CN115316196B - Mutagenesis and cultivation method of wild locust Fan Shikong bacteria, locust Fan Shikong bacteria and application - Google Patents

Mutagenesis and cultivation method of wild locust Fan Shikong bacteria, locust Fan Shikong bacteria and application Download PDF

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CN115316196B
CN115316196B CN202210981857.0A CN202210981857A CN115316196B CN 115316196 B CN115316196 B CN 115316196B CN 202210981857 A CN202210981857 A CN 202210981857A CN 115316196 B CN115316196 B CN 115316196B
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庄磊
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Abstract

The invention provides a mutagenesis method based on wild locust Fan Shikong bacteria, a cultivation method of locust Fan Shikong bacteria, locust Fan Shikong bacteria and application thereof, and belongs to the technical field of locust Fan Shikong bacteria. In order to solve the problems that the fruiting body of the locust Fan Shikong fungus is difficult to artificially cultivate and the mutagenesis effect is poor. By passing through 60 The Co-gamma is used for mutagenesis of a wild locust Fan Shikong bacterial spore suspension containing a wild locust Fan Shikong bacterial stock solution, and the locust Fan Shikong bacterial after mutagenesis is cultivated in a mother culture medium to obtain a new variety of locust Fan Shikong bacterial. Realizing artificial cultivation of the fruiting bodies of the locust Fan Shikong bacteria; the tolerance is improved after the wild locust Fan Shikong bacterial stock solution is added, so that the mutagenesis effect is improved; the antagonism test shows that the obtained locust Fan Shikong bacteria are new variety locust Fan Shikong bacteria with the preservation number of CGMCC No.40140; the test result of continuous two-year cultivation shows that the new strain of the locust Fan Shikong bacteria has stable character, and the per mu yield of fruiting bodies can reach 1000 jin.

Description

Mutagenesis and cultivation method of wild locust Fan Shikong bacteria, locust Fan Shikong bacteria and application
Technical Field
The invention relates to the technical field of locust Fan Shikong bacteria, in particular to a mutagenesis method based on wild locust Fan Shikong bacteria, a cultivation method of locust Fan Shikong bacteria, locust Fan Shikong bacteria and application thereof.
Background
In China fungus (science Press, first edition, 2021, 2 nd edition, pages 205-206), it is mentioned that the main characteristic of the fungus Sophora japonica Fan Shikong is annual basidiomycetes. Robinia pseudoacacia Fan Shikong is grown on the trunks of Robinia pseudoacacia, pterocarpus Indicus, etc. The fungi are distributed in Beijing, jiangsu, liaoning, shanxi, shandong, sichuan and other places in China and generally grow on living trees of locust.
The locust Fan Shikong bacteria has extremely high medicinal value, and can be used as a targeted antitumor drug for treating liver cancer, lung cancer, breast cancer, gastric cancer and the like. Wild resources of the locust Fan Shikong bacteria are scarce, and fruiting bodies are difficult to artificially cultivate. At present, the solid fermentation method is only used for producing the locust Fan Shikong mycoplasm, and the liquid culture method is used for producing mycelium for medical use. However, the medicinal ingredient raw material of the locust Fan Shikong fungus is only the fermentation product of the locust ear hyphae, and the medicinal efficacy of the fruiting body of the locust Fan Shikong fungus is far higher than that of the mycelium. In terms of components, the fruiting body has many secondary metabolites (such as locust Fan Shikong mycolic acid and adenosine) besides polysaccharide and immunoregulatory protein, and the mycelium has few secondary metabolites, and the components are mainly polysaccharide. Therefore, the mycelium is not comparable to the fruiting body in terms of the "kind" of active ingredient. In the research of the locust Fan Shikong bacteria, the following problems were found:
1. many of the fruiting bodies of the fungus Acacia Fan Shikong are picked directly from their host tree species. However, the host tree species of the locust Fan Shikong bacteria are sparse, so that the number of the fruiting bodies of the locust Fan Shikong bacteria is limited, and the huge demand of the medicinal material market for the fruiting bodies of the locust Fan Shikong bacteria is difficult to meet.
