol lysis buffer;

3) vortexing the tube containing the leaf tissue and culturing at room temperature;

4) The primer set of SEQ ID NO: 1 and SEQ ID NO: 2, the primer set of SEQ ID NO: 3 and SEQ ID NO: 4, the primer set of SEQ ID NO: 5 and SEQ ID NO: 6, the primer set of SEQ ID NO: 7 and SEQ ID NO: The primer set of SEQ ID NO: 9 and SEQ ID NO: 10, the primer set of SEQ ID NO: 11 and SEQ ID NO: 12, the primer set of SEQ ID NO: 13 and SEQ ID NO: 14, the primer set of SEQ ID NO: The primer set of SEQ ID NO: 18, the primer set of SEQ ID NO: 19 and SEQ ID NO: 20, the primer set of SEQ ID NO: 21 and SEQ ID NO: 22, the primer set of SEQ ID NO: 23 and SEQ ID NO: (Direct PCR) reaction using any one of the primer set of the group consisting of the primers set;

5) identifying the amplified band by electrophoresis analysis of the reactant, and providing a method for analyzing a genotype in a plant.

The primer set of SEQ ID No. 1 and SEQ ID No. 2, the primer set of SEQ ID No. 3 and 4, the primer set of SEQ ID No. 5 and SEQ ID No. 6, the primer set of SEQ ID No. 7 and SEQ ID No. 8, The primer set of SEQ ID NO: 10, the primer set of SEQ ID NO: 11 and the primer set of SEQ ID NO: 12, and the primer set of SEQ ID NO: 13 and SEQ ID NO: 14 are preferably primers for amplifying an endogenous gene, but are not limited thereto.

The primer set of SEQ ID NO: 15 and SEQ ID NO: 16, the primer set of SEQ ID NO: 17 and SEQ ID NO: 18, the primer set of SEQ ID NO: 19 and SEQ ID NO: 20, the primer set of SEQ ID NO: 21 and SEQ ID NO: The primer set of SEQ ID NO: 24 and the primer set of SEQ ID NO: 25 and SEQ ID NO: 26 are preferably primers for amplifying an exogenous gene, but are not limited thereto.

The primer sets of SEQ ID NO: 1 and SEQ ID NO: 2 are OsTPI , The primer set of SEQ ID NO: 15 and SEQ ID NO: 16 are prepared by amplifying the primer set of Cry1Ac , SEQ ID NO: 23 and SEQ ID NO: 24 and the primer set of SEQ ID NO: 25 and SEQ ID NO: 26, But is not limited thereto.

In the primer set of SEQ ID NO: 3 and SEQ ID NO: 4, AtActin , the endogenous gene of Arabidopsis , and the primer set of SEQ ID NO: 17 and SEQ ID NO: 18 amplify Hyg which is an exogenous gene of Arabidopsis thaliana.

The primer sets of SEQ ID NO: 5 and SEQ ID NO: 6 are preferably NtActin , the endogenous gene of tobacco , and the primer set of SEQ ID NO: 19 and SEQ ID NO: 20, but are not limited to, amplify Bar ( ppt ) .

The SEQ ID NO: 7 and a primer set of SEQ ID NO: 8 is the primer set of the endogenous gene of potato StActin, SEQ ID NO: 21 and SEQ ID NO: 22 is a preferred to amplify the exogenous gene of potato Km (NptⅠⅠ), are not limited to, .

The primer set of SEQ ID NO: 9 and SEQ ID NO: 10 is preferably, but not limited to, amplifying StActin , the endogenous gene of tomato.

The primer set of SEQ ID NO: 11 and SEQ ID NO: 12 is preferably a primer set of CmActin which is an endogenous gene of Chrysanthemum , and the primer set of SEQ ID NO: 17 and SEQ ID NO: 18 amplifies Hyg which is an exogenous gene of Chrysanthemum.

