JPH0436183A - Production of mutant having genetic marker and differing in genetic nature from parental strain - Google Patents

Abstract

PURPOSE:To obtain the title mutant for breeding, etc., by mutual mating of siitake mushroom mycelia with (respective) genetic marker(s) followed by culture of the resulting binuclear mycelia and by separating and incubating basidiospores from the resultant basidiocarps to effect reducing the risk of the death of genetic marker strain. CONSTITUTION:Mononuclear mycelia of siitake mushroom is either made into protoplast or subjected to a mixer together with an appropriate amount of sterilized water into mycelia pieces each made up of one to several cells. The product is then irradiated with ultraviolet rays or treated with a chemical into genetic marker strain. Thence, mutual mating or cell fusion is made between two kinds of siitake mushroom mycelia with said genetic markers respectively or such two kinds of mycelia with one of them having said genetic marker, and the resulting binuclear mycelia is incubated using the fungal bed of a mushroom medium such as material wood, and basidiospores are separated from the resulting basidiocarps and incubated, thus obtaining the objective mutant.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is used for breeding of shiitake mushrooms, etc., and produces a large number of mutant strains having genetic markers and different genetic properties from their parent strains at once. Regarding the method.

(Conventional technology) Recently, with the development of biotechnology, research on cell fusion is being conducted as a technique for improving mushroom varieties.

In particular, there have been no reports that the fruiting bodies of shiitake mushrooms, which are highly valued as food, can be formed through cell fusion, and cell fusion technology has not been applied to breed improvement.

In order to advance cell fusion within one species, it is necessary to create a strain with genetic markers to enable selection of a fused strain.

In order to create a genetic marker strain, the target hyphae are converted into protoplasts or into hyphal pieces of one to several cells.
Methods of irradiating these protoplasts or hyphal pieces with ultraviolet rays or treating them with chemicals have been used.

However, with ultraviolet irradiation or drug treatment, protoplasts (viable cells) that are not killed but regenerated have only the target metabolic system completely inhibited, and the other parts are normal (in other words, there is a probability that they can be used as a genetic marker strain). The number of surviving cells is extremely small, at most 1 out of every 10 to 10 cells, and it has been extremely difficult to obtain a large amount of genetic marker strains containing the desired genetic elements.

There is also the problem of reverse mutations, and a method to create strains with stable genetic markers has been strongly desired.

[Spy H that invention tries to solve]

Therefore, an object of the present invention is to provide a method for producing a large number of shiitake mushroom mutant strains that have stable genetic markers and have different genetic properties from their parent strains, with little effort and with certainty.

[Means to solve problems]

The present invention is characterized by mating or cell fusion of shiitake mushroom hyphae with genetic markers on both or one side, culturing the resulting dinuclear mycelia, and separating and culturing basidiospores from the fruiting bodies that have developed. This method creates mutant strains that have genetic markers that differ from their parent strains in genetic properties.

The genetic marker referred to in the present invention refers to a physiological characteristic marker (auxotrophy, drug resistance, etc.) in mycelium.

Examples of the shiitake mycelia used in the present invention include Kanebo's preserved strains KBL-08 (Feiko Kenzonoyori No. 11436), KBL-14 (Feiko Kenzonoyori No. 11435), and the like. Shiitake mushroom hyphae are not limited to these, and may be used as long as they can form fruiting bodies, but the above-mentioned KBL-O
8, KBL, -14 is preferable because it can be easily cultivated in fungal bed cultivation and fruiting bodies can be obtained early.

In addition to shiitake mushrooms, there are also arage mushrooms, enoki mushrooms, wood ear mushrooms, kuritake mushrooms, and shirotamogitake mushrooms.

The present invention can also be applied to other mushrooms that can be cultivated artificially, such as Tamogitake mushrooms, Tsukuritake mushrooms, Nameko mushrooms, Maitake mushrooms, and Mushroom mushrooms.

The mutant strain production method of the present invention can be carried out, for example, as follows.

The desired monokaryotic hyphae are converted into protoplasts or mixed with an appropriate amount of sterile water to form hyphal pieces of one to several cells.

This is irradiated with ultraviolet rays or treated with chemicals (N-methyl N'-nitro N-nitrosoguanidine, ethyl methanesulfonate, etc.) to create a genetic marker strain.

