CN103270945B - Tremella fuciformis strain ion beam injection mutation breeding method and Tremella fuciformis strain bred therethrough - Google Patents
Tremella fuciformis strain ion beam injection mutation breeding method and Tremella fuciformis strain bred therethrough Download PDFInfo
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- CN103270945B CN103270945B CN201310228604.7A CN201310228604A CN103270945B CN 103270945 B CN103270945 B CN 103270945B CN 201310228604 A CN201310228604 A CN 201310228604A CN 103270945 B CN103270945 B CN 103270945B
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- white fungus
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Abstract
The invention relates to a Tremella fuciformis strain ion beam injection mutation breeding method and a Tremella fuciformis strain bred therethrough. The breeding method comprises the following steps: 1, washing fresh auricles of an original Tremella fuciformis strain for obtaining basidiospores on the fresh auricles, coating the basidiospores onto a metal culture dish, and carrying out aseptic air blow drying; 2, carrying out low-energy N<+> ion beam injection mutation of the Tremella fuciformis basidiospores on the metal culture dish under a vacuum condition, wherein the radiation induced mutation dosage is 5*10<14>-5*10<15>ions/cm<2>; 3, culturing the mutated Tremella fuciformis basidiospores, and screening Tremella fuciformis strains with the thalli having high dry weights; and 4, pairing the screened strains with a Hypoxylon sp. strain, cultivating, and screening a Tremella fuciformis strain having a high output. The invention also provides the Tremella fuciformis strain obtained by breeding through the method. The Tremella fuciformis strain provided by the invention has a preservation number of CGMCC No.7458, the output of the Tremella fuciformis strain is 13.5% higher than that of the original strain, and the Tremella fuciformis polysaccharide content of the Tremella fuciformis strain is 5.41% higher than that of the original strain.
Description
Technical field
The invention belongs to breed of edible fungus field, the white fungus bacterial strain that particularly white fungus bacterial strain ion beam injects mutagenic breeding method and educates.
Background technology
White fungus claims again tremella, belongs to fungus circle, Basidiomycota, and white fungus guiding principle, Tremellales, Tremellaceae, Tremella, is a kind of jelly fungus of dietotherapeutic of preciousness, nutritive value is very abundant.China is the major country of production of white fungus, and export volume accounts for the more than 90% of world's white fungus volume of trade.At present the existing cultivar of white fungus mainly contains two kinds, yellow white fungus and white white fungus, mainly come from long-term domestication and the artificial selection of wild white fungus bacterial classification, the white fungus bacterial classification output that this traditional white fungus strain breeding method is cultivated is not high, and easily degenerates, unstable.In the urgent need to excavating high yield, stable, colory white fungus microorganism resource, when improving white fungus output, promote white fungus quality at present.
Ion beam injects induced-mutation technique, and be first the eighties in 20th century be applied to crop breeding by people such as blasts more than the Chinese Academy of Sciences, for crops such as breeding rice, wheat, corns, along with the development of technology, be applied to gradually microorganism mutation breeding research, and successful seed selection arachidonic acid is produced bacterial strain etc., it is a kind of effectively breeding technique.Ion beam injects induced-mutation technique compared with traditional ultraviolet ray, X ray, gamma-rays etc., except having energy deposition, also has Momentum Transfer, the effects such as quality deposition and charge-exchange.In addition can, according to different mutation breeding needs, select different types of ion, energy, dosage to combine, thereby there is the advantages such as mutation spectrum is wide, mutation rate is high, mutant genetics stability is high, reverse mutation rate is low.Existing bibliographical information, the patent CN102067788B of people's inventions such as Quan Weifeng discloses a kind of low energy N
+ion beam injects the method for mutagenic and breeding ganoderma strain capable, and has successfully selected the ganoderma strain capable that a plant height produces.
There is not yet any report about adopting ion beam injection technique mutagenic and breeding white fungus.In general different types of microorganism, the size of bacterium bacteroid cell is different with the thin and thick of cell wall, Mycophyta spore size is different with conidial cell wall thin and thick, therefore to ion beam Implantation Energy, the tolerance degree difference of dosage, survival rate and mutation rate are also inconsistent, can make different types of microorganism there is different best mutagenic conditions, can obtain with this understanding higher mutation rate, can screen and obtain more mutant strain, especially we want the direct mutation bacterial strain obtaining, this method can not be indiscriminately imitated another microorganism from a kind of microorganism.
Therefore the present invention is directed to white fungus bacterial strain, to carry out the mutagenesis of ion beam injection technique be a creative work, and the bacterial strain that seed selection obtains is not obtainable bacterial strain in the market.