The primer sets of SEQ ID NO: 13 and SEQ ID NO: 14 are preferably BnActin , which is an endogenous gene of rapeseed , and the primer sets of SEQ ID NO: 19 and SEQ ID NO: 20, amplify Bar ( ppt ) .

Preferably, the alkaline polyethylene glycol lysis buffer of step 2) comprises 6% PEG (polyethylene glycol) and 20 mM NaOH, but is not limited thereto.

The PCR reaction of step 4) was repeated at 35 cycles in 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds in step 1 and 72 ° C for 5 minutes in step 3 However, the present invention is not limited thereto.

But the present invention is not limited thereto, but it is preferable to identify a single copy insertion gene in the transgenic plant through the amplified band of step 5).

When the transgene introduced into a plant transgene is inserted into a gene sequence existing in a plant or a regulatory gene, it is difficult to verify that the transgene exhibits any expression trait because it may abnormally induce gene expression of the plant. Thus, it is most desirable that transgenes are introduced into the intergenic sequence between the gene and the gene, which is the easiest single gene insertion and easy analysis of the gene.

Therefore, when the method of analyzing the genotype in the plant of the present invention is used, it can be clearly confirmed that transgenes are introduced into a single gene insertion and an intergenic sequence between the gene and the gene, useful.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Materials and methods

<1-1> Plant material

The major plants used in this study were rice cultivars Dongjin and Haima , Arabidopsis species Columbia- 0, tobacco Nitotiana Six selected species of transgenic plants were used except for seven kinds of plants and tomatoes as benthamiana , potato varieties, chrysanthemum varieties, tomato varieties, and rapeseed varieties.

<1-2> Preparation of new plant dissolution buffer

0.2 N NaOH solution was prepared freshly before the experiment so as to have a concentration of 0, 1, 5, 10, 20, 50, 100 mM and 6% PEG 200 (Sigma-Aldrich Co. USA) Experiments were performed to determine the optimum condition of Alkaline PEG lysis buffer from 6% PEG / 20 mM NaOH solution from various plants.

<1-3> direct polymerase chain reaction Direct PCR ) Way

The leaves of each plant from 7 different plants including rice, which is the main crop, were cut and harvested using a 1.0 mm diameter Harris Uni-core punch (Ted Pella Inc. USA) Was added to a PCR tube containing 50 [mu] l of an Alkaline PEG lysis buffer. The solution was vortexed for 15 seconds in a well-immersed solution, stored on ice until all samples were ready, briefly centrifuged, and left at room temperature for 1 minute. The PCR was carried out on ice again for about 5 minutes. The remaining samples were stored at -20 ° C and used again whenever necessary. PCR was carried out using 2 μl of lysate solution in 50 μl and using AccuPower PCR premix (Bioneer co. Korea). PCR was carried out using the primer sets described in Table 1 according to plant species. PCR conditions were 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles, and 3 ° C for 5 minutes. After the PCR reaction, electrophoresis was performed using 1% agarose gel. The schematic diagram for Direct PCR is shown in FIG. 1, and the specific sequences of primers according to 7 plants and genes are shown in Table 1 below.