2. In order to meet the requirement of mass cultivation of the locust Fan Shikong bacteria, the mycelia can be artificially cultivated when artificial cultivation is carried out, only wood-pinned porous fungus meat tissues can be grown by bag cultivation, and normal fruiting bodies are difficult to artificially cultivate.
3. When the mushroom is subjected to mutation breeding, in particular by 60 When Co-gamma is subjected to mutation breeding, the mutation rate is effectively improved when the mutation agent amount is usually 100Gy-1000Gy and exceeds 1000Gy, but the death rate of the mushrooms is close to 100%, and beneficial mutation breeding still cannot be obtained, so that the mutation effect is poor. Thus, control of death under conditions effective to increase mutagenesis rateThe rate of apoptosis is 90% -95% critical.
Disclosure of Invention
In order to solve the problems that the fruiting bodies of the locust Fan Shikong bacteria are difficult to artificially cultivate, the wild collection quantity is small, the market demand is difficult to meet, and the mutagenesis effect is poor.
The invention provides a mutagenesis method based on wild locust Fan Shikong bacteria, which comprises the following steps:
step one, dissolving spores of wild locust Fan Shikong bacteria in sterile physiological saline, counting selected wild locust Fan Shikong bacteria as shown in figure 1 by using a blood cell counting plate, and diluting until the concentration of spores in spore suspension is 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 5-15 parts of wild locust Fan Shikong bacterial stock solution, 35-40 parts of corn starch, 15-25 parts of wheat bran, 8-12 parts of sucrose, 8-12 parts of glucose, 1.8-2.2 parts of monopotassium phosphate, 0.9-1.1 part of magnesium sulfate, 5-7 parts of yeast powder, 0.008-0.012 part of VB1, 16-20 parts of agar and 800-1200 parts of water, and controlling the ph value of the mutagenesis medium to be 6.3-6.5;
mixing the fruiting body of wild locust Fan Shikong bacteria of the same strain as the spores with water according to the mass ratio of 1 (5-10), boiling, and filtering to obtain a wild locust Fan Shikong bacteria stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of spores in the step one into the same mutagenesis medium at the same time, wherein the volume ratio of the spore suspension to the mutagenesis medium is 1: (2-3), then culturing for 2d at 26 ℃, then irradiating with 60 Co-gamma rays, wherein the irradiation dose is 1150Gy-1200Gy, the irradiation is performed three times at 2 hours intervals, and the irradiation time of the three times is sequentially 20s, 40s and 60s, so as to obtain the locust Fan Shikong bacteria after mutagenesis.
Further, culturing the post-mutagenesis locust Fan Shikong bacteria in the mutagenesis medium in the second step again, and continuously passaging the selected post-mutagenesis locust Fan Shikong bacteria for at least 3 times to obtain the post-mutagenesis locust Fan Shikong bacteria with more stable inheritance in order to obtain the post-mutagenesis locust Fan Shikong bacteria with stable inheritance.
And (3) selecting a colony which grows faster, is full and has better color and luster for culture, wherein the colony is the post-mutagenesis locust Fan Shikong bacteria in the third step or the post-mutagenesis locust Fan Shikong bacteria which are genetically stable in the fourth step, and the colony and the wild Fan Shikong bacteria can be subjected to culture at 25 ℃ for 6 hours so as to observe the obvious antagonistic line as shown in figure 5. According to the antagonism test, the locust Fan Shikong bacteria after mutagenesis can be obtained as a new variety.
The length of the mycelium can be 2mm-4mm or other longer length, and the mycelium can be one or more than two.
Preferably, the mortality rate reaches 94.68% at 1175Gy, and 90% -95% is advantageous for mutant strain production according to mutagenesis theory studies, so the assay selects 1175Gy as mutagen for the corresponding irradiation dose at 94.68% in order to increase the probability of screening for mutant strains.
