JPH01269500A - Production of brassinosteroid through plant cultured cell - Google Patents

Abstract

PURPOSE:To obtain in place of chemical synthetic process, the title steroid having plant growth-regulating effect, useful as a drug for the medical industry, by culture of the crown gall cells in Dicotyledoneae followed by isolation and purification from the resulting cultured product. CONSTITUTION:The crown gall cells in Dicotyledoneae is put to culture, and the objective steroid is isolated from the resulting cultured product. It is suggested that said be produced by infecting the seedlings of Catharanthus roseus L. with plant oncogenic bacteria, taking the tumor site on an artificial culture medium, carrying out sterilizing treatment and growing through liquid culture.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing brassinosteroids by culturing plant cells.

[Background of the invention]

Brassinosteroid is a plant growth regulator with a steroid skeleton, and was recognized by the U.S. Department of Agriculture in 1979 as a plant growth regulator.
ve, Mandava et al. isolated brassinolide (1) from oilseed rape pollen in Myanmar.
The structure was determined 1). Subsequently, castasterone (2) was isolated from chestnut plants (2), as well as brassinolide and brassinolide from tea leaves, Istuki spp., Fuji pea immature seeds, French bean immature seeds, rice stems and leaves, corn pollen, etc. Various homologs of castasterone have been isolated one after another, and to date 22 types of homologs have been identified.

These congeners are also mistakenly referred to as brassinosteroids.

1) M, D, Grove, G, P, 5pe
ncer, K., W., Rohwedder, N.
Mandava, J.F., Worley, J.

D, l'1arthen Jr, G, L,
5teffens, J.L.

Flippen-Handerson, J. C., Co.
Ok Jr. Nature.

281, 216 (1979> 2) Yokota et al., Tetrahedron Lett.
23. 12753) Takao Yokota, Chemical Regulation of Plants, 22.10-From these research results, it has become clear that brassinosteroids are plant growth regulators produced by higher plants; The content in the body is extremely small at 1 μg to 100 μg/kg live weight.

As for its physiological effects on plants, it shows activity at a concentration of 0.000 lppm in the rice lamina joint test, and also exhibits an elongation-promoting effect on the hypocotyl epicotyl and hypocotyl cucumber.

Various congeners of brassinosteroids have been chemically synthesized, and samples for testing have become available 4), and their physiological effects on agricultural crops have now been investigated over a wide range of areas.

To date, the following effects have been confirmed.

In other words, yield increasing effect on wheat, corn, and cucumber, enhancement of cold tolerance on rice, cucumber, and eggplant,
It has been revealed that in addition to enhancing disease resistance against Chinese cabbage, it has useful effects such as increasing drug resistance and salt tolerance5).

4) Kenji Mori: Journal of the Society of Synthetic Organic Chemistry, 43.5) Fumio Fujita: Chemistry and Biology, 23.717 As mentioned above, the usefulness of brassinosteroids as agricultural chemicals is being demonstrated, but their As for the manufacturing method, chemical synthesis is currently the only method available. The chemical synthesis of brassinosteroids consists of a complex combination of chemical reactions.
Requires advanced technology such as separation of by-product isomers. Although it is now possible to prepare samples for testing, chemical synthesis is said to have limitations as a method for producing agricultural chemicals. Therefore, attempts have been made to prepare it through biological production, such as searching for microorganisms that produce brassinosteroids, but this has not yet been successful.

[Problem to be solved by the invention]

Therefore, an object of the present invention is to provide a new method for producing brassinosteroids that can be substituted for the chemical synthesis method described above.

[Means to solve the problem]

In the process of searching for the presence or absence of brassinosteroid production in various cultured plant cells, the present inventors discovered that one type of crown gall cell in dicotyledonous plants produces brassinolide and castasterone. I found it. The present invention was completed based on this discovery.

That is, the present invention is a method for producing brassinosteroids, which is characterized by culturing crown gall cells of dicotyledonous plants and isolating brassinosteroids from the culture.

The crown gall cells used in the present invention include dicotyledonous plants,
For example, it can be obtained from periwinkle, tobacco, Jerusalem artichoke, etc. An example of this is the periwinkle (Vinca ros).
ea) seedlings are infected with plant tumor pathogens, such as Agrobacterium tumefaciens (Agrobacterium tumefaciens).
Rium tumefaciens) A 208, the resulting tumor was placed on an artificial medium, sterilized, and then treated with Murashige-Skoog.
Examples include crown gall cells named VNC', which are obtained by proliferating in liquid culture in e-5koog (MS) medium.

The crown gall cells obtained in this way are cultured with shaking in MS medium, etc., and the proliferated cells are ground with a homogenizer, etc., extracted with methanol, chloroform, etc., and the crude extract is subjected to conventional methods such as chromatography. Separate and purify to obtain the desired brassinosteroid.