plant Cultivated variety gene
name Amplification
Size (bp) GI No.
(NCBI) Primer sequences (5 ' - > 3 ') order
number Endogenous gene Endogenous genes ) rice plant Dongjin OsTPI 360 115434515 AGCTAGCCTGCCTTCACTTG One CGTTATCCCCAGGTTTGGCT 2 Arabic pole Columbia-0 AtActin 491 145361675 GGCGATGAAGCTCAATCCAAAC 3 GGTCACGACCAGCAAGATCAAG 4 tobacco Xanthi NtActin 524 20038 CCCTCCCACATGCTATTCT 5 AGAGCCTCCAATCCAGACA 6 potato Superior StActin 320 460408873 TCCTCCATTGAAAAGAACTATG 7 CCAGACACTGTACTTTCTCTC 8 tomato Microtome StActin 247 460378622 CTCGAGCAGTGTTTCCCAGT 9 GGTGCCTCAGTCAGGAGAAC 10 Chrysanthemum Shinma CmActin 243 475393035 TGCTGACAGGATGAGCAAGG 11 GGGCCAGACTCGTCGTATTC 12 Rapeseed Youngsan BnActin 512 4139263 TCACCATCGGAGCTGAGAGA 13 TGGACCCGACTCATCGTACT 14 Exogenous gene Exogenous genes ) rice plant
Dongjin
Cry1Ac
658
22415750
TTGTGGCTCTCTTCCCGAAC 15 AACTCGGCAGAACGGTGAAT 16 Arabic pole
Chrysanthemum Columbia-0 Hyg
510
34596493
CGTCTGCTGCTCCATACAAG 17 Shinma GTGCTTGACATTGGGGAGTT 18 tobacco
Rapeseed Xanthi Bar ( ppt )
170
375153555
TACACCCACCTGCTGAAGTC 19 Youngsan AAACCCACGTCATGCCAGTT 20 potato
Superior
Km  ( NptII ) 249
19569229 CAAGATGGATTGCACGCAGG 21 TTCAGTGACAACGTCGAGCA 22 rice plant
Dongjin
35S P
644
28193426
CTGATCTCCTTTGCCCCGGA 23 AATCCGAGGAGGTTTCCCGA 24 rice plant
Dongjin
35S P
173
28193426
ATCACCATGGACGACTTTCTCT 25 ATCGTCTTGATGAGACCTGCC 26

< Example  2> Alkaline PEG lysis buffer  Optimal condition analysis

5, 10, 20, 50, and 50% solution of 6% PEG 200 (Sigma-Aldrich Co. USA) solution to establish the optimal concentration of NaOH in 6% PEG 200 (Sigma- A 0.2 N NaOH solution was prepared to have a concentration of 100 mM, and a direct-PCR kit using a general control was prepared.

Specifically, as in the case of Example 1-1, the rice, Arabidopsis and potato leaves were perforated with a puncher to a diameter of 1.0 mm. Next, 50 μl of a solution containing 6% PEG 200 (Sigma-Aldrich Co. USA) and 0.2 N NaOH solutions having the concentrations of 0, 1, 5, 10, 20, 50 and 100 mM were added to the tubes, The direct-PCR kit, which also contains the rice, Arabidopsis and potato plants, is vortexed and incubated at room temperature for 1 minute. 2 μl of the cultured lysate and each of the primers set forth in SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 7 and 8, which are primers for rice, Arabidopsis and potato, were mixed with AccuPower PCR premix (Bioneer Co., To perform PCR reaction. PCR conditions were 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles, and 3 ° C for 5 minutes. After the PCR reaction, electrophoresis was performed using 1% agarose gel.

As a result, direct PCR using 6% PEG 200 (Sigma-Aldrich Co. USA) solution and Alkaline PEG lysis buffer containing 0.2 N NaOH solution to have concentrations of 5 mM, 10 mM, 15 mM and 20 mM resulted in TPI of 360 bp ), Arabinophilic Actin (491bp), and Potentia Actin (320bp). A 6% PEG 200 (Sigma-Aldrich Co. USA) solution and an alkaline PEG lysis buffer containing 0.2 N NaOH solution (Fig. 2).

< Example  3> Alkaline PEG lysis buffer Using direct - PCR  Transformant analysis by method

<3-1> Alkaline PEG lysis buffer The endogenous gene direct - PCR  Transformant analysis by method

Using a nucleic acid extraction buffer containing 6% PEG 200 (Sigma-Aldrich Co. USA) solution prepared in Example 2 and 0.2 N NaOH solution to have a concentration of 20 mM, direct-PCR was performed from various species of plants The transformants were amplified.