A cultivation method of locust Fan Shikong bacteria comprises the following steps:
culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong, as shown in figure 2;
the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 24-28 ℃ when the mother culture medium is used for tissue culture, white villiated mycelium grows out after 3-4 d culture, after the mycelium extends to the mother culture medium, a fungus inoculating needle is used for picking the top part of the mycelium, and the mycelium is inoculated to a new mother culture medium;
the mother culture medium is a composite CPDA culture medium added with pagodatree bark juice and peptone;
(2) Preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room at 25-28 ℃ for 25-30 d to obtain stock seeds of the locust Fan Shikong bacteria;
(3) Preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at the temperature of 25-28 ℃ for 25-30 days to obtain a cultivated species of the locust Fan Shikong bacteria;
(4) Ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 10d-12d, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, harvesting when a large number of white spores are ejected from fruiting bodies to grow and the fungus covers are not increased any more, wherein the increase of the fungus covers is lower than 1mm within three days, and the increase of the fungus covers is not increased any more.
Further, the composite CPDA culture medium consists of the following raw materials in parts by weight: 200-250 parts of fresh potatoes, 20-25 parts of pagodatree bark, 1000-1500 parts of water, 18-22 parts of glucose, 18-22 parts of agar, 1.8-2.2 parts of monopotassium phosphate and 0.8-1.5 parts of magnesium sulfate.
Further, the preparation method of the composite CPDA culture medium comprises the following steps: washing 200g-250g of fresh potatoes, peeling, cutting up, adding 1L-1.5L of water into 20g-25g of pagodatree bark, boiling for 25min-35min, adding water into filtrate obtained after gauze filtration to supplement 1L, adding 18g-22g of glucose, 18g-22g of agar, 1.8g-2.2g of monopotassium phosphate and 0.8g-1.5g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized composite CPDA culture medium, subpackaging the unsterilized composite CPDA culture medium into test tubes, and subpackaging each test tube with about 5mL-10mL; and (3) sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use.
Further, the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 24-28 ℃ when the mother culture medium is used for tissue culture, white villiated mycelium grows out after 3-4 d culture, when the mycelium extends to the mother culture medium, a fungus inoculating needle is used for picking the top part of the mycelium, and the mycelium is inoculated to a new mother culture medium for subsequent steps.
Further, the temperature is kept at 24-28 ℃ during the culture period of the mother strain, the relative air humidity is controlled below 75%, and the mother strain is cultured in a dark place.
Further, the stock culture medium and the cultivar culture medium each comprise: 75-80 parts of miscellaneous wood chips, 13-17 parts of bran, 4-5 parts of soybean meal, 1-1.5 parts of sucrose, 0.5-1.5 parts of gypsum powder, wherein the stock culture medium comprises pH value of 5-6 and water content of 55% -60%.
Sophora japonica Fan Shikong strainVanderbylia robiniophila) The preservation number is CGMCC No.40140, the Chinese microorganism strain preservation management committee common microorganism center is preserved, the preservation address is North Chen Xili No. 1 and No. 3 in the Korean area of Beijing city, and the preservation time is 2022, 4 months and 24 days.
An application of a locust Fan Shikong bacterium, wherein fruiting bodies of the locust Fan Shikong bacterium are used for preparing antitumor drugs.
Further, the antitumor drugs include drugs for treating liver cancer, lung cancer, bone cancer, breast cancer and pancreatic cancer.
The invention relates to a mutagenesis method based on wild locust Fan Shikong bacteria and a cultivation method of locust Fan Shikong bacteria, which are implemented by 60 The Co-gamma carries out mutagenesis treatment on wild locust Fan Shikong bacteria, the obtained mutagenesis locust Fan Shikong bacteria are purified and cultured by using a mother culture medium to obtain pure mother seeds of locust Fan Shikong bacteria, the pure mother seeds of locust Fan Shikong bacteria are cultured by using a stock culture medium and a cultivar culture medium 25d-30d to obtain locust Fan Shikong bacteria cultivars, the cultivated seeds of locust Fan Shikong bacteria are moved to a fruiting room for fruiting, a large number of white spores are ejected when fruiting bodies grow, and a bacterial cover is not enlarged any more, and the mother culture medium is a composite CPDA (CPDA) culture medium added with pagodatree bark juice and peptone;
