JP2001275667A - Method of enhancing gene-transferring efficiency for monocotyledon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for enhancing the transferring efficiency of an objective gene for a monocotyledon that is important as a food resource for human being upon the gene transfer by the genetic engineering technique. SOLUTION: This method for enhancing a gene-transferring efficiency for a monocotyledon (e.g. Oryza sativa) in obtaining a tissue of plant body of transformed plant comprises transferring a cytokinin-related gene (e.g. ipt = isopenenyltransferase) into the monocotyledon together with the objective gene followed by making the monocotyledonous cell grow, or grow and redifferentiate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for improving the efficiency of introducing a target gene when a transformant is prepared by introducing the target gene into a monocotyledonous plant by a genetic engineering technique.

[0002]

2. Description of the Related Art Gene transfer into plants using genetic engineering technology makes it possible to directly introduce a target gene into a target plant. Therefore, (a) only a trait to be modified can be introduced. (B) traits of species other than plants (microorganisms, etc.) can be introduced into plants, and (c) breeding period can be significantly shortened. are doing. Therefore, its application is expected to bring about dramatic progress in plant breeding, and research is progressing mainly in Europe and the United States. As a result, many useful transformed plants have been produced and are now on the market.

[0003] Specifically, in order to introduce a target gene into a target plant to prepare a transformed plant, (1) introduction of the target gene into a plant cell (including a case where the target gene is introduced into a chromosome, a nucleus, or the like); (2) Selection of a plant tissue consisting only of cells into which the target gene has been introduced, and (3) Regeneration of a plant from the selected plant tissue. However, the target gene-introduced tissue obtained through such a process,
Alternatively, in many cases, the target gene-introduced individual does not necessarily exhibit the expected properties and / or level of transformation by the target gene transfer. With the current technology, even though the target gene can be introduced into the plant chromosome, it is difficult to control it so that it can be introduced into the appropriate position on the chromosome in an appropriate number, and this is uncertainty. Is one of the causes.

Therefore, in order to obtain a target gene-introduced tissue / individual exhibiting expected properties at an expected level, a number of target gene-introduced tissues / individuals must be prepared and selected. Under such circumstances, a method for obtaining as many target gene-transfected tissues as possible from the tissue after the gene transfer treatment, that is, a method for improving the target gene transfer efficiency, is required. In terms of these preparation methods, gene introduction conditions, selection conditions for target gene-introduced tissues, regeneration conditions, etc.,
Various studies have been made.

[0005]

SUMMARY OF THE INVENTION Therefore, the present invention
An object of the present invention is to improve the efficiency of gene transfer in monocotyledonous plants including crop varieties such as rice, corn, and wheat, which are important as human food resources, when the genes are transferred by genetic engineering techniques.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that when a gene is introduced into a monocotyledonous plant, the cytokinin-related gene is introduced into the same cell together with the gene of interest. It has been found that the introduction efficiency of the compound is greatly improved, and the present invention has been completed.

[0007] That is, the object of the present invention is achieved by introducing a cytokinin-related gene into a monocotyledonous plant cell together with a target gene.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, genes capable of imparting industrially excellent traits, and not necessarily industrially excellent traits, include genes required for studying gene expression mechanisms. Various genes can be selected and used as target genes.

[0009] The cytokinin-related gene used in the present invention is a gene that acts to increase the effect of cytokinin in the introduced plant cells. Examples of such a gene include ip, which is a cytokinin synthesis gene derived from Agrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens).
t gene (ACSmigocki, LDOwens, Proc. Natl. Acad.
Sci. USA 85: 5131, 1988), β-glucuronidase from Escherichia coli, a gene that activates inactive cytokinins
Genes (Morten Joersbo and Finn T. Okkels, Plant Ce
ll Reports 16: 219-221, 1996), CKI derived from Arabidopsis thaliana and considered to be a cytokinin receptor gene
One gene (Kakimoto T. Science 274: 982-985, 1996) and the like are typical. Among them, the ipt gene used in the examples of the present application is the most well-known gene whose function has been elucidated.