Monokaryotic hyphae carrying this genetic marker are transformed into protoplasts again using a cell wall lytic enzyme that combines cellulase and chitinase. (In addition to the cell wall-dissolving enzymes, combinations of zymolyase, β-glucuronidase, etc. can also be used.) Cell fusion or hybridization is performed using the obtained protoplasts, and examples of the methods are shown below.

Electrofusion method; suspending protoplasts between parallel electrodes,
After forming a crowbar chain by applying an alternating current voltage, the protoplasts are fused by applying a high voltage pulsed direct current voltage.

Polyethylene glycol fusion method (PEG method); Appl, Microbiol Biotec at room temperature
hnol.

Fusion is performed by the method described in 22.121-127.1985.

In addition, cell fusion can also be performed by a high Ca'-high PH method or the like.

Mycelium can be regenerated by culturing the fused cells in an agar medium containing an osmotic pressure regulator such as neuucrose.

There are natural mating, Dai-Mon mating, etc.

Natural hybridization: Two types of monokaryotic mycelium of shiitake mushrooms are inoculated onto an agar medium at a distance of about 1 cm, and cultured at a constant temperature in the dark for 1 to 2 weeks. It has grown from the area where two types of hyphae came into contact,
Dikaryotic hyphae with clamp connections are isolated and cultured again.

These markers for cell fusion and hybridization may be attached to both of the two types of hyphae, or may be attached to only one of them.

(In the case of cell fusion, the fused strain can be selected by Mayupurenokun.) The dinucleated hyphae obtained as described above are prepared using log 1, a commonly used mushroom culture medium, or a fungal bed such as an artificial crosspiece. It is cultivated to form fruiting bodies.

Spores are isolated from this fruiting body, and strains with genetic markers are selected.

If we examine the mating types (incompatibility factors) and other genetic properties of strains with these genetic markers, we will find that many mutant strains with characteristics different from their parent strains have been obtained.

The present invention will be explained in more detail with reference to Examples below.

Example I Fruiting bodies were formed from dikaryotic hyphae of KBL-08, and monospore separation was performed to obtain monokaryotic hyphae. This nuclear hyphae was irradiated with ultraviolet rays, and an inositol-requiring mutant strain (hereinafter referred to as X (ino-)) was created by a conventional method.

(Dikaryotic mycelia KBL-08 are monospores isolated from the fruiting bodies of shiitake mushrooms preserved by our company. The fruiting bodies are mountain-shaped, medium in size, and fleshy.

For medium thickness and naturally occurring temperature range for log cultivation,
It is a mesopsychrotrophic bacterium that grows from November to May. The hyphae form white fluff-like colonies on the PDA medium.

The optimal culture temperature is 25°C, and the growth rate is 5.50/
It is day. ] On the other hand, monokaryotic mycelia KB obtained by separating monospores from fresh shiitake mushrooms
L-14 was irradiated with ultraviolet light to create a leucine auxotrophic mutant strain (hereinafter referred to as Y (1eu-)).

(The fruiting body of this raw shiitake mushroom is hard, umbrella-shaped, slightly chevron-shaped, medium-sized, and medium in size. The natural temperature range for log cultivation is 1.
It is a psychrotrophic bacterium that grows from January to April. The hyphae form white fluff-like colonies on the PDA medium.

The optimal culture temperature is 25°C and the growth rate is 5.0mm.
/day. ) X (ino-) and Y (j! eu-) are a combination that can form dikaryotic hyphae by mating.

X (ino-) and Y (leu-), respectively, are M
YG agar medium (1.0% malt extract, 0.4% yeast extract, 0.4% glucose, 1.5% agar; pH 4
, 5) and cultured for 1 to 2 weeks.

Cut the cultured mycelia into small pieces and place them into 100mff1 flask.
MYG liquid medium (1.0% malt extract, 0.4% yeast extract, 0.4% glucose, P
H4,5) and statically cultured for 4 days.

The obtained mycelium was excavated aseptically and treated with 50mM succinic acid.
NaOH buffer (containing 0.58M mannitol, pH 4)
, 5) several times, put 100 mg of mycelium into a test tube with a cap, and add enzyme solution (Cellulase Onozuka R3 (manufactured by Yakult) 30 mg/mj2. Chitinase (manufactured by Sigma) 1 mg/mE to t0. 6M mannitol 50mM NaOHIl succinate solution, pH 4,5)
Add 1 ml of X (i
No-) and Y (leu-) protoplasts were obtained.