The present invention will be explained in detail below using examples.

Example (1) Preparation of crown gall cells and VNC' cells of periwinkle Agrobacterium tumefaciens
A 208 was cultured on nutrient broth agar for 16 hours, the resulting colonies were scraped off, and these were
It was applied to the stem of a periwinkle seedling (stem length 15-20 cm), where a 1-2 mm wound was made with a scalpel. The seedlings were grown for one month at 27-28°C in a glass greenhouse. A tumor, crown gall, approximately 1 cm in size occurred at the inoculation site.

This crown gall was cut out, the surface was sterilized with 10% Salashco solution, a section of about 3 mm square was taken from inside the tumor tissue, and antibiotics (200 mg/β-carbenin phosphorus and 10%
The cells were placed in an MS liquid medium with the following composition containing 0 mg/β vancomycin) and cultured with shaking (26°C, 10 Qrp
m, dark).

MS medium composition (Murashige-3koog medium)
(mg/ffl) MgSO4・7H20370 CaC12= 2H20440 KNO3190O NH4N○3 1650 KH2P○4 170 Fe S 04' 7820 27.8Na
2E DTA 37.3Mn S
04 ・4 H2C223 ZnSO4・ 7H208,6 CuS O= ・5 H2C0,024CoCβ2・
6H20' 0.025K I
0.83H3B
O36,2 NazMoO4・2 H2C0,25 Sucrose
30000 Myo-inositol 100 Nicotinic acid 0.5 Pyridoxine hydrochloride 0.5 Thiamine hydrochloride
0.1 Glycine 2 One week later, the sterilized tissue grown in the liquid medium was taken out and transplanted onto an MS agar medium prepared by adding 2% agar to the above MS medium. The cultured cells thus obtained, V2O
3 grew rapidly on MS agar and was subcultured every 20 days on the same agar. V2O3 cells were transplanted into MS liquid medium, cultured with shaking, and subcultured every week.
VNC' cells were obtained as cells suspended in liquid.

V2O3 cells were subcultured on agar medium every 20 days.
It has been stored for years and was transferred to liquid culture 4 years after preparation. While transferring to liquid culture, subculture every week 4
Completely suspended cells in liquid were obtained in the second generation.

It was confirmed that the brassinosteroid production function of these VNC' cells was stable and reproducible even after one year of continuous subculturing.

(2) Culture of VNC' cells The VNC' cells thus obtained were cultured at 150-M
500mf including S medium! Using 4 Erlenmeyer flasks,
Preculture was performed at 27°C for 8 days using a shaking method (100 rpm/ml). From this preculture, each 151n1. The culture solution was transferred to 24 500 m 13 square flasks containing 150 ml of MS medium, and cultured with shaking at 27°C for 12 days (10
0 rpm/m1n). After harvesting, the culture solution was filtered to obtain 1059 g of cells.

(3) Extraction of brassinosteroid fraction 41 methanol was added to 1059 g of cells, and the cells were ground with a homogenizer and extracted. After filtering the methanol extract,
The residue was re-extracted with 4β methanol and filtered to obtain a total of 8β methanol extract.

This extract was concentrated under reduced pressure to approximately 1 [) Oml! This was extracted three times with 53 mg of chloroform. The chloroform extract was combined and chloroform was distilled off under reduced pressure, and the resulting residue was dissolved in 50 volumes of hexane. The hexane solution was extracted three times with 50 ml of 85% methanol,
The 5% methanol extracts were collected and concentrated under reduced pressure. This concentrate was dissolved in 50 mg of ethyl acetate, and the ethyl acetate solution was washed three times with 20 volumes of 0.2M K2HPO4.
After dehydrating the ethyl acetate fraction with anhydrous sodium sulfate, it was concentrated under reduced pressure to obtain a Franz steroid (BS) fraction of 480 m
I got g.

This BS classification was determined in the rice lamina point test.
When an amount equivalent to 7 g of VNC' cell live weight was used, as shown in FIG. 1, the activity was equivalent to 0.0005 ppm of Franz steroid.

Good lamina joint test (Rice leaf blade bending test)
was done as follows.

1) Rice (Koshihikari) seeds were stored at 28-30℃ for 2 days.
Soak in water and germinate.

2) Sow the germinated seeds in a mesh hunt, soak the mesh hunt in water, and incubate at 28 to 30°C in the dark for 6 to 7 days.
Hydroponically cultivated for days.

3) From the depleted rice seedlings obtained in this way, select a seedling with a straight and uniform leaf blade that has not yet produced the third leaf, and cut off the leaf blade and leaf sheath approximately 1 cm above and below the lamina joint of the second leaf. .

