KR20030041463A - Plant transformation vector prcv2 containing hygromycin resistance gene for rescue cloning - Google Patents

Abstract

PURPOSE: Provided is a plant transformation vector, pRCV2, containing hygromycin resistance gene for rescue cloning. In particular, functions of genes in plants are detected and abnormal expression of an introduced gene in transformed plants are analyzed by using rescue cloning. CONSTITUTION: A plant transformation vector, pRCV2, is characterized by inserting ampicillin resistant gene and a replication origin of bacteria into pCAMBIA 1301 vector containing hygromycin resistant gene and beta-glucuronidase gene.

Description

Plant transformation vector pRCV2 containing hygromycin resistance gene for rescue cloning}

The present invention relates to a plant transformation vector pRCV2 for rescue cloning comprising a hygromycin resistance gene, and more specifically, to search for the function of the genes present in the plant using Rescue cloning technology and appear in various transgenic crops. The present invention relates to the development of plant transformation vectors for studying abnormal expression patterns of introduced genes.

Rescue cloning is Agrobacterium (Agrobacterium) of the T-DNA is by using a developer to be introduced to random in the plant genome, by securing a large number of mutant transgenic, and compares them with transgenic non-transformed normal plants can be compared to the function of the gene It is similar to T-DNA tagging technology, but it is a more effective way to separate plant flanking DNA of various pathways by tagging it once when T-DNA is transferred to multiple. For such Rescue cloning, a vector designed to enable recloning should be inserted into the genome of the target crop by a transformation technique using Agrobacterium, and the phenotype of the transformant obtained is normal. If there is a difference, re-cloned the inserted T-DNA to obtain sequencing information of fragments and flanking portions on both sides of the fragments. Genetic sequencing information should be comparable. As described above, in order to reclonal the T-DNA portion inserted into the genome of the plant and the plant genome portion flanking to both sides of the inserted T-DNA portion of the transformant when transforming with Agrobacterium, the genomic DNA of the transformant When cleaving with a restriction enzyme, an appropriate restriction enzyme should be selected that can adequately contain the transferred gene, ie the flanking portion without cleaving the T-DNA portion. In addition, the genome fragments digested by restriction enzymes were re- ligation, transformed with E. coli , cultured, and cultured in LB medium to insert only antibiotic fragments within the T-DNA to select only the desired fragments. There must be a bacterial replication origin to facilitate growth in the condition. Based on the information obtained by recloning and sequencing the parts that caused the phenotypic differences from normal individuals, the method can be compared with the entire sequencing data of the individuals obtained through the genome project or used as RFLP markers to determine the location on the chromosome. Can be. Rescue cloning is a method for studying the function of individual genes. It is easy to obtain relatively large numbers of transformants, and is widely used in Arabidopsis where genome sequences of whole individuals are decoded. It is becoming.

Rescue cloning can also be used to solve the problem of transgene aberration, which occurs frequently in transformation with Agrobacterium . Transgene expression aberration problem is a phenomenon that the expression of the introduced gene is not normal, which is called gene silencing phenomenon, and it has recently attracted a lot of attention. The silencing of transgenes that occur in transformants can occur depending on the type or number of copies of the genes introduced. Also, when multiple transgenes are introduced at the same time, they may occur between the genome and the transgenes of a crop or between multiple transgenes. It is assumed to be caused by DNA methylation. Recently, researches on the cause of the silencing phenomenon have been actively conducted. As the mechanism is revealed, it is divided into three representative hypotheses. Representative hypotheses so far found are primarily at the nucleic acid level and can be divided into DNA-DNA, RNA-RNA and DNA-RNA interrelationships. The term 'homology-dependent gene silencing' is used to include all of these hypotheses, which are caused by homology between transgenes and transgenes that already exist in the plant itself or between transgenes with multiple copies. It can be seen as including all.

The T-DNA tagging method can be used to confirm how transgenes are introduced into the genome of a plant, thereby identifying the orientation, copy number, and transgene phenomena, such as DNA methylation, when transgenes are introduced into the genome. Furthermore, it has an important function of determining similarity or association in the position of introduction into the genome.

The present invention has been proposed to develop an effective vector for rescue cloning and to use it to explore the functions of genes present in plants and to study abnormal expression patterns of introduced genes in various transgenic crops. In addition, the present invention has made the primer easily sequencing the sequence inside and outside the T-DNA added to the ease of study and unlike the existing studies to allow the use of restriction enzymes to obtain flanking parts of both sides at the same time There is an advantage that the vector construction process has already been properly manipulated.

