JPH0690771A - Method fr introducing gene into umbelliferous plant - Google Patents

Abstract

PURPOSE:To introduce a gene into an umbelliferous plant to transform the plant by adding DNA to a protoplast of an Umbelliferae plant and applying electric pulse, etc., to the protoplast. CONSTITUTION:A DNA is added to a protoplast of an umbelliferous plant and electric pulse is applied to the protoplast or a cell fusing agent is added thereto to introduce a gene into the umbelliferous plant. A cell transformed by a saponin synthesizing gene can produce saponin.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the introduction of foreign genes into protoplasts of plants belonging to the family Asteraceae, and more particularly to a method for transforming the genus Bupleurum such as P. persicae. Bu
Pleurum spp. is one of the important medicinal plants that have been used for a long time as a crude drug having anti-inflammatory, antipyretic and analgesic effects. Preparations containing Mishima-Psycho are generally called so-called Psycho-agents, and are used as the main therapeutic agent for small intestinal diseases in Kampo (medical science).

[0002]

2. Description of the Related Art Generally, as a method for introducing a foreign gene into a plant cell, in addition to an indirect introduction method in which a gene is introduced via Agrobacterium, an electroporation method,
Direct introduction methods such as PEG method and PEG method are known. This direct introduction method is applicable to both monocotyledonous and dicotyledonous plants, and is also being used as a promoter expression analysis system. It is used for the purpose of expression analysis of cloned genes and preparation of transformants. The electroporation method is a method in which a high-strength electric field is applied to a permeable lipid bilayer to directly introduce DNA into cells. In the PEG method, protoplasts are converted into polyethylene glycol (PE
G) was incubated with DNA in the presence of
This is a method of directly introducing NA. As a practical example of the direct method, for example, there is a report on gene transfer by a direct transfer method into tobacco (M. Fromm, et al. P.
roc. Natl. Acad. Sci. US
A). On the other hand, as a method for introducing a gene into the genus Bupleurum by the indirect method, a method for inducing a transformed root by Agrobacterium of the present applicant (Japanese Patent Laid-Open No. 62-11169).
No. 6). As a premise of the present invention, it is necessary to establish a protoplast culture technique for agaricaceae such as the genus Bupleurum, but a protoplast culture technique for Mishima Psycho has been established by the present inventors (see JP-A-2-245180). .

[0003]

SUMMARY OF THE INVENTION In the indirect gene transfer method using Agrobacterium into plants such as Mishima Psyllico and other herbaceous plants, the disadvantage that a non-target gene such as T-DNA is also transferred, and There is a drawback that bacterial contamination occurs. In order to avoid this, it is desirable to directly introduce the gene, but a method for introducing the gene into the agaraceae plant by the direct method has not been known so far. Although there is an example in which the direct method is applied to tobacco, in general, different plant species have different culture methods and culture conditions, and it is not always possible to apply successful methods to other types. Although not bound by theory, the present inventor seems to be concerned with differences in cell membrane stability among plant species. Cervidae plants, such as the white-necked peach (Bupleurum), are one of the important medicinal plants that have long been used as crude drugs with anti-inflammatory, antipyretic and analgesic effects. Until now, there has been no example of successful introduction into a botanical plant, in particular, Mishima Psycho cells.

[0004]

As a result of repeated studies to solve the above problems, the present inventor has found a method for efficiently introducing a gene into a botanical plant and completed the present invention. The present invention provides a method for introducing a gene into cells of a serpentine plant using a direct introduction method of DNA. Also provided is a method for introducing a gene into a cucumber plant cell by adding DNA to a protoplast of a cetacean plant and applying an electric pulse or adding a cell fusion agent. Further, it provides a method for introducing a gene into a plant cell in the case where the arid plant is of the genus Bupleurum.

As the target ceriaceae, Anges
lica, Anethum, Anthriscus, A
Pium, Apodicarpum, Bupleuru
m, Carm, Caucalis, Centella,
Chamaele, Cicuta, Cnidium, C
oelopleurum, Conioselinum,
Coriandrum, Cryptotania, C
ryptotaeniopsis, Daucus, Fo
eniculum, Glenhnia, Heracleu
m, Hydrocotyl, Ligusticum,
Nothommyrnium, Osmorhiza, O
stericum, Pastinaca, Peuced
anum, Phellopterus, Pleuros
permum, Pimpinella, Pternop
etalum, Saniculla, Seseli, S
iler, Sium, Spuriopimpinell
a, Tilingia, Toris, etc. Of these, Bupleurum is particularly preferable. Particularly preferred B
The genus uplerum is the Pacific flesh (Bupleur
um falcatum L. ), B. chinese
DC. , B. scorzoneraefolium
Wild. , B. komarovianumLinc
z. , B. sibiricum Vest. , B. f
alcatum L. subsp. marginatu
m, C.I. B. Clarke, B .; longicaul
e Wall, Brongicaule Wall. v
ar. giralduWall, B .; tenueBuc
h. et Ham. , B. multipleveDC. ，
B. longeradiatum Turcz.