the invention is realized by 60 The Co-gamma carries out mutagenesis treatment on wild locust Fan Shikong bacteria, so that the fruiting bodies of the mutagenized locust Fan Shikong bacteria can be artificially cultivated; the used mother culture medium simulates the growth environment of wild locust Fan Shikong bacteria by adding locust tree bark juice and peptone, so that the locust Fan Shikong bacteria can be cultivated in the mother culture medium to obtain pure mother strain of locust Fan Shikong bacteria, and after subsequent treatment, a new variety of locust Fan Shikong bacterial fruiting body with better growth vigor is obtained, thereby not only solving the defect that the wild locust Fan Shikong bacterial fruiting body is difficult to artificially cultivate, but also increasing the yield of artificially cultivated locust Fan Shikong bacterial fruiting body and meeting the great demand of medicinal material market on locust Fan Shikong bacteria;
placing a suspension of spores of wild locust Fan Shikong bacteria in a solution containing wild locust shellCulturing the original solution of Phellinus in a mutagenesis medium, and then 60 The mutagenesis treatment of Co-gamma improves the comparison of the wild locust Fan Shikong bacterial stock solution 60 The tolerance of Co-gamma ensures that the death rate is 90-95% under the condition that the irradiation dose is 1150-1200 Gy, and the mutagenesis effect is better;
as shown in FIG. 5, the locust Fan Shikong bacteria prepared by the scheme is a new variety of locust Fan Shikong bacteria and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.40140;
the result of the continuous two-year cultivation test of the novel strain of the locust Fan Shikong bacteria is shown in fig. 6, and the average acre yield of the novel strain of the locust Fan Shikong bacteria for two continuous years can reach 1000 jin/year, so that the artificial cultivation of the novel strain of the locust Fan Shikong bacteria is further proved, and the yield is high.
Drawings
FIG. 1 is a diagram of wild locust Fan Shikong bacteria in the present invention;
FIG. 2 is a physical diagram of a pure mother strain of Robinia pseudoacacia Fan Shikong in the invention;
FIG. 3 shows the mycelium of Robinia pseudoacacia Fan Shikong under 1600 times magnification of an optical microscope;
FIG. 4 is a physical view of a fruiting body with a large number of white spores ejected in the invention;
FIG. 5 is a graph showing the results of antagonism experiments of wild Fan Shikong bacteria and post-mutagenesis locust Fan Shikong bacteria in the present invention;
FIG. 6 is a graphical representation of the results of the second year cultivation test of the fruiting body of Robinia pseudoacacia Fan Shikong in the present invention;
FIG. 7 is a graphical representation of the results of the second year cultivation test of the fruiting body of Robinia pseudoacacia Fan Shikong obtained in example 7;
FIG. 8 is a graphical representation of the results of the second year cultivation test of the fruiting body of Robinia pseudoacacia Fan Shikong obtained in example 8;
FIG. 9 is a graph showing the preservation of the strain of Robinia pseudoacacia Fan Shikong.
Detailed Description
The components of the culture medium selected in the invention can be purchased through public channels, and the equipment and instruments adopted in the process are all common equipment in the field.
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
Examples
(1) Dissolving spores of wild locust Fan Shikong bacteria in sterile physiological saline, counting the selected wild locust Fan Shikong bacteria by using a blood cell counting plate, and diluting until the concentration order of spores in the spore suspension is 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 5 parts of wild locust Fan Shikong bacteria stock solution, 35 parts of corn starch, 15 parts of wheat bran, 8 parts of sucrose, 8 parts of glucose, 1.8 parts of monopotassium phosphate, 0.9 part of magnesium sulfate, 5 parts of yeast powder, 0.008 part of VB1, 16 parts of agar and 800 parts of water, and controlling the ph value of the mutagenesis medium to be 6.3;
mixing a wild locust Fan Shikong bacterial fruiting body of the same strain as the spores with water according to a mass ratio of 1:5, boiling, and filtering to obtain a wild locust Fan Shikong bacterial stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of the spores in the same mutagenesis medium at the same time, wherein the length of the hyphae is 2mm, one hyphae is selected, and the volume ratio of the spore suspension to the mutagenesis medium is 1:2, culturing for 2d at 26 ℃, then irradiating with 60 Co-gamma rays with the irradiation dose of 1150Gy for three times at 2 hours intervals, wherein the three irradiation times are sequentially 20s, 40s and 60s, so as to obtain the locust Fan Shikong bacteria after mutagenesis;