[0010] These target genes and cytokinin-related genes are introduced into the same monocot plant cell. There is no particular limitation on the method of introduction. Generally, these are incorporated into an appropriate vector, and then geminivirus, brom mosaic virus, A. Claws,
Physical and chemical methods such as microinjection method, electroporation method, polyethylene glycol method, cell fusion method, high-speed ballistic penetration method, etc. indirectly via viruses and bacteria such as Agrobacterium rhizogenes Is introduced directly (I. Potrykus, Annu. Rev. Plant Physio
l.Plant Mol. Biol., 42: 205, 1991). By the way, conventionally, Agrobacterium does not infect monocotyledonous plants,
It has been said that it cannot be used for gene transfer to monocotyledonous plants because it infects only dicotyledonous plants.In recent years, however, gene transfer to monocotyledonous plants using such Agrobacterium has been carried out. (For example, see International Publication WO94 / 00977).

The target gene and the cytokinin-related gene may be integrated on the same vector or on different vectors as long as they are introduced into the same cell. Alternatively, a selectable marker gene may be incorporated into the vector into which the target gene has been incorporated, and the expression of the selectable marker gene may be used as an index for introducing the target gene into the target plant cell. However, cytokinin-related genes, when expressed in plant cells,
It upsets the growth and differentiation of the plant cells and causes morphological abnormalities such as redifferentiation of adventitious buds and formation of polyblasts. Therefore, if the target gene and the cytokinin-related gene are integrated on the same vector and introduced into a plant cell, the target gene can be introduced using the morphological abnormality caused by the expression of the cytokinin-related gene as an index without using a selection marker gene. In some such cases, it is not necessary to use a selectable marker gene. In any case, the target gene, cytokinin-related gene and / or
Alternatively, when the selectable marker genes are integrated on the same vector, it is necessary to arrange them so that the presence of any of the genes does not inhibit the expression of other genes.

Generally, in order to produce a protein such as an enzyme from a gene, in addition to the structural gene (hereinafter also referred to simply as "gene" in the present application) sequence encoding the amino acid sequence information, the promoter of the structural gene is used. Regulatory sequences such as (expression initiation sequence) and terminator (expression termination sequence) are required. In the present invention, as long as it functions in the plant, such regulatory sequences can be used together with the above-mentioned target gene and cytokinin-related gene. Can be used without restrictions. For example, as a promoter, a cauliflower mosaic virus 35S promoter (JTOdell
et al., Nature (London), 313: 810, 1985), the nopaline synthase promoter (WHRLangridge et al.,
Plant Cell Rep., 4: 355, 1985), as well as a promoter of a glutathione-S-transferase I gene, which is a chemical-inducible promoter (Japanese Patent Laid-Open No. 5-268).
965), glutathione-S-transferase II
(GST-II) gene promoter (International Publication W
O93 / 01294), Tet repressor fusion type cauliflower mosaic virus 35S (CaMV35)
S) promoter (C. Gatz et al., Mol. Gen. Genet., 2
27: 229, 1991), Lac operator / repressor promoter (R. Wilde et al., The EMBO Journal, 1
1: 1251, 1992), alcR / alcA promoter (International Publication WO94 / 03619), glucocorticoid promoter (Takashi Aoyama, protein nucleic acid enzyme, 41: 2559, 1996), par promoter (T. Sakai)
et al., Plant Cell Physiol., 37: 906, 1996) and the light-inducible promoter ribulose 2-phosphate carboxylase small subunit such as the heat-inducible promoter hsp80 promoter (JP-A-5-276951). (rbcS) gene promoter (R.Fluhr et
USA, 83: 2358, 1986), fructose-1,6-bisphosphatase gene promoter (Japanese Patent Publication No. 7-501921), light-harvesting chlorophyll a / b binding Various inducible promoters such as a protein gene promoter (JP-A-5-89) can be used. On the other hand, the terminator sequence includes a polyadenylation signal of nopaline synthase (A.
picker et al., J. Mol. Appl. Gen., 1: 561, 1982), polyadenylation signal of octopine synthase (J. Gielen
et al., EMBO J., 3: 835, 1984) and the like.

The above genes and regulatory sequences used in the present invention can be obtained by cloning cDNA or genomic DNA. If the sequence is known in advance, it may be obtained by chemical synthesis.