Summary of the invention
For the new way of developing white fungus strain improvement, excavate high yield, stable, colory white fungus bacterial classification, a white fungus bacterial strain that the object of the present invention is to provide white fungus bacterial strain ion beam to inject mutagenic breeding method and educate.
The invention provides a kind of white fungus bacterial strain ion beam and inject mutagenic breeding method, comprise the steps:
1) with the basidiospore on the fresh auricle of white fungus starting strain under aseptic washing, be applied on metal culture dish, sterile wind dries up;
2), under vacuum condition, the white fungus basidiospore on metal culture dish is carried out to low energy N
+ion beam injects mutagenesis, and energy is 30kev, and vacuum is 10
-3pa, adopts continuous injection mode, and radioinduction dosage is 1 × 10
15-2.5 × 10
15ions/cm
2;
3) cultivate the white fungus basidiospore after mutagenesis, the screening dry cell weight white fungus bacterial strain higher than white fungus starting strain;
4) cultivation after white fungus bacterial strain step 3) being screened and the pairing of incense ashes bacteria strain, the output after mensuration cultivation, the bacterial strain that screening output improves, and separate preservation bacterial strain.
Described in step 1), starting strain is that output is high, the existing white fungus bacterial strain of quality better.
The fresh auricle of white fungus starting strain described in step 1), for starting strain bottle is planted the fresh clean white fungus auricle obtaining.
Described in step 1), be coated with, with glass bar or metal bar coating.
The present invention also provides described white fungus bacterial strain ion beam to inject the application of mutagenic breeding method at white fungus strain breeding thereof.
Inject a white fungus bacterial strain (Tremella fuciformis) of mutagenic breeding method seed selection with white fungus bacterial strain ion beam of the present invention, its preserving number is CGMCC No.7458, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Described preserving number is the white fungus bacterial strain stabilization characteristics of genetics of CGMCC No.7458, and output improves 13.5% than former bacterial strain, and tremella polysaccharides content improves 5.41%.
The present invention also provides the application of described white fungus bacterial strain as cultivated strains.
Beneficial effect of the present invention:
White fungus bacterial strain ion beam of the present invention injects mutagenic breeding method, grope to inject for the ion beam of white fungus bacterial strain the optimal dose of mutagenesis, with method mutagenesis white fungus bacterial strain of the present invention, can screen the white fungus mutant strain of more high yields, greatly widen the scope of white fungus strain breeding thereof.
Preserving number of the present invention is the white fungus bacterial strain stabilization characteristics of genetics of CGMCC No.7458, and output improves 13.5% than former bacterial strain, and tremella polysaccharides content improves 5.41%, is good white fungus bacterial strain.
Brief description of the drawings
Fig. 1 is the influence curve figure of the embodiment of the present invention 1 ion beam implantation dosage to tremella spore survival rate.
Fig. 2 is the influence curve figures of the embodiment of the present invention 2 ion beam implantation dosages to white fungus bacterial strain positive mutation rate.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment that the inventive method, step or condition are done or replacement, all belong to protection scope of the present invention.
If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art.
Embodiment of the present invention used medium formula is as follows:
Murphy juice: 200g potato liquor 1000ml.
PDA medium: glucose 20g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, agar powder 15g, murphy juice is settled to 1000ml, pH value nature, 121 DEG C, 30min sterilizing.
PDB medium: glucose 20g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, murphy juice is settled to 1000ml, pH value nature, 121 DEG C, 30min sterilizing.
Wood chip bottle is planted medium: wood chip 67%, and wheat bran 32%, land plaster 1%, water is 1.2-1.5:1 with material ratio.
Cultivating in bag medium: cotton seed hulls 83%, wheat bran 15%, land plaster 2%, water is 1.0-1.2:1 with material ratio.
Sterile water: 121 DEG C of sterilizing 30min of ultra-pure water (ultra-pure water of being made by Mi Libo water purification machine MiliQ A10).
Embodiment of the present invention chemical reagent used is except agar is biological level, and it is pure that other is analysis, purchased from chemical reagents corporation of traditional Chinese medicines group.Potato, wood chip, wheat bran, land plaster, cotton seed hulls etc. are all commercially available.
Determining of embodiment 1 ion implantation dosage
Starting strain: the white fungus bacterial strain (Tremella fuciformis BRC010) that preserve in Beijing City Radiation Centre's Microbial Breeding laboratory, the wild white fungus that this white fungus bacterial strain is gathered by Fujian separates and obtains, through Laboratory Acclimation, function admirable, carry out cultivation experiments checking by Gutian Zhouyang Agricultural Technology Co., Ltd. of Fujian Province, and preserved bacterial classification.