culturing the mutation-treated locust Fan Shikong bacteria in the mutation medium in the second step, and continuously passaging the selected mutation-treated locust Fan Shikong bacteria for at least 3 times to obtain the mutation-treated locust Fan Shikong bacteria with more stable inheritance;
culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong;
the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 24 ℃ when the mother culture medium is subjected to tissue culture, white villiated mycelium grows out after 3d of culture, and after the mycelium extends to the mother culture medium, the top end part of the mycelium is picked by a fungus grafting needle, and the mycelium is inoculated to a new mother culture medium;
the preparation method of the mother culture medium comprises the following steps: washing 200g of fresh potatoes, peeling, cutting up, adding 1L of water into 20g of pagodatree bark, boiling for 25min, adding water into the filtrate filtered by gauze to supplement 1L, adding 18g of glucose, 18g of agar, 1.8g of monopotassium phosphate and 0.8g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized compound CPDA culture medium, subpackaging the unsterilized compound CPDA culture medium into test tubes, and subpackaging each test tube for about 5mL; sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use;
(3) Preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room at 25 ℃ for 25 days to obtain stock seeds of the locust Fan Shikong bacteria;
the stock culture medium comprises 75 parts of wood dust, 13 parts of bran, 4 parts of soybean powder, 1 part of sucrose and 0.5 part of gypsum powder, wherein the pH value of the stock culture medium is 5, and the water content of the stock culture medium is 55%;
(4) Preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at 25 ℃ for 25 days to obtain cultivated species of the locust Fan Shikong bacteria;
the culture medium for the cultivars comprises 75 parts of wood dust, 13 parts of bran, 4 parts of soybean powder, 1 part of sucrose and 0.5 part of gypsum powder, wherein the pH value of the stock culture medium is 5, and the water content is 55%;
(5) Ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 10 days, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, and harvesting when a large number of white spores are ejected from fruiting bodies and the fungus covers are not increased.
Examples
(1) Dissolving spores of wild locust Fan Shikong bacteria in sterile physiological saline, counting the selected wild locust Fan Shikong bacteria by using a blood cell counting plate, and diluting until the concentration order of spores in the spore suspension is 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 7 parts of wild locust Fan Shikong bacterial stock solution, 36 parts of corn starch, 17 parts of wheat bran, 9 parts of sucrose, 9 parts of glucose, 1.85 parts of monopotassium phosphate, 0.95 part of magnesium sulfate, 5.5 parts of yeast powder, 0.009 part of VB1, 17 parts of agar and 900 parts of water, and controlling the ph value of the mutagenesis medium to be 6.3;
mixing a wild locust Fan Shikong bacterial fruiting body of the same strain as the spores with water according to a mass ratio of 1:6, boiling, and filtering to obtain a wild locust Fan Shikong bacterial stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of spores in the step one into the same mutagenesis medium at the same time, wherein the volume ratio of the spore suspension to the mutagenesis medium is 1:2, culturing for 2d at 26 ℃, then irradiating with 60 Co-gamma rays with the irradiation dose of 1160Gy for three times at 2 hours intervals, wherein the three irradiation times are sequentially 20s, 40s and 60s to obtain the locust Fan Shikong bacteria after mutagenesis;
culturing the mutation-treated locust Fan Shikong bacteria in the mutation medium in the second step, and continuously passaging the selected mutation-treated locust Fan Shikong bacteria for at least 3 times to obtain the mutation-treated locust Fan Shikong bacteria with more stable inheritance;
(2) Culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong;
the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 25 ℃ when the mother culture medium is subjected to tissue culture, white villiated mycelium grows out after 3d of culture, and after the mycelium extends to the mother culture medium, the top end part of the mycelium is picked by a fungus grafting needle, and the mycelium is inoculated to a new mother culture medium;
the preparation method of the mother culture medium comprises the following steps: cleaning 210g of fresh potatoes, peeling, cutting up, adding 1.1L of water into 21g of pagodatree bark, boiling for 27min, adding water into the filtrate filtered by gauze to supplement 1L, adding 19g of glucose, 19g of agar, 1.9g of monopotassium phosphate and 0.9g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized composite CPDA culture medium, subpackaging the unsterilized composite CPDA culture medium into test tubes, and subpackaging each test tube for about 6mL; sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use;