The target gene-introduced tissue is grown by culturing the plant cells into which the target gene and the cytokinin-related gene and the like have been introduced as described above under appropriate conditions according to the type of the plant, or It can be obtained by expansion and redifferentiation. For example, when a commonly used hygromycin resistance gene (HPT gene) is used as a selection marker gene to perform gene transfer into rice callus cells, the cells after the gene transfer operation are treated with sucrose at 1 to 60 g / l. , 2.4 as a plant hormone
-D6 containing hygromycin containing 1 to 5 mg / l
Medium (CCChu, Proc. Symp. Plant Tissue Culture, Sci.
ence Press Peking, pp. 43-50, 1978) to proliferate the cells into which the gene of interest has been introduced, select a tissue consisting of such cells, and then sucrose 1-60 g of sucrose. / L, NAA as plant hormone
0.01 to 5 mg / l and BAP 0.01 to 10 mg / l
1. Modified MS medium containing hygromycin (MS basic medium (T. Murashige and F. skoog, Physio) containing 1 to 60 g / l of a sugar alcohol such as sorbitol as an osmotic pressure regulator
l.Plant., 15: 473, 1962)) 0.1-3 g of casamino acid
/ L is added) to regenerate the bud into which the target gene has been introduced. Furthermore, in order to obtain a target gene-transferred individual, the bud into which the target gene has been introduced is redifferentiated, and this is cultured in a known rooting medium to differentiate the root.

The present invention is directed to monocotyledonous plants. Any type of monocotyledonous plant can be used. For example, rice,
The present invention can be applied to corn, barley, wheat, asparagus, and the like.

In the present invention, since a cytokinin-related gene is introduced into a plant cell together with a target gene, the resulting transformant may exhibit an abnormal form such as a polyblast due to the effect of the cytokinin-related gene. is there. However, such a problem can be circumvented by using an inducible promoter as the promoter of the cytokinin-related gene and controlling the expression of this gene, and in fact, the glucocorticoid system, which is an inducible promoter, is used as the promoter. After the introduction of the ipt gene using a promoter into tobacco and cabbage, it is possible to differentiate the tissue that finally shows a normal morphology from the polyblasts once formed in these tissues by controlling the expression. It has been reported that it was possible (T. Kunkel et al., Nature Biotech., 17:91
6, 1999). Furthermore, as disclosed in JP-A-9-154580, if this cytokinin-related gene is used in combination with a transposon, a site-specific recombination system, or the like, it can be removed after introduction of the target gene. Problems can be solved fundamentally.

[0017]

[Function] Cytokinin, a plant hormone, is known to promote plant cell division. Also, ipt
It has also been known that introduction of a cytokinin-related gene such as a gene into a plant chromosome promotes division and differentiation of the plant cell.

However, if the function of the cytokinin-related gene merely promotes the division or differentiation of the plant cell, the gene of interest is introduced when this gene is introduced into the plant cell together with the gene of interest. Although the growth and regeneration rate of cells and tissues may be improved, the efficiency of target gene transfer itself should not be improved.

On the other hand, the present inventors have found that when a cytokinin-related gene is introduced into a monocot plant cell together with a target gene, the transfer efficiency of the target gene is greatly improved. The reason for this is not clear. However, it is thought that the cytokinin-related gene's ability to promote cell division is involved in some way.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on embodiments. In the following examples, more detailed experimental procedures for genetic engineering techniques, unless otherwise stated,
Molecular Cloning Second Edition (Sambrook et al.e
ds., Cold Spring Harbar Laboratory Pres, New Yor
k, 1989), or according to the manufacturer's instructions.

Example 1 I. Creating plasmid pIPT5HM plasmid pNPI123 from (JP-A 9-80821), cut ipt gene linked to the CaMV35S promoter with restriction enzyme Hind III, plasmid pIG121Hm (Nakamura et al., Modern chemical Extra Edition, plant biotechnology II, Pp.123 -132, 1991) Hind II
Plasmid pIPT5Hm inserted into the I restriction enzyme site and used as a gene transfer vector in this example
I got This plasmid pIPT5Hm is used for E. coli ( Es
cherichia coli ) JM109 strain, and this E. coli was coli JM109 (pIPT5Hm)
Deposited in Japan (Accession number: FERM P-1779)
No. 0).