Specifically, the rice cultivar Dongjin, Arabidopsis Columbia-0, tobacco Nitotiana The leaves of seven plants of benethamiana , potato varieties, tomato microtome, chrysanthemum varieties, and rapeseed varieties were drilled with a puncher at a diameter of 1.0 mm. Then, 50 μL of a solution containing 6% PEG 200 (Sigma-Aldrich Co. USA) and 0.2 N NaOH solution having a concentration of 20 mM was added to the tube, the tissue of the plant was submerged in buffer, And incubate at room temperature for 1 minute. 2 μl of cultured lysate and rice OsTPI , Arabidopsis Actin , tobacco NtActin , potato StActin , tomato StActin Chrysanthemum CmActin And the primer of SEQ ID NO: 1 and for the endogenous gene of rapeseed BnActin 2, of SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10 primers of SEQ ID NOS: 11 and 12 of the primer, and Each of the primer sets of SEQ ID NOS: 13 and 14 was added to AccuPower PCR premix (Bioneer Co., Korea) to perform PCR reaction. PCR conditions were 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles, and 3 ° C for 5 minutes. After the PCR reaction, electrophoresis was performed using 1% agarose gel.

As a result, compared with the control (wild type, line 1) plants and the no-template for PCR reaction, rice (360bp), Arabidopsis (491bp), cigarette (524bp), potato (320bp) 243 bp) and rapeseed (512 bp) (FIG. 3).

<3-2> Alkaline PEG lysis buffer And the exogenous gene direct - PCR  Transformant analysis by method

Using a nucleic acid extraction buffer containing 6% PEG 200 (Sigma-Aldrich Co. USA) solution prepared in Example 2 and 0.2 N NaOH solution to have a concentration of 20 mM, direct-PCR was performed from various species of plants The transformants were amplified.

Specifically, the rice cultivar Dongjin, Arabidopsis Columbia-0, tobacco Nitotiana The leaves of six plants of benethamiana , potato varieties, chrysanthemum varieties, and rapeseed varieties were punched at a diameter of 1.0 mm using a puncher. Then, 50 μL of a solution containing 6% PEG 200 (Sigma-Aldrich Co. USA) and 0.2 N NaOH solution having a concentration of 20 mM was added to the tube, the tissue of the plant was submerged in buffer, And incubate at room temperature for 1 minute. In the cultured lysates, 2 and rice Cry1Ac , Cryptobacia Hyg , Tobacco Bar ( ppt ) , Potato Km ( NPTII ) , Chrysanthemum Hyg And primer sets of SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, which are primers for exogenous genes of rapeseed bar , were added to AccuPower PCR premix (Bioneer Co., PCR reaction. PCR conditions were 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles, and 3 ° C for 5 minutes. After the PCR reaction, electrophoresis was performed using 1% agarose gel.

As a result, compared to the control (wild type, line 1) plants and the no-template for PCR reaction, rice (658 bp), Arabidopsis (510 bp), tobacco (170 bp), potato (249 bp), chrysanthemum 170 bp) (Fig. 4).

< Example  4> Semi - quentitative Direct - PCR And transgenic analysis using Southern hybridization (Southern hybridization)

The results of the semi-quentitative direct-PCR analysis were compared with the results of Southern hybridization using a buffer for effectively extracting nucleic acids from the Alkaline PEG lysis buffer established in Example 2 at all times.

Specifically, in order to analyze Southern hybridization, an exogenous gene such as Cry1Ac (FIG. 5), Hyg (FIG. 6), 35S promoter (FIG. 7) and 35S promoter The conversion was analyzed. Transgenic plants overexpressing the internal gene, which is a super family of rice genes, and transgenic plants overexpressing general internal genes were selected to extract 10 의 of genomic DNA, The expression vector of the present invention was treated with appropriate restriction enzymes that did not break down the vector DNA, and then electrophoresed and blotted on a membarane using a capillary method. Southern hybridization was performed using the introduced gene, the selection marker gene, or the DNA portion of the appropriate promoter region as a probe.