(3) Preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room with the temperature of 25.5 ℃ for 26d to obtain stock seeds of the locust Fan Shikong bacteria;
the stock culture medium comprises 76 parts of wood dust, 14 parts of bran, 4.2 parts of soybean powder, 1.1 parts of sucrose and 0.7 part of gypsum powder, wherein the pH value of the stock culture medium is 5.2, and the water content of the stock culture medium is 56%;
(4) Preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at 25.5 ℃ for 26 days to obtain cultivated species of the locust Fan Shikong bacteria;
the culture medium for the cultivated species comprises 76 parts of wood dust, 14 parts of bran, 4.2 parts of soybean powder, 1.1 parts of sucrose and 0.7 part of gypsum powder, wherein the pH value of the stock culture medium is 5.2, and the water content is 56%;
(5) Ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 10.4 days, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, and harvesting when a large number of white spores are ejected from fruiting bodies and the caps are not increased;
examples
(1) Step one, taking wild locust vanDissolving spore of porus in sterile physiological saline, selecting wild Robinia pseudoacacia Fan Shikong, counting with blood cell counting plate, and diluting to spore concentration of 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 9 parts of wild locust Fan Shikong bacteria stock solution, 37 parts of corn starch, 19 parts of wheat bran, 10 parts of sucrose, 10 parts of glucose, 1.9 parts of monopotassium phosphate, 0.95 part of magnesium sulfate, 6 parts of yeast powder, 0.010 part of VB1, 18 parts of agar and 1000 parts of water, and controlling the ph value of the mutagenesis medium to be 6.4;
mixing a wild locust Fan Shikong bacterial fruiting body of the same strain as the spores with water according to a mass ratio of 1:7, boiling, and filtering to obtain a wild locust Fan Shikong bacterial stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of spores in the step one into the same mutagenesis medium, wherein the length of the hyphae is 2.8mm, the number of the hyphae is one, and the volume ratio of the spore suspension to the mutagenesis medium is 1:2, culturing for 2d at 26 ℃, then irradiating with 60 Co-gamma rays with 1170Gy of irradiation dose for three times at 2 hours intervals, wherein the three irradiation times are sequentially 20s, 40s and 60s to obtain the locust Fan Shikong bacteria after mutagenesis;
culturing the mutation-treated locust Fan Shikong bacteria in the mutation medium in the second step, and continuously passaging the selected mutation-treated locust Fan Shikong bacteria for at least 3 times to obtain the mutation-treated locust Fan Shikong bacteria with more stable inheritance;
culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong;
the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 25 ℃ when the mother culture medium is subjected to tissue culture, white villiated mycelium grows out after 3d of culture, and after the mycelium extends to the mother culture medium, the top end part of the mycelium is picked by a fungus grafting needle, and the mycelium is inoculated to a new mother culture medium;
the preparation method of the mother culture medium comprises the following steps: cleaning 220g of fresh potatoes, peeling, cutting up, adding 1.2L of water into 22g of pagodatree bark, boiling for 29min, adding water into the filtrate filtered by gauze to supplement 1L, adding 20g of glucose, 20g of agar, 2g of monopotassium phosphate and 1g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized compound CPDA culture medium, subpackaging the unsterilized compound CPDA culture medium into test tubes, and subpackaging each test tube for about 7mL; sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use;
(3) Preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room at 26 ℃ for 27 days to obtain stock seeds of the locust Fan Shikong bacteria;
the stock culture medium comprises 77 parts of wood dust, 15 parts of bran, 4.4 parts of soybean powder, 1.2 parts of sucrose and 0.9 part of gypsum powder, wherein the pH value of the stock culture medium is 5.4, and the water content of the stock culture medium is 57%;
(4) Preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at 26 ℃ for 27 days to obtain cultivated species of the locust Fan Shikong bacteria;
the culture medium for the cultivated species comprises 77 parts of wood dust, 15 parts of bran, 4.4 parts of soybean powder, 1.2 parts of sucrose and 0.9 part of gypsum powder, wherein the pH value of the stock culture medium is 5.4, and the water content of the stock culture medium is 57%;
(5) Ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 10.8 days, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, and harvesting when a large number of white spores are ejected from fruiting bodies and the caps are not increased.