A scheme for preparing pIPT5Hm and its pI
FIG. 1 shows a restriction enzyme map of a region (T-DNA region) to be integrated into a plant chromosome in PT5Hm. In FIG. 1, NOS-P is a promoter of nopaline synthase, NPTII is a kanamycin resistance gene, T is a polyadenylation signal of nopaline synthase, 35S
-P is CaMV35S promoter, circled T is i
polyadenylation signal of pt gene itself, intGU
S is β-glucuronidase (GU containing an intron)
S) Gene and small black triangle are T-DN, respectively.
The RB site and the LB site that define the A region are shown.

As is clear from FIG. 1, this plasmid contains a GUS gene having an intron inserted therein as a model of a target gene, an ipt gene which is a cytokinin-related gene, and an NPTII gene as a selection marker gene in the T-DNA region. It has the HPT gene.
Therefore, when a gene is introduced into a plant using this plasmid, the ipt gene, the target gene, and the selection marker gene are introduced into the same plant chromosome. The GUS gene is a gene widely used for analysis of gene expression in plants, since cells having the gene in the chromosome metabolize a special substrate to produce a blue pigment. The purpose of inserting the intron was to suppress the expression of this gene in Agrobacterium, which mediates introduction into plants,
This is for stably expressing this in the introduced plant cells.

II. PIPT5 for Agrobacterium
Introduction of Hm Tumefaciens EHA105 strain was added to 10 ml of Y
EB liquid medium (beef extract 5 g / l, yeast extract 1 g)
/ L, peptone 1 g / l, sucrose 5 g / l, 2 mM M
gSO ₄ , pH 7.2 at 22 ° C. (hereinafter, unless otherwise specified, pH at 22 ° C.)) and inoculate OD.
Cultured at 28 ° C. until ₆₃₀ ranged from 0.4 to 0.6. The culture was centrifuged at 6900 × g at 4 ° C. for 10 minutes to collect the cells, and then the cells were cultivated in 20 ml of 10 mM HEPES.
(PH 8.0), and again 6900 × g at 4 ° C.
The cells are collected by centrifugation for 10 minutes, and the cells
Was suspended in a YEB liquid medium and used as a bacterial solution for plasmid introduction.

Plasmid pIP into Agrobacterium
T5Hm was introduced by an electroporation method. That is, by applying an electric pulse of 2.5 kV, 25 μF, and 200 Ω at a distance of 0.2 cm between the electrodes to 50 μl of the plasmid-introduced bacterial solution mixed with 3 μg of pIPT5Hm prepared in I, pIPT5Hm was transformed into Agrobacterium. (Bio-Rad GENE
Use PULSER II. ). The cells after the electroporation operation were added to 200 μl of YEB liquid medium, cultured at 25 ° C. for 1 hour with shaking, and then added to 50 mg / l kanamycin-added YEB solid medium (agar 1.5 w / v%).
, And cultured at 28 ° C for 2 days to obtain pIPT.
5Hm-introduced strains were selected. The final confirmation of the introduction of pIPT5Hm was performed by cutting a plasmid extracted from the selected strain by an alkaline method with a restriction enzyme and comparing the electrophoresis patterns of the cut fragments.

III. Introduction and introduction of pIPT5Hm into rice
PIPT5Hm introduction A. that were selected in the analysis II of fine gene transfer organization Tumefaciens was cultured again in YEB solid medium at 28 ° C. for 2 days, then in YEB liquid medium at 25 ° C. and 180 rpm overnight, and centrifuged at 300 rpm for 20 minutes to collect the cells. Syringon 10mg / l, 2.4-
An Agrobacterium for infection was suspended in an N6 liquid medium containing 2 mg / l of D and 30 g / l of sucrose so that OD ₆₀₀ = 0.1.