Therefore, an object of the present invention includes a hygromycin resistance gene as a selection marker, a β-glucuronidase gene and an ampicillin resistance gene as a reporter gene, and a bacterial replication origin, and rescue cloning is performed more efficiently. Rescue cloning including a restriction site that cleaves genomic DNA of the individual to be tagged to be appropriately contained in the T-DNA and flanking sections, and a primer site that can sequence the outer and inner sections of the T-border immediately To provide a plant transformation vector pRCV2.

Another object of the present invention is to provide a transformant Agrobacterium tumer Pession LBA4404 / pRCV2 introduced with the plant transformation vector pRCV2 and a primer capable of sequencing the T-border outside, the inside of the vector.

Still another object of the present invention is to provide a method for analyzing genes of plant transformants into which the vector is introduced.

The object of the present invention is to establish the ampicillin resistance gene and bacterial replication origin in the T-DNA of the plant transformation vector pCAMBIA1301 equipped with a hygromycin resistance gene and a reporter gene as a selection marker in plant transformation. The genomic DNA of the individual to be tagged with the T-DNA part is removed by inserting the part of the cell and inserting and removing the restriction enzyme site that can cut the genomic DNA of the plant into appropriate fragments. Restriction enzymes that can be cleaved are selected to properly include flanking parts, and primers that can directly sequence the outer part of the T-DNA border and the inside of the T-DNA are prepared to sequence the developed vector in the plasmid DNA state. As a result, it was achieved by confirming the validity of the function.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a schematic diagram showing a cleavage map of a plant transformation vector pRCV2 constructed by recombining pBluescriptII KS (+) at multiple cloning sites of pCAMBIA1301.

Figure 2 is a schematic diagram showing the location of BamH I, Hind III, Pst I in multiple cloning sites of pCAMBIA1301 and pBluescriptII KS (+).

3 is a result of electrophoresis of the present invention vector pRCV2 treated with Pst I.

Figure 4 shows the double elution of pCAMBIA1301 and pBluescriptII KS (+) with BamH I and Hind III and gel elution in gel.

Figure 5 shows the results of mapping the pRCV2 clone and PCR with hpt primer and gus primer.

Figure 6 shows the results of analyzing the base sequence with a primer made to sequence the T-border outside (right) of pRCV2.

Figure 7 shows the results of sequencing the primers made to sequence the T-border outside (left) of pRCV2.

Figure 8 shows the results of sequencing with primers made to sequence the inside of the T-border (right) of pRCV2.

Figure 9 shows the results of sequencing with primers made for sequencing the T-border inside (left) of pRCV2.

Figure 10 shows the result of PCR with hpt primer and gus primer prep pRCV2 introduced in A. tumefaciens LBA4404.

Figure 11 shows the results confirmed by Southern blotting the number of transgene copy transferred to the transformant tobacco.

12 is a result of PCR reaction of DNA extracted from the transformant tobacco using HPT primer and GUS primer.

Figure 13 is a result of measuring the size of the flanking fragment outside the T-border by restriction enzyme treatment to the plasmid DNA extracted from the transformant tobacco.

14 is a result of confirming that the plasmid DNA extracted from the transformant tobacco contains a part of the genome of the tobacco by reacting with a primer prepared in advance to sequencing the outside of the T-border.

The present invention provides a tagging vector (pRCV2) by inserting pBluescriptII KS (+) having an ampicillin resistance gene and a bacterial replication point into pCAMBIA1301 carrying a hygromycin resistance gene (hpt) among plant transformation vectors. Determining a restriction enzyme to be used to cleave the genomic DNA of the individual to be cleaved; Mapping the cloned vector (pRCV2) to confirm whether the cloning is correct and confirming by PCR whether the selection marker and the reporter gene are present in the vector; Based on the entire nucleotide sequence of pRCV2, a primer that can sequence the outside and the inside of both borders of T-DNA was prepared to amplify the prepared primer by PCR and analyze the sequencing to correctly sequence the outside and inside. Confirming; pRCV2 was inserted into A. tumefaciens LBA4404, and PCR was used to confirm whether the selected marker and reporter gene were present in the inserted pRCV2.