Examples
(1) Dissolving spores of wild locust Fan Shikong bacteria in sterile physiological saline, counting the selected wild locust Fan Shikong bacteria by a blood cell counting plate, and diluting to the concentration of spores in spore suspensionOf the order of 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 11 parts of wild locust Fan Shikong bacteria stock solution, 38 parts of corn starch, 21 parts of wheat bran, 11 parts of sucrose, 11 parts of glucose, 2 parts of monopotassium phosphate, 1 part of magnesium sulfate, 6.5 parts of yeast powder, 0.011 part of VB1, 19 parts of agar and 1100 parts of water, and controlling the ph value of the mutagenesis medium to be 6.4;
mixing a wild locust Fan Shikong bacterial fruiting body of the same strain as the spores with water according to a mass ratio of 1:8, boiling, and filtering to obtain a wild locust Fan Shikong bacterial stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of spores in the step one into the same mutagenesis medium at the same time, wherein the volume ratio of the spore suspension to the mutagenesis medium is 1:3, culturing for 2d at 26 ℃, then irradiating with 60 Co-gamma rays with the irradiation dose of 1175Gy for three times at 2 hours intervals, wherein the three irradiation times are sequentially 20s, 40s and 60s, so as to obtain the locust Fan Shikong bacteria after mutagenesis;
culturing the mutation-treated locust Fan Shikong bacteria in the mutation medium in the second step, and continuously passaging the selected mutation-treated locust Fan Shikong bacteria for at least 3 times to obtain the mutation-treated locust Fan Shikong bacteria with more stable inheritance;
(2) Culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong;
the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 27 ℃ when the mother culture medium is subjected to tissue culture, white villiated mycelium grows out after 4d of culture, and after the mycelium extends to the mother culture medium, the top end part of the mycelium is picked by a fungus grafting needle, and the mycelium is inoculated to a new mother culture medium;
the preparation method of the mother culture medium comprises the following steps: washing 230g of fresh potatoes, peeling, cutting up, adding 1.3L of water into 23g of pagodatree bark, boiling for 31min, adding water into the filtrate filtered by gauze to supplement 1L, adding 21g of glucose, 21g of agar, 2.1g of monopotassium phosphate and 1.1g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized composite CPDA culture medium, subpackaging the unsterilized composite CPDA culture medium into test tubes, and subpackaging about 8mL of each test tube; sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use;
(3) Preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room with the temperature of 26.5 ℃ for 28 days to obtain stock seeds of the locust Fan Shikong bacteria;
the stock culture medium comprises 78 parts of wood dust, 16 parts of bran, 4.6 parts of soybean powder, 1.3 parts of sucrose and 1.1 parts of gypsum powder, wherein the pH value of the stock culture medium is 5.6, and the water content of the stock culture medium is 58%;
(4) Preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at 26.5 ℃ for 28 days to obtain cultivated species of the locust Fan Shikong bacteria;
the culture medium for the cultivated species comprises 78 parts of wood dust, 16 parts of bran, 4.6 parts of soybean powder, 1.3 parts of sucrose and 1.1 parts of gypsum powder, wherein the pH value of the stock culture medium is 5.6, and the water content is 58%;
(5) Ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 11.2 days, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, and harvesting when a large number of white spores are ejected from fruiting bodies and the caps are not increased.