4) Float this rice section in distilled water at 28-30°C.
When placed under dark conditions, a slight bend appears between the leaf blade and leaf sheath after 24 hours. A section bent at an angle of about 30 to 40 degrees is selected from among these and used for the test.

5) Put the test substance into a 2.5 cm diameter Zambre tube or Nyare, dissolve it in one bottle of 2.5 mM potassium maleate buffer, float 10 of the above sections, and place 28-3
Place in the dark at 0°C for 48 hours.

6) Take out the rice section and measure the resulting leaf blade inclination angle with a protractor.

(References: IE, Maeda, Phys'i
o'l, Plant.

(4) Purification of Brassinosteroids Purification of brassinosteroid active substances from the BS section was carried out as shown in the following scheme.

First, VNC' cell live weight 30'g phase divisible sample 4'8
0"mg was subjected to silica gel column chromatography (Merck, 5g), and each fraction of 1"5mff was collected while increasing the methanol concentration of chloroform/methanol, and the activity of each fraction was measured by rice lamina joint test. . The results are shown in Figure 2.

This chloroform/methanol 4'''/96~7/9
3 fraction (1'6 mg') was added to Cef'7denox L''
Column chromatography with H""20 [bed volume 2007, developing solvent methanol/dichloroform = 4/1 (v/V)] was applied to □, and 15 ml aliquots were collected for each column, and the activity of each column was determined. was measured by the rice liner joint test. The results are shown in Figure 3.

This relative elution amount (V'e',"V't) O,'6""
25-0.7”75 fraction (34 mg) was treated with activated charcoal (
Methanol/water (40/6
0 to 10010), then eluted with methanol/chloroform (9/1 to 1/9), aliquots of 15 mp each were collected, and the activity of each aliquot was measured by the rice lamina joint test. The results are shown in Figure 4.

This methanol/water elution fraction (1'Omg) was again subjected to activated carbon adsorption column chromatography (Nacalai, 60-1
50 mesh, 230 mg), methanol/water (
40/60 to 10010), then eluted with methanol/chloroform (9/1 to 1/9), and the X fraction 1'5F
The activity of each aliquot was measured by the Inelamib Beyond India Test.

The results are shown in Figure 5.

This methanol/chloroform (9/1-515) elution fraction (2.5 mg') and the methanol/chloroform (9/1-5/'5') elution fraction shown in Figure 4.
(3.4 mg of "'1") were combined, dissolved in methanol, and filtered through a Niodesox 6T ED-13CR filter unit to obtain an active fraction (13.8 mg).

This active fraction was subjected to high performance liquid chromatography (Senshu Pack 0DS-3251-D, developing solvent acetonitrile/water-45155, flow rate lrd/min), and the activity of each fraction was measured by the rice lamina joint test.
tR (retention time) 16-18 minutes activity division Δ and t, 24
An active segment B of ˜27 minutes was obtained. The results are shown in Figure 6. The retention times of standard bulla/nosteroids (driphrite, planolide, homodrifsterone, casksterone, homobrassinolide, and ethylbrassinone) are also shown. This revealed that VNC' cells produce at least two types of planosteroids.

(5) Identification of brassinosteroids Activity category A (tR16-18 minutes) and activity category B (tR2
4 to 27 minutes> respectively in methanoboronic acid and pyridine to obtain methanoborate derivatives, and then subjected to a gas chromatography mass spectrometer (GC-MS, JASC
ODX-303).

Analysis conditions: Column, DB-1 silica capillary column (0.25 mm x 15 m) Column temperature, 17
After 2 minutes, the temperature was increased to 275°C (32°C/min), and then the temperature was constant at 275°C; inlet temperature, 2
90℃.

For activity category A, a peak was observed at t, 21.24 minutes (7th
It was identified as brassinosteroid from its mass spectrum (Figure 8). For active category B, a peak was observed at 17.50 minutes (Fig. 9), and its mass spectrum (10th
It was identified as castasterone from Figure).

〔Effect of the invention〕

According to the present invention, brassinosteroids having useful physiological activity can be mass-produced at low cost.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the rice lamina joint test results for brassinosteroid classification, FIGS. 2 to 6 are graphs showing the elution curve of brassinosteroid, and FIG.
The figure shows the gas chromatogram for the active section, Fig. 8 shows the mass spectrum of the peak at minute 2124 in Fig. 7 (brassinolide), Fig. 9 shows the gas chromatogram for the active section B, and Fig. 9 shows the gas chromatogram for the active section B.
Figure 0 shows the mass spectrum (castasterone) of the peak at 17.50 minutes in Figure 9. JP 1-269500 (1,1)

Claims (1)

[Claims] A method for producing brassinosteroids, which comprises culturing crown gall cells of dicotyledonous plants and isolating brassinosteroids from the culture.