The acquisition of white fungus basidiospore
White fungus starting strain is adopted sawdust medium to carry out bottle in laboratory and plant, turn out ear for 20-22 DEG C, obtain fresh Tremella fructification.
By fresh 20-30g clean white fungus auricle (fruit body), take off with tweezers and scalpel, put into the aseptic centrifuge tube of 50ml that 35ml sterile water is housed, build lid, be placed on vortex oscillation device concuss repeatedly, also can with hand-held live centrifuge tube two ends, upper and lower concuss number minute, basidiospore on white fungus auricle is fully eluted in sterile water, obtains white fungus basidiospore suspension.
Under aseptic condition, adopt aseptic pipette or liquid-transfering gun to draw 0.1-0.5ml white fungus basidiospore suspension, adopt glass or metal spreading rod to be evenly applied to metal culture dish surface, in super-clean bench, dry up with sterile wind.Described metal culture dish is that custom-designed special equipment is tested for carrying out ion beam injection mutagenesis by our unit, ion beam injects mutagenesis to carry out under vacuum state, therefore we have designed double-level-metal culture dish, lower floor is the metal level of edge with air-vent, upper strata is the metal level of edge seal, white fungus basidiospore need to be applied to respectively two-layer on, it is used for doing ion beam injection experiments at the middle and upper levels, lower floor contrasts as vacuum with air-vent, ensure that vacuum contrasts and ion beam injection sample stops in vacuum target chamber time and condition are in full accord, get rid of the impact of vacuum on basidiospore survival rate, simultaneously, metal culture dish has certain electric conductivity, also can prevent the electric discharge causing because of the accumulation of charged ion electric charge simultaneously.
The low energy N of white fungus bacterial strain
+ion beam mutagenesis
It is the low energy ion implanter BNU-400 of Beijing City Radiation Centre (Nuclear Science and Technology institute of Beijing Normal University) that experiment is used instrument and equipment, and white fungus basidiospore ion beam injects mutagenesis and adopts N
+ion, vacuum is 10
-3pa, adopts continuous injection mode, and carries out preferably for the condition of different-energy and different implantation dosages, and energy range is 30-100kev, and implantation dosage scope is 1 × 10
12-1 × 10
16ions/cm
2, concrete implantation dosage is made as 1 × 10
12, 5 × 10
12, 1 × 10
13, 5 × 10
13, 1 × 10
14, 5 × 10
14, 1 × 10
15, 5 × 10
15, 1 × 10
16ions/cm
2.
Low energy N
+the impact of ion beam mutagenesis on bacterial strain survival rate
Metal culture dish after mutagenesis is added to 1ml sterile water, with the basidiospore of aseptic glass or the coating of metal spreading rod scraping, by abundant basidiospore wash-out, eluent is drawn and transferred in aseptic EP pipe with aseptic pipette or liquid-transfering gun, carry out again gradient dilution, draw respectively 0.1ml coating PDA solid plate, 3 parallel laboratory tests of each sample, 20-25 DEG C of constant temperature culture 5d, add up each dilution factor basidiospore amount of survival, the basidiospore number obtaining taking spread plate after vacuum control sample wash-out, as contrast, calculates survival rate, calculates publicity as follows:
Survival rate (%)=Implantation sample basidiospore number/vacuum control sample basidiospore number × 100%
Statistics 1 × 10
12, 5 × 10
12, 1 × 10
13, 5 × 10
13, 1 × 10
14, 5 × 10
14, 1 × 10
15, 5 × 10
15, 1 × 10
16ions/cm
2survival rate under these 9 dosage, obtain mutagenesis preferably dosage range be 5 × 10
14-5 × 10
15ions/cm
2.In dosage range preferably, respectively 5 × 10
14, 10 × 10
14, 15 × 10
14, 20 × 10
14, 25 × 10
14, 30 × 10
14, 35 × 10
14, 40 × 10
14, 45 × 10
14, 50 × 10
14ions/cm
2these 10 dosage repeat mutagenesis experiment, and add up survival rate under various dose, obtain survival rate curve as shown in Figure 1.
What survival rate curve as shown in Figure 1 reacted is the variation relation between white fungus basidiospore survival rate and ion beam implantation dosage, present " saddle-shaped curve ", along with the increase of implantation dosage, the survival of white fungus basidiospore takes the lead in declining rapidly, then have after a rising and continue to decline, it is generally acknowledged at the flex point place of saddle type curve and the ion implantation dosage of near zone better (in Fig. 1, corner position is 1.5 × 10
15ions/cm
2, at this position and near zone thereof), can obtain more direct mutation bacterial strain.