On the other hand, as a material to be introduced with pIPT5Hm, callus derived from rice scutellum was used. In other words, according to “Cell Engineering Separate Volume, Plant Cell Engineering Series 4, Experimental Protocol for Model Plants” (pp.93-98), calli were induced from the scutellum using rice seeds as a material, and this was placed in a cylinder with a mesh. Immerse in Agrobacterium for infection for 3 minutes and introduce pIPT5Hm. PIPT5Hm was introduced into rice cells by infecting Tumefaciens.

After the above treatment, the callus is placed on a filter paper together with the mesh cylinder to remove excess water, taken out of the cylinder, and treated with N6 containing 10 mg / l acetosyringone and 30 g / l sucrose. Solid medium (Gellite 4g)
/ L) and cultured for 2 days in the dark at 28 ° C.
After washing with N6 liquid medium containing carbenicillin 500 mg / l, N6 solid medium containing carbenicillin 500 mg / l, hygromycin 50 mg / l, 2.4-D2 mg / l, and sucrose 30 g / l (Gerlite 4 g
/ L) and culture was observed two months later.

As a result, pIPT5Hm was introduced. Callus (2-4 mm in size) after treatment with Tumefaciens infection 1
New callus growth was observed in 17 out of 02,
These calli were shown to be hygromycin resistant. Furthermore, when GUS activity was examined by selecting a part of each of these 17 calli, the activity was detected in 6 calli, and these calli showed pIP
It was confirmed that the target gene was introduced by T5Hm. Table 1 shows the results.

IV. Regeneration of buds and the hygromycin-resistant callus obtained in Analysis III were cultured again in the same hygromycin-added N6 solid medium, and then carbenicillin 250 mg /
1, hygromycin 50 mg / l, NAA 1 mg /
l, BAP 2 mg / l, sorbitol 30 g / l, casamino acid 2 g / l, MS solid medium (4 g / l gellite) containing sucrose 30 g / l, to induce regeneration of buds. As a result, buds were regenerated from 11 calli, and buds showing GUS activity could be obtained from 9 of them. Table 1 shows the results.

Comparative Example 1 Transfection of rice, analysis of transgenic tissue, regeneration of buds in the same manner as in Example 1 except that plasmid pIG121Hm (see FIG. 1) was used as a gene transfer vector. And analyzed.

As a result, pIG121Hm was introduced. Callus after treatment with Tumefaciens infection (2-4 mm in size)
For 86, the number of the obtained hygromycin-resistant calli was 3, and the number of GUS-active calli was 2. In addition, among the hygromycin-resistant calli obtained here, the buds were redifferentiated in two, and both of them regenerated the buds showing GUS activity.

As is clear from FIG. 1, this pIG12
1Hm contains the target gene (intGU) in the T-DNA region.
S gene) and selectable marker genes (NPTII gene and HPT gene), but not cytokinin-related gene ( ipt gene). Therefore, when a gene is introduced into a plant using this plasmid, only the target gene and the selectable marker gene are introduced into the same plant chromosome. Table 1 shows the results.

Example 2 I. Creating plasmid pGSTIPT3 plasmid pNPI300 from (JP-A 9-80821), using the restriction enzyme Xba 1 and Hind III GST
-II-27 promoter was excised and plasmid pUC
Xba 1 -Hind I of 18 (purchased from Takara Shuzo Co., Ltd.)
The plasmid pUC-G is inserted between the II restriction sites.
Give the STP, the Bam HI restriction enzyme site of the pUC-GSTP, restricted from plasmid pNPI123 enzyme Ba
Insert the ipt gene were cut at m HI, resulting in plasmid pUC-GSTPIPT1.

Next, the obtained pUC-GSTPIPT
1 and a Hind III linker were inserted into the Sma I restriction enzyme site to prepare plasmid pUC-GSTPIPT2. From this pUC-GSTPIPT2, GST-II
Excised ipt gene linked to the -27 promoter with restriction enzyme Hind III, which plasmid pIG
Inserting into the Hind III restriction enzyme site of 121Hm,
In this example, a plasmid pGSTIPT3 used as a vector for gene introduction was obtained.

The creation scheme of pGSTIPT3 is shown in FIGS.
FIG. 4 shows a restriction map of the T-DNA region of pGSTIPT3. The meanings of the symbols in the figure are the same as those in FIG.