The plasmid pCAMBIA1301 used in this step was purchased from CAMBIA.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Determination of restriction enzymes capable of simultaneously obtaining flanking portions of both T-DNA during rescue cloning in plant transformation vector (pRCV2) for rescue cloning

Rescue cloning common between FIG from the multi-cloning site of the multiple cloning site and pBluescriptII KS (+) of pCAMBIA1301 BamH Ⅰ and Hind Ⅲ site, as shown in FIG. 2 in order to determine an appropriate restriction enzyme to cut the genomic DNA of the object to One restriction enzyme, Pst I site, was removed. Multiple cloning sites of pCAMBIA1301 and multiple cloning sites of pBluescriptII KS (+) have only one restriction site in each vector. If one of these sites is removed, the restriction site in pRCV2 completely removes the site. To lose. Therefore, when pRCV2 is transferred to a plant and then the genomic DNA of the plant transformed with the corresponding restriction enzyme is cut, the T-DNA portion and the flanking portion on both sides can be obtained at the same time.

Example 2 Construction and Transformant of Plant Transformation Vector (pRCV2) for Rescue Cloning Escherichia coli Preparation of pRCV2 (DH5α)

As shown in FIG. 1, to construct a vector for rescue cloning, an ampicillin resistance gene and a bacterial replication origin were placed at a multiple cloning site in a T-border of pCAMBIA1301 carrying a hygromycin resistance gene (hpt) among plant transformation vectors. PBluescriptII KS (+) was recombined. First, pCAMBIA1301 and pBluescriptII KS (+) were cleaved with BamH I and Hind III to remove the Pst I site, a restriction enzyme recognition site common to the multiple cloning site of pCAMBIA1301 and the multiple cloning site of pBluescriptII KS (+). After treatment with gel eluted (gel elution) in the gel (Fig. 4). In order to transform the Ligation reaction solution into E. coli , DH5α competent cells were incubated at 37 ° C. until OD ₅₇₀ became 0.375 in LB liquid medium. The grown cell was precipitated by centrifugation, and the pellet was immersed in CaCl ₂ solution and repeated twice. Put ligation reaction solution into competent cell prepared in this way, heat shock in 42 ℃ water bath for 1 minute 30 seconds, and then add LB liquid medium and incubate it for 1 hour, then 50 mg / L of hygromycin (Hygromycin) and ampicillin 50 Colonies grown the next day were selected in LB solid medium with mg / L added. To confirm whether the cloning was successful, the selected colonies were miniprep and mapped to select the correct clones. PCRs were performed with hpt primers and gus primers to confirm the presence of selection markers and reporter genes in the recombinant vector. 5).

The Agrobacterium tumer Passion LBA4404 / pRCV2 transformed with the plant transformation vector pRCV2 was deposited on November 19, 2001 with the Korean Culture Center of Microorganisms under accession number KFCC11283.

Example 3: Preparation of primers capable of sequencing outside the T-border in plant transformation vector (pRCV2) for rescue cloning

Primers were prepared based on the entire nucleotide sequence of pCAMBIA1301 to sequence outside the T-DNA border (SEQ ID NO: 1, SEQ ID NO: 2). The entire nucleotide sequence of pCAMBIA1301 was obtained from NCBI genebank, and the T- of pCAMBIA1301 was obtained using the primer3 program (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) which can prepare primers from DNA sequences. Based on the sequences inside the left border and inside the T-right border, primers were read outwards. This primer was PCR amplified with the prepared primer to check whether the T-border outside of pRCV2 can be properly sequenced and compared with the entire nucleotide sequence of pCAMBIA1301 and pBluescriptII KS (+). It was confirmed that it can (Fig. 6, 7).

Example 4 Preparation of a Primer capable of Sequencing T-Border in Plant Transformation Vector (pRCV2) for Rescue Cloning

To sequence the inside of the T-DNA border, primers were prepared based on the entire nucleotide sequence of pCAMBIA1301 (SEQ ID NO: 3, SEQ ID NO: 4). The entire nucleotide sequence of pBluescriptII KS (+) was obtained from NCBI genebank, using primer3 program (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi), which can prepare primers from DNA sequences. Based on the nucleotide sequence near the center of the ampicillin resistance gene of pBluescriptII KS (+), primers were read outward. In order to check whether the prepared primers can sequence the T-border inside of pRCV2 properly, PCR amplification was performed with the prepared primers, and the entire sequences of pCAMBIA1301 and pBluescriptII KS (+) were compared. It was confirmed that it can (Fig. 8, 9).