The screening of embodiment 2 white fungus bacterial strains
The cultivation of white fungus bacterial strain
To in embodiment 1, add up the basidiospore difference picking of having sprouted on the white fungus PDA flat board of amount of survival to new PDA flat board, the basidiospore of 1 sprouting of each plating, white fungus basidiospore after sprouting is exactly white fungus bacterium, white fungus bacterium is put into 20-25 DEG C of constant incubator to be cultivated, can around white fungus bacterium, form white mycelia, some also can form yeast sample gemma.
Selecting of white fungus bacterial strain
White fungus bacterium forms circle or subcircular bacterium colony after sprouting mycelia, and mensuration colony diameter size can tentatively judge the speed of white fungus bacteria growing, picks out growth rate white fungus bacterium faster.
The liquid culture of white fungus bacterial strain
By the white fungus bacterium colony of picking out, liquid culture is carried out in switching, adopts PDB medium, and triangular flask shaking table is cultivated, and shaking speed is turn/min of 180-220,20-25 DEG C of constant temperature culture 10-15d.By liquid culture 8000rpm, centrifugal 5min, outwells supernatant, and sediment is transferred to freeze drying in culture dish, weighs dry weight.Compared with starting strain, after mutagenesis, the increase by 5% of white fungus bacterial strain dry cell weight and above bacterial strain are direct mutation bacterial strain, add up the positive mutation rate of white fungus bacterial strain under different implantation dosages according to this method, find the ion beam implantation dosage that positive mutation rate is higher, the relation of white fungus bacterial strain positive mutation rate and ion beam implantation dosage as shown in Figure 2,1 × 10
15-2.5 × 10
15ions/cm
2in scope time, positive mutation rate is higher, for ion beam injects the optimum condition of mutagenesis.
Obtaining thus white fungus ion beam injection mutagenesis optimum condition is energy 30kev, and implantation dosage is 1 × 10
15-2.5 × 10
15ions/cm
2, repeatedly repeat with this understanding ion beam and inject mutagenesis experiment, and screen high yield white fungus bacterial strain.
The pairing of white fungus bacterium and incense ashes bacterium
The formation of Tremella fructification is more special, must white fungus bacterium and incense ashes bacterium (source is Gutian Zhouyang Agricultural Technology Co., Ltd. of Fujian Province) exist simultaneously, can form former base and grow Tremella fructification, therefore the white fungus bacterium that output primary dcreening operation being obtained improves and the general incense ashes bacterium of cultivation match, and make and cultivate female kind and cultivated species.Matching method is conventional method.
Cultivating white fungus experiment
Cultivating white fungus kind after pairing is carried out to cultivating in bag experiment, and the white fungus bacterial strain that screening is obtained all adopts the conditions such as identical cultivation Bag Material, cultivation temperature, humidity, ventilation, statistics white fungus output, and measure Tremella fructification polyoses content.
The white fungus bacterial strain that embodiment 3 filters out with method of the present invention
(concrete grammar is shown in embodiment 1 to the white fungus bacterial strain filtering out with the inventive method, 2), it is carried out to mitotic stability experiment, go down to posterity 15 times, carry out cultivation experiments respectively with after the pairing of incense ashes bacterium at every turn, compared with the first generation, fruiting body yield is stable, output there was no significant difference (P>0.05) between each generation, and white fungus is all stablized unchanged at aspects such as a type size and glossiness, this white fungus bacterial strain is to inject by ion beam the superior strain that mutagenesis screening obtains, output improves 13.5% than former bacterial strain, tremella polysaccharides content improves 5.41%, and stabilization characteristics of genetics, this white fungus bacterial strain is stored in and is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 24th, 2013, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. a white fungus bacterial strain (Tremella fuciformis), its preserving number is CGMCC No.7458.
Described in claim 1 white fungus bacterial strain as the application of cultivated strains.
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| CN103503693B (en) * | 2013-09-30 | 2015-09-23 | 东北林业大学 | A kind of method utilizing dry auricle quick separating edible fungus |
| CN106834270A (en) * | 2017-02-27 | 2017-06-13 | 新疆大学 | A kind of ion beam mutation splits that lid saddle fungus is full genome mutated and method of orthogenesis |
| CN110373409A (en) * | 2019-07-02 | 2019-10-25 | 昆明旭日丰华农业科技有限公司 | A kind of tremella strain breeding method |
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| CN102067788B (en) * | 2010-10-25 | 2012-04-25 | 江苏安惠生物科技有限公司 | Method for breeding ganoderma lucidum strain by low-energy N<+> implantation induced mutation and bred strain thereof |
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