In addition, the transformants were analyzed by semi-quentitative direct-PCR method. Rice seedlings were punched at the leaf of Dongjin using a puncher with a diameter of 1.0 mm. Then, 50 μL of a solution containing 6% PEG 200 (Sigma-Aldrich Co. USA) and 0.2 N NaOH solution having a concentration of 20 mM was added to the tube, the tissue of the plant was submerged in buffer, And incubate at room temperature for 1 minute. 2 &lt; RTI ID = 0.0 &gt; ul &lt; / RTI &gt; in the cultured lysate and primers SEQ ID Nos. 15 and 16 for the exogenous gene of rice Cry1Ac , Primers SEQ ID Nos. 17 and 18 for Hyg, primer sequences 19 and 20 for Bar, primer sequences 21 and 22 for Km, primer sequences 23 and 24 for 35S, primer sequences 25 and 26 for 35S, Each Was added to AccuPower PCR premix (Bioneer co. Korea) to perform PCR reaction. PCR conditions were 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, 35 cycles, and 3 ° C for 5 minutes. After the PCR reaction, electrophoresis was performed using 1% agarose gel.

As a result, it was confirmed that Semi-quentitative Direct-PCR and Southern hybridization showed 90% The technology can be applied to the analysis of gene number when the endogenous gene is introduced as well as the exogenous gene. Therefore, if it is utilized in the first selection of the transformant through the semi-quentitative direct-PCR analysis, It was confirmed that the time and cost can be greatly reduced by greatly reducing the number of blot hybridization analysis samples (FIGS. 5, 6, 7, and 8).

<110> REPUBLIC OF KOREA <120> Genotyping method for various plants using a new plant cell lysis          buffer <130> p14r12d0365 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Oryza sativa <400> 1 agctagcctg ccttcacttg 20 <210> 2 <211> 20 <212> DNA <213> Oryza sativa <400> 2 cgttatcccc aggtttggct 20 <210> 3 <211> 22 <212> DNA <213> Arabidopsis thaliana (L.) Heynh. <400> 3 ggcgatgaag ctcaatccaa ac 22 <210> 4 <211> 22 <212> DNA <213> Arabidopsis thaliana (L.) Heynh. <400> 4 ggtcacgacc agcaagatca ag 22 <210> 5 <211> 19 <212> DNA <213> Nicotiana benthamiana <400> 5 ccctcccaca tgctattct 19 <210> 6 <211> 19 <212> DNA <213> Nicotiana benthamiana <400> 6 agagcctcca atccagaca 19 <210> 7 <211> 22 <212> DNA <213> Solanum tuberosum L <400> 7 tcctccattg aaaagaacta tg 22 <210> 8 <211> 21 <212> DNA <213> Solanum tuberosum L <400> 8 ccagacactg tactttctct c 21 <210> 9 <211> 20 <212> DNA <213> Lycopersicon esculentum <400> 9 ctcgagcagt gtttcccagt 20 <210> 10 <211> 20 <212> DNA <213> Lycopersicon esculentum <400> 10 ggtgcctcag tcaggagaac 20 <210> 11 <211> 20 <212> DNA <213> Dendranthema grandiflorum <400> 11 tgctgacagg atgagcaagg 20 <210> 12 <211> 20 <212> DNA <213> Dendranthema grandiflorum <400> 12 gggccagact cgtcgtattc 20 <210> 13 <211> 20 <212> DNA <213> Brassica napus <400> 13 tcaccatcgg agctgagaga 20 <210> 14 <211> 20 <212> DNA <213> Brassica napus <400> 14 tggacccgac tcatcgtact 20 <210> 15 <211> 20 <212> DNA <213> Oryza sativa <400> 15 ttgtggctct cttcccgaac 20 <210> 16 <211> 20 <212> DNA <213> Oryza sativa <400> 16 aactcggcag aacggtgaat 20 <210> 17 <211> 20 <212> DNA <213> Arabidopsis thaliana (L.) Heynh. <400> 17 cgtctgctgc tccatacaag 20 <210> 18 <211> 20 <212> DNA <213> Dendranthema grandiflorum <400> 18 gtgcttgaca ttggggagtt 20 <210> 19 <211> 22 <212> DNA <213> Nitotiana benthamiana <400> 19 ggcgatgaag ctcaatccaa ac 22 <210> 20 <211> 20 <212> DNA <213> Brassica napus <400> 20 aaacccacgt catgccagtt 20 <210> 21 <211> 20 <212> DNA <213> Solanum tuberosum L <400> 21 caagatggat tgcacgcagg 20 <210> 22 <211> 20 <212> DNA <213> Solanum tuberosum L <400> 22 ttcagtgaca acgtcgagca 20 <210> 23 <211> 20 <212> DNA <213> Oryza sativa <400> 23 ctgatctcct ttgccccgga 20 <210> 24 <211> 20 <212> DNA <213> Oryza sativa <400> 24 aatccgagga ggtttcccga 20 <210> 25 <211> 22 <212> DNA <213> Oryza sativa <400> 25 atcaccatgg acgactttct ct 22 <210> 26 <211> 21 <212> DNA <213> Oryza sativa <400> 26 atcgtcttga tgagacctgc c 21