Next, after filtration through 120 μm and 60 μm nylon membranes, the mixture is centrifuged at 800 rpm for 5 ml to remove mycelium residue. Pour the supernatant containing protoplasts again at 280 Orp.
Purified protoplasts were obtained by centrifugation at 5 ml.

This precipitate was suspended in 1 mf of 0.6 M mannitol,
Measure the number of protoplasts using a hemocytometer.

Then, these two types of purified protoplasts were mixed at a ratio of 1=1, centrifuged again at 280 Orpm for 5 minutes, and then 0.6M mannitol solution (1,0mM CaC
ff12-containing) at a concentration of about 3.0 x 10' cells/ml, and the fused chitenhar C-
After connecting the 0 body added to 02, the AC voltage VAc""4
Create a crowbar chain by applying current at 0V for about 1 minute, then pulse DC = 40kV/cm, during pulse! 1PW
Fusion was performed under the condition that the number of pulse applications was n=3. After application of the final pulse, let stand for about 20 minutes, dilute the second fusion suspension to 10'-10' cells/ml, and add 100 μl to a recirculating agar medium (mannitol 0.0.
6M, grape 112g, ammonium tartrate 0.2g
, Mg50.7 HzO: 0.05 g, KH2PO
, :0. l g, NazcOs: 0.1 g,
Fumaric acid: 0.1 gFez(Son)=: 1
．． Omg, Zns○, -78,01, Omg, Mn5O
a・48zO: 1. Omg, Thiamine-HCl:
1 Oag, 1.2 g of agar, 100 mj of water). The cells were cultured in the dark at 25° C. and 70% relative humidity for 10 days, and the grown colonies were collected.

Since the agar medium for regeneration does not contain inositol or leucine, the regenerated colonies are composed of X (ino-) and Y (
j! It is considered to be a fusion strain of eu-).

When the 13 fused strains thus obtained were cultivated for 3 months on artificial crossbars in a medium made of sawdust and rice bran, fruiting bodies were obtained for 6 of the strains.

One fruiting body was selected from each of the four strains (■ to ■), and the spores were separated by a conventional method.

The isolated spores were diluted to 10 ~ 103 cells/m4 and added to MYG.
Spread on agar medium and culture in the dark at 25°C for about 6 days.
227 mycelia that had germinated were isolated and their nutritional requirements were examined.

Table 1 (Unit: Strains) As shown in Table 1, auxotrophic markers (ino-,
A total of 127 strains with j2eu- or 1no- feu-) were obtained.

Next, in order to investigate whether strains with different biparental tastes and genetic properties have been produced, the mating types (incompatibility factors) of these auxotrophic marker strains can be used to determine the presence or absence of clamp conexitan formation. This was considered.

Table 2 shows the results of the investigation of inositol auxotrophs (15 strains) and leucine auxotrophs (8 strains) obtained from fruiting body ■.

In addition, the mating type of the fusion parent strain X (ino-) is set to AIBIY
The junction type of (leu-) is assumed to be 42B2.

As shown in Table 2, among the same auxotrophic strains,
There are 4 types of mating types, and mutant strains that have genetic markers and differ in both taste and genetic sex t (mating type) are:
Thirteen strains (A2B1; 6 strains, AlB2; 7 strains) were obtained.

In addition, 10 strains with the same mating type as the amphiphiles (AIBI; 4
If we investigate other genetic properties of the A2B2 strain (A2B2; 6 strain), it is possible to find strains that have different tastes from both parents.

Example 2 KBL-08 inositol auxotrophic mutant strain X (i
no-) and a normal KBL-14 strain without auxotrophy (hereinafter referred to as 1
z) was inoculated onto a MYG''N medium at a concentration of about 1 cmM and cultured at 25° C. in the dark for 1 to 2 weeks.

Dikaryotic hyphae with clamp coerules extending from the area where X (ino-) and Z hyphae came into contact were isolated and cultured in PDA medium (20% potato extract, 2% glucose, 2% agar). After culturing again for about 10 days, it was cultured using an artificial crosspiece.

Table 3 shows the results of testing the auxotrophy of 109 monokaryotic hyphae derived from spores obtained from the formed fruiting bodies.

Table 4 shows the results of examining the mating types of 16 of the 79 mucoinositol-requiring strains.

In addition, the mating type of the parent strain X (ino-) used for mating was
Using a medium consisting of BI sawdust and rice bran, 8 mutant strains (43 BI;
4 strains, AlB5; 4 strains) were obtained.