Isolation of protoplasts Callus derived from Saiko, preferably Mishima Saiko, suspension cultured cells or plant tissue is collected from Saiko by a conventional method, and cell wall degrading enzyme, in the presence of an osmotic agent,
Preferably, it is treated with cellulase or pectinase. As the osmotic pressure adjusting agent, mannitol, glucose or sodium chloride is preferable.

Suspension of Protoplasts The isolated protoplasts are suspended in a buffer. The buffer preferably contains a cation and an osmotic pressure adjusting agent. As the cation, a divalent cation, particularly Ca ²⁺ , M
g ²⁺ is preferred. As the osmotic pressure adjusting agent, mannitol, glucose or sodium chloride is preferable. Osmotic pressure is preferably adjusted to _{300-900 m Osm / kg H 2 0} .

[0008] The addition of DNA to be introduced to this introduction by fusion-inducing agent, the addition of a fusion inducing agent. Any kind of DNA may be used as the DNA to be introduced, but if a reporter gene such as CAT gene, GUS gene, beta-galactosidase gene is used, it can be confirmed in which tissue and when the confirmation of the gene introduction into the plant cell was expressed. Moreover, the expression level can be accurately examined quantitatively. Furthermore, genes related to the production of secondary metabolites such as saponin such as saponin synthesis gene can be used, and useful substances can be produced.

The fusion-inducing agent to be added is preferably PEG (polyethylene glycol), PVA (polyvinyl alcohol) or PL (poly-L-lysine), especially P.
EG is preferable, and PEG having an average molecular weight of 1,000 to 10,000 is preferable. The addition amount is DNA 1-10
00 mg / ml and PEG 5-30% (w / v) are preferred. Incubation is then preferably 1-
Do it for 60 minutes. Then, it wash | cleans and carries out protoplast culture.

Electroporation The isolated protoplasts were suspended in a buffer to give DN
Add A. The DNA to be added is as described above. Next, an electric pulse is applied. Preferred application conditions are a temperature of about 4 ° C., a time constant of about 2 ms, an initial electric field strength (E) of preferably about 500 to about 900 V / cm, and particularly preferably about 500 V / cm. Damping wave electric pulses are preferred. After the pulse application, the cells are left for about 10 minutes, and the cells are collected by centrifugation.

Determination of Gene Transfer Efficiency After introducing a reporter gene such as CAT gene or GUS gene into cells and culturing for a short period of time usually about 1-2 days, CAT activity and GUS activity are determined by a conventional method. Find out by. This shows the efficiency of gene transfer.

[0012]

EXAMPLES Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention. Example 1 Aseptically germinated and grown, B.
eurum falcatum L. ) 2,
LS agar medium containing 4-D 1 mg / l and kinetin 0.1 mg / l:

NH ₄ NO ₃ 1650, KH ₂ PO ₄ 170, NH ₄ NO ₃ 600, KNO ₃ 1900, CaCl ₂ .2H ₂ 0 440, MgSO ₄ .7H ₂ O 370, FeSO ₄ .7H ₂ O 27.8 , Na ₂ EDTA 37.3, H ₃ BO ₃ 6.2, MnSO ₄ · 4H ₂ 0 22.3, ZnSO ₄ · 7H ₂ O 8.6, Na ₂ MoO ₂ · 2H ₂ O 0.25 CuSO ₄ · 5H ₂ O 0.025, CoCl ₂ .6H ₂ 0 0.025, KI 0.83, myo-inositol 100, thiamine-HCl 0.4, (above unit mg / l) saccharose 30 g / l, 0.8% agar pH 5.8

After culturing on the above, callus induction was performed, and this was suspended in an LS liquid medium containing 2,4-D 0.5 mg / l and shake-cultured at 105 rpm and 27 ° C. This liquid suspension-cultured cell was subcultured by transferring 1/10 amount to a new medium every week. This 4-5th day was used as the material for protoplast isolation. As an osmotic pressure regulator,
2% of cellulase Onozuka RS (Yakult) and pectriase Y-23 in hormone-free protoplast medium (glucose free) containing 0.5 M mannitol.
(Sorijin Pharmaceutical Co., Ltd.) was dissolved in 0.1% and the cultured cells were added to the prepared enzyme solution, and the mixture was stored in the dark at 27 ° C. and 50 ° C.
Shake at rpm for about 5 hours. Then, it was filtered with an 82 μm nylon mesh to remove undigested cell mass and cell debris. The resulting protoplasts were buffered with buffer A:

1.65 mM CaCl ₂ , 5 mM M
After washing with ES {2- (N-morpholino) ethane sulphonic acid}, 530 mM mannitol, pH 5.8, it was used for gene transfer. Washed protoplasts with buffer B: 25 mM CaCl ₂ , 5 mM MES, 500 mM
Suspended in mannitol, pH 5.8 to 2 × 10 ⁶ cells / ml. To 1 ml of this suspension, 20 μg of CAT gene (pCaMVCN, manufactured by Pharmacia) and 50 μg of calf thymus DNA (manufactured by Sigma) were added. Next, F medium:

125 mM CaCl ₂ , 140 mM N
aCl, 5 mM KCL, 0.75 mM Na ₂ HPO ⁴ ,
5 mM glucose, pH 7.05

PEG solution dissolved in (PEG # 600
0, Nakarai Tesque) slowly add 0.5 ml,
After incubating for 30 minutes, dilute slowly with 5 ml of F medium, and further add 10 ml of protoplast medium.
Was added and washed, and the protoplasts were collected by centrifugation at 500 rpm. A part of the collected protoplasts is a medium for protoplasts:

NH ₄ NO ₃ 600, KNO ₃ 1900, CaCl ₂ · 2H ₂ 0 600, MgSO ₄ · 7H ₂ O 300, KH ₂ PO ₄ 170, KCl 300, FeSO ₄ · 7H ₂ O 28, Na ₂ EDTA 37 , H ₃ BO ₃ 3, MnSO ₄ .H ₂ 0 10, ZnSO ₄ .7H ₂ O 2, Na ² MoO ² .2H ₂ O 0.25 CuSO ₄ .5H ₂ O 0.025, CoCl ² .6H ₂ 0 0. 025, KI 0.75, inositol 100, nicotinic acid 1, pyridoxine-HCl 1, thiamine-HCl 10, calcium pantothenate 1, folic acid 0.4, p-aminobenzoic acid 0.02, biotin 0.01, choline chloride. 1, ascorbic acid 2, vitamin A 0.01 vitamin D ₃ 0.01 vitamin D ₁₂ 0.02, glycine 2 pyruvic Sodium 20, citric acid 40, sodium malate 40, fumarate 40, casein hydrolyzate 250, NAA 1.5mg / l, (or more units mg / ml) Glucose 30 g / l, mannitol 0.4 M, pH 5.8

After suspending in C and culturing for 2 days, C
Gene transfer efficiency was determined by measuring AT activity. The remaining protoplasts were embedded in a protoplast medium containing 1% low-melting point agarose to perform bead type culture, and after 2 weeks of culture, cell viability was determined by microscopic observation. The results are shown in Table 1. In Table 1,
The PEG concentration indicates the concentration during incubation, and C
The AT activity is expressed as a relative value per the same number of protoplasts, and the viability is shown as the ratio of cells dividing at 2 weeks of culture (the same applies to Tables 2 and 3). As shown in Table 1,
Good results were obtained for both CAT activity and survival rate. Example 2 5 mM KH2PO4 in F medium was added to 5 mM MES
Was carried out, the (Modified F Medium) PEG concentration was changed to 10-20%, and the other conditions were the same as in Example 1. The results are shown in Table 2. As shown in Table 2,
Good results were obtained for both CAT activity and survival rate.

Example 3 Protoplasts isolated as in Example 1 were suspended in buffer to 2 × 10 4 cells / ml and pCaMVC was prepared.
N 20 μg / ml, calf thymus DNA 50 μg / ml
Was added. Next, a decay wave electric pulse having a time constant of about 2 ms and an initial electric field intensity (E) of 500 to 900 V / cm was applied at 4 ° C. After application of the pulse, the cells were left for about 10 minutes, and the cells were collected by centrifugation. The cells recovered were obtained from Example 1.
The cells were cultured in the same manner as above, part of which was used for measuring CAT activity, and the rest was used for measuring cell viability. The results are shown in Table 3. As shown in Table 3, when E = 500, good results were obtained for both CAT activity and survival rate. At E = 600 or higher, CAT activity was good, but survival rate was 0.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, it was possible to efficiently introduce a gene into Mishima Psychoprotoplasts and transform it. Cells transformed with the saponin synthesis gene can be used for efficient saponin production. In addition, the plant body can be redifferentiated from the transformed cells to improve the breeding of Mishima Psycho.

Table 1 PEG concentration CAT activity Survival rate (%) (relative value) (%) 0 0.17 18 13 19 14 14 13 15 15

[Table 2] PEG concentration CAT activity survival rate (%) (relative value) (%) 0 0.17 16 10 20 27 13 13 18 23 17 17 18 22 20 19 19 19 Reference F medium case PEG concentration CAT activity survival rate (%) ) (Relative value) (%) 13 18 27

Table 3 E (V / cm) CAT activity Survival rate (relative value) (%) 0 0.17 18 500 2.3 18 600 3.6 0 700 700 4.7 0 800 3.6 0 900 100 0

Claims (1)

[Claims] 1. A method of introducing a gene into a cucumber plant cell by adding DNA to a protoplast of a cetacean plant and applying an electric pulse thereto or by adding a cell fusion agent.