Claims (13)

1) collecting leaf tissue using a punch;
2) adding the leaf tissue to a tube containing an alkaline polyethylene glycol lysis buffer;
3) vortexing the tube containing the leaf tissue and culturing at room temperature;
4) a primer set of amplifying an endogenous gene OsTPI of rice in the culture lysate SEQ ID NO: 1 and SEQ ID NO: 2, the primer set for amplifying the endogenous gene of Arabidopsis AtActin SEQ ID NO: 3 and SEQ ID NO: 4, cigarette of SEQ ID NO: amplifying an endogenous gene NtActin 5 and SEQ ID NO: 6 primers, sequences for amplifying an endogenous gene StActin of potato No. 7 and SEQ ID NO: primers 8, for amplifying an endogenous gene StActin of tomato sequence number of 9 and a primer set of SEQ ID NO: 10, a primer set of SEQ ID NO: 11 and SEQ ID NO: 12 to amplify the endogenous gene CmActin of chrysanthemum, SEQ ID NO: amplifying an endogenous gene BnActin rape 13 and SEQ ID NO: primers 14, rice the primer set of the exogenous gene of SEQ ID NO: 15 and SEQ ID NO: 16 to amplify the Cry1Ac, the exogenous gene of Arabidopsis thaliana or chrysanthemum Hyg Primers amplify SEQ ID NO: 17 and SEQ ID NO: 18 primer sets, tobacco, or SEQ ID NO: 19 and SEQ ID NO: 20 to amplify the exogenous gene Bar (ppt) of rapeseed in that, for amplifying an exogenous gene for potato Km (NptⅠⅠ) A primer set of SEQ ID NO: 21 and SEQ ID NO: 22, a primer set of SEQ ID NO: 23 and SEQ ID NO: 24 which amplify the CaMV 35S promoter which is an exogenous gene of rice, and SEQ ID NO: 25 which amplifies the CaMV 35S promoter which is an exogenous gene of rice, A direct PCR reaction using any one primer set of the primer set # 26;
5) analyzing the reaction product by electrophoresis to identify an amplified band,
Wherein the alkaline polyethylene glycol lysis buffer of step 2) comprises 6% PEG (polyethylene glycol) 200 and 5 to 50 mM NaOH.
delete delete delete delete delete delete delete delete delete delete The method according to claim 1, wherein the PCR reaction in step 4) is repeated at 35 cycles in 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds in step 1, 72 &lt; 0 &gt; C for 5 minutes.
The method according to claim 1, wherein the single-copy insert gene is identified in the transgenic plant through the amplified band of step 5).

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