In addition, the remaining 8 stocks (AIBI; 2 stocks, ^3B3i6
If we investigate other genetic properties of the strain (Japanese strain), it is possible to find a strain that has different characteristics from its parent strains.

Example 3 KBL-08-karyotic hyphae were irradiated with ultraviolet light, and the inositol auxotrophic mutant strain (hereinafter referred to as X (ino-)) and the leucine auxotrophic mutant strain [hereinafter referred to as P(j!eu-)] Two types of auxotrophic mutant strains were created.

By crossing X (ino-) and P (ffieu-),
This is a combination that can form dikaryotic hyphae.

Cell fusion was performed in the same manner as in Example 1 using X (ino-) and P (feu-), and the resulting fusion strain 1o strain was cultivated for a month, and fruiting bodies were obtained for three strains.

Select one fruiting body per plant from the two ■～
(2) Spores were separated using a conventional method.

The isolated spores were diluted to 10-103 spores/ml.
It was applied to a YG''IM heaven medium and cultured in the dark at 25°C for about 6 days.
0 strains (total 150) were isolated and their nutritional requirements were examined.

As shown in Table 5, auxotrophic markers (ilo-,
l eu- or 1no-! A total of 97 strains with V, eu-) were obtained.

Table 6 shows the results of examining the mating types of inositol auxotrophs (16 strains) and leucine auxotrophs (7 strains) obtained from fruiting body ■.

The mating type of the parent strain X (ino-) for the fusion is AIBI.

As shown in Table 6, among the same auxotrophic strains,
There are 4 types of mating types, and mutant strains that have genetic markers and have a different genetic sex f (mating type) from their parents are:
Thirteen strains (A481: 6 strains, AIB4 near strain) were obtained.

In addition, 10 strains with the same mating type as the parent strains (AIB!: 4
If we examine other genetic properties of the A4B4 strain (A4B4; 6 strain), there is a possibility that we will find strains that have different characteristics from their parent strains.

As can be seen from the results of Examples 1 to 3, by cell fusion or crossbreeding of strains that have genetic markers on both sides or on the - side and pass through the fruiting bodies, it is possible to differentiate the genetic characteristics from the parent strains that have genetic markers. It was possible to easily create a large number of different mutant strains.

〔Effect of the invention〕

In the method of the present invention, genetic marking only needs to be done once at the beginning.

In addition, by passing through the fruiting bodies, we can produce a wide variety of mutant strains that have genetic markers and differ in genetic properties from their parent strains.
It is possible to obtain a large quantity and reliably.

In addition, this genetic marker is a stable genetic marker that is retained even after nuclear fusion in the basidium, meiosis, etc., and reverse mutations are unlikely to occur.

Furthermore, since it can be stored in the form of secondary hyphae, there is less risk of the genetic marker strain dying.

From the above, it is clear that the present invention is a method for reliably producing mutant strains with stable genetic markers, which are useful for research on cultivar improvement of shiitake mushrooms.

Patent application 2 filed on May 30, 1990, name of the invention 3, relationship with the amended case Patent applicant address 5-17-4 Sumida, Sumida-ku, Tokyo 534 Tomobuchi, Bejima-ku, Osaka-shi No. 5-90 Town-chome Kanebo Co., Ltd. Patent Department Correction 6 in the "Explanation" column, Contents of the correction (1) The specification page 1 O, line 2 r40VJ is corrected to "30v".

(2) "Mannitol" on page 10, line 8 of the specification is amended to "sucrose."

(3) Page 10, line 11 of the specification “Fumaric acid: 0.1 g,
", then add "Sunpearl CP: 0.4g, J.

(4) Specification page 10 line 13 rMn S O4' 4
HzOJ rMncjl! Correct as = H4HzOJ.

(5) On page 19 of the specification, lines 6 to 7, "I can get it" is amended to "I can get it."

7. List of attached documents Document certifying that the invention is stipulated in Article 30, Paragraph 1 of the Patent Act; 1 copy

Claims (1)

[Claims] (1) Crossing or cell fusion of shiitake mushroom hyphae with genetic markers, or shiitake mushroom hyphae with genetic markers on only one side, and culturing the resulting dikaryotic hyphae,
A method for producing mutant strains that have genetic markers and differ in genetic properties from their parent strains, which is characterized by separating and culturing basidiospores from the generated fruiting bodies.