In this plasmid, the GST-II-27 promoter used as a promoter of the ipt gene was a promoter isolated from maize, which is a monocotyledonous plant like rice, and is known as a chemical-inducible promoter. However, it has been confirmed by the present inventors that it is also activated in callus and causes the expression of genes under its control.

This plasmid pGSTIPT3 was introduced into Escherichia coli JM109 strain, and this E. coli was transformed into E. coli. coli JM109 (pGSTIPT3)
(Accession number: FERM P-
17791).

II. Introduction of pGSTIPT3 to rice,
Analysis of transgenic tissue and redifferentiation of buds Using the plasmid pGSTIPT3 prepared in Analysis I as a vector for transfection, gene transfection, analysis of transgenic tissue, and redifferentiation of buds in the same manner as in II to IV of Example 2. And analyzed.

As a result, pGSTIPT3 was introduced. Callus after treatment with Tumefaciens infection (2-4 mm in size)
Of the 52 cells, 12 obtained hygromycin-resistant calli and 8 GUS-active calli. In addition, among the hygromycin-resistant calli obtained here, the buds were redifferentiated in six, and all of them regenerated the buds showing GUS activity. Table 1 shows the results.

[0041]

[Table 1]

As is clear from Table 1, together with the cytokinin-related gene ( ipt gene) and the target gene (GUS
By introducing the gene into rice callus, the efficiency of introduction of the target gene (GUS active callus / gene-transferred callus × 100, compared with the case of introducing the target gene alone)
Alternatively, callus obtained by redifferentiating GUS-active buds / gene-transduced callus × 100) was improved from 2 times to nearly 7 times.

[0043]

According to the present invention, the efficiency of introducing a target gene into monocotyledonous plants is greatly improved. For this reason, according to the present invention, since many target gene-transfected tissues and target gene-transferred individuals can be obtained from the tissue after the gene transfer treatment, a transformant exhibiting the expected properties at the expected level can be obtained using these target genes. The possibility of existence in the introducing organization etc. also increases.

Therefore, according to the present invention, for monocotyledonous plants including crop varieties such as rice, maize and wheat which are important as human food resources, a transformant with further improved usefulness can be efficiently produced. can do.

[Brief description of the drawings]

FIG. 1 is a diagram showing a pIPT5Hm creation scheme.

[Fig. 2] Among pGSTIPT3 creation schemes, pUC-
FIG. 4 is a diagram showing up to creation of GSTPIPT1.

[FIG. 3] Among pGSTIPT3 creation schemes, pUC-
It is a figure which shows until GUCPIPT1 to creation of pUC-GSTPIPT2.

[FIG. 4] Among pGSTIPT3 creation schemes, pUC-
It is a figure which shows from GSTPIPT2 to completion of pGSTIPT3.

Continuation of front page (72) Inventor Hiroyasu Ebinuma 5-21-1, Oji, Kita-ku, Tokyo Nippon Paper Industries Central Research Laboratory F-term (reference) 2B030 AA02 AB04 AD08 CA06 CA17 CA19 CB03 CD13 CD17 4B024 AA08 BA80 CA04 DA01 DA05 EA10 GA11 HA01 4B065 AA11X AA26X AA99Y AB01 BA02 CA53

Claims (4)

[Claims] 1. A method for obtaining a transgenic monocotyledon, which comprises introducing a cytokinin-related gene into a cell of the monocotyledon together with a gene of interest upon obtaining a transformed monocotyledon tissue or individual. How to improve. 2. The monocotyledon plant gene transfer efficiency according to claim 1, wherein the cytokinin-related gene is introduced into the monocotyledon plant cell together with the target gene, and then the monocotyledon plant cell is grown or proliferated and redifferentiated. How to improve. 3. The method for improving gene transfer efficiency of a monocotyledonous plant according to claim 1, wherein the method is performed on rice among monocotyledonous plants. 4. ipt as cytokinin-related gene
The method for improving gene transfer efficiency of a monocotyledonous plant according to claim 1, 2 or 3, wherein the (isopentenyltransferase) gene is used.