Example 5 Introducing a Plant Transformation Vector (pRCV2) for Rescue Cloning A. tumefaciens LBA4404 / pRCV2

PRCV2 identified by sequencing was inserted into A. tumefaciens LBA4404 cells using the Freeze-thaw method. First, LBA4404 competent cells were cultured at 28 ° C. until OD ₆₀₀ became 1.0 in YEP liquid medium. The grown cells were centrifuged down, and the pellets were immersed in 0.02M CaCl ₂ and divided into 0.1 ml portions. PRCV2 plasmid DNA was added to competent cells prepared in this manner and frozen in liquid nitrogen. Next, heat shock for 5 minutes in a 37 ℃ water bath was added to the YEP liquid medium and incubated for about 2 hours, the colonies grown on the next day by dispensing in a YEP solid medium to which 50 mg / L of hygromycin was added Selected. In order to check whether or not the insertion was properly inserted, the selected colonies were prep and confirmed by PCR with hpt primer and gus primer (Fig. 10).

Example 6 Preparation of Transgenic Plant Incorporated with a Plant Transformation Vector (pRCV2)

Sections of 1 cm diameter tobacco leaf were inoculated into Agrobacterium containing pRCV2 vector and co-cultivated for 3 days in coculture medium (MS, 1.5 mg / L BA) (3 days). After co-culture, the sections were cultured with hygromycin (transformation selection marker) and cefatoxime (Agrobacterium removal) (MS, 1.5 mg / L BA, 20 mg / L hygromycin, 200 mg / L cefotaxime, 0.8%). agar). Root differentiation from the selection medium was transferred to root differentiation medium (1/2 MS, 200 mg / L cefotaxime, 0.8% agar) to induce rooting. The roots were stabilized (1-2 weeks) and then grown in soil.

Example 7 Rescue Cloning from Plants Incorporating a Plant Transformation Vector (pRCV2) for Rescue Cloning

For rescue cloning, the number of copies of the transgene transferred to the transformant was first confirmed by Southern blotting (FIG. 11). After confirmation, the different numbers of lines were selected and grown in a greenhouse. High-purity genomic DNA was extracted from each strain using plant genomic isolation kit (Boehringer mannheim, USA). The extracted genomic DNA was treated with Pst I and self-ligation of the cut DNA fragments with T4DNA ligase and then electroporation into supercompetent cells (Stratagene, USA) using gene-pulser (Bio-Rad, USA). This was dispensed into LB medium to which ampicillin was added and cultured at 37 ° C. to obtain desired colonies into which plant DNA fragments were inserted.

Plasmid DNA was extracted from the colonies obtained by the above method using a plasmid miniprep kit (Qiagen, Germany). The extracted DNA was subjected to PCR reaction using HPT primer and GUS primer to confirm whether pRCV2 was included in the extracted plasmid DNA (FIG. 12), and the size of the flanking fragment flanked outside the T-border through restriction enzyme treatment. Predicted (FIG. 13). In addition, the extracted plasmid DNA was sequencing by reacting with a prefabricated primer for sequencing outside the T-border, thereby obtaining a sequence including a part of the genome of tobacco (FIG. 14).

The plant transformation vector pRCV2 for rescue cloning of the present invention constructed by the above method can be effectively used for T-DNA tagging studies for searching gene functions in various plants and for studying abnormal expression patterns of introduced genes.

As is apparent from the above examples, the present invention is a recloning of β-glucuronidase and bacteria as a hygromycin resistance gene and a reporter gene as a selection marker that can be used for transformation using A. tumefaciens . Ampicillin-resistant genes and bacterial origins for replication are available in plants and E. coli , which can be used to explore the function of genes or to study the stability of introduced genes and the mechanism of introduction itself. In addition, the tagging method is usefully used to explore the function of genes. In addition, the present invention can separate and secure the mutated and plant genomic DNA in the transferred T-DNA as well as T-DNA. Can be analyzed even when multiple copies have been transferred to multiple copies, making it easier to analyze the function of genes. This is a very useful invention in the genetic engineering industry.

Claims (5)

A plant transformation vector pRCV2, wherein an ampicillin resistance gene and a bacterial replication origin are inserted into a pCAMBIA 1301 vector comprising a hygromycin resistance gene and a β-glucuronidase gene. Agrobacterium tumer facsimile LBA4404 / pRCV2 ( A. tumefaciens LBA4404 / pRCV2) having a deposit number KFCC11283, which introduces the plant transformation vector pRCV2 of claim 1 A primer capable of sequencing outside the T-border of pRCV2 represented by SEQ ID NO: 1 and SEQ ID NO: 2. The method of claim 1, wherein genomic DNA is extracted from the plant transformed with the pRCV2 vector, digested with Pst I, and then ligation to rescue cloning. Kit for plant transformation and analysis, characterized in that consisting of the vector of claim 1 and the primers of SEQ ID NO: 1 to SEQ ID NO: 4.