Examples
(1) Dissolving spores of wild locust Fan Shikong bacteria in sterile physiological saline, counting the selected wild locust Fan Shikong bacteria by using a blood cell counting plate, and diluting until the concentration order of spores in the spore suspension is 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 13 parts of wild locust Fan Shikong bacteria stock solution, 39 parts of corn starch, 23 parts of wheat bran, 11 parts of sucrose, 11 parts of glucose, 2.1 parts of monopotassium phosphate, 1 part of magnesium sulfate, 6.8 parts of yeast powder, 0.011 part of VB1, 19 parts of agar and 1100 parts of water, and controlling the ph value of the mutagenesis medium to be 6.4;
mixing a wild locust Fan Shikong bacterial fruiting body of the same strain as the spores with water according to a mass ratio of 1:9, boiling, and filtering to obtain a wild locust Fan Shikong bacterial stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of the spores in the same mutagenesis medium at the same time, wherein the length of the hyphae is 3.6mm, the number of the hyphae is 2, and the volume ratio of the spore suspension to the mutagenesis medium is 1:3, culturing for 2d at 26 ℃, then irradiating with 60 Co-gamma rays with the irradiation dose of 1185Gy for three times at 2 hours intervals, wherein the three irradiation times are sequentially 20s, 40s and 60s to obtain the locust Fan Shikong bacteria after mutagenesis;
culturing the mutation-treated locust Fan Shikong bacteria in the mutation medium in the second step, and continuously passaging the selected mutation-treated locust Fan Shikong bacteria for at least 3 times to obtain the mutation-treated locust Fan Shikong bacteria with more stable inheritance;
culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong;
the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 24 ℃ when the mother culture medium is subjected to tissue culture, white villiated mycelium grows out after 3d of culture, and after the mycelium extends to the mother culture medium, the top end part of the mycelium is picked by a fungus grafting needle, and the mycelium is inoculated to a new mother culture medium;
the preparation method of the mother culture medium comprises the following steps: cleaning 240g of fresh potatoes, peeling, cutting up, adding 1.4L of water into 24g of pagodatree bark, boiling for 33min, adding water into the filtrate filtered by gauze to supplement 1L, adding 22g of glucose, 22g of agar, 2.2g of monopotassium phosphate and 1.3g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized composite CPDA culture medium, subpackaging the unsterilized composite CPDA culture medium into test tubes, and subpackaging each test tube for about 9mL; sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use;
(3) Preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room at the temperature of 27 ℃ for 29 days to obtain stock seeds of the locust Fan Shikong bacteria;
the stock culture medium comprises 79 parts of wood dust, 17 parts of bran, 4.8 parts of soybean powder, 1.4 parts of sucrose and 1.3 parts of gypsum powder, wherein the pH value of the stock culture medium is 5.8, and the water content of the stock culture medium is 59%;
(4) Preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at the temperature of 27 ℃ for 29 days to obtain cultivated species of the locust Fan Shikong bacteria;
the culture medium for the cultivated species comprises 79 parts of wood dust, 17 parts of bran, 4.8 parts of soybean powder, 1.4 parts of sucrose and 1.3 parts of gypsum powder, wherein the pH value of the stock culture medium is 5.8, and the water content is 59%;
(5) Ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 11.6 days, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, and harvesting when a large number of white spores are ejected from fruiting bodies and the caps are not increased.
Examples
(1) Dissolving spores of wild locust Fan Shikong bacteria in sterile physiological saline, counting the selected wild locust Fan Shikong bacteria by using a blood cell counting plate, and diluting until the concentration order of spores in the spore suspension is 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 15 parts of wild locust Fan Shikong bacteria stock solution, 40 parts of corn starch, 25 parts of wheat bran, 12 parts of sucrose, 12 parts of glucose, 2.2 parts of monopotassium phosphate, 1.1 parts of magnesium sulfate, 7 parts of yeast powder, 0.012 part of VB1, 20 parts of agar and 1200 parts of water, and controlling the ph value of the mutagenesis medium to be 6.5;
mixing a wild locust Fan Shikong bacterial fruiting body of the same strain as the spores with water according to a mass ratio of 1:10, boiling, and filtering to obtain a wild locust Fan Shikong bacterial stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of spores in the step one into the same mutagenesis medium at the same time, wherein the volume ratio of the spore suspension to the mutagenesis medium is 1:3, culturing for 2d at 26 ℃, then irradiating with 60 Co-gamma rays at a dose of 1200Gy for three times at a time interval of 2 hours for 20s, 40s and 60s in sequence to obtain the locust Fan Shikong bacteria after mutagenesis.
Culturing the post-mutagenesis locust Fan Shikong bacteria in the mutagenesis medium in the second step, and continuously passaging the selected post-mutagenesis locust Fan Shikong bacteria for at least 3 times to obtain the post-mutagenesis locust Fan Shikong bacteria with more stable inheritance.
The preparation method of the mother culture medium comprises the following steps: washing 250g of fresh potatoes, peeling, cutting up, adding 1.5L of water into 25g of pagodatree bark, boiling for 35min, adding water into the filtrate filtered by gauze to supplement 1L, adding 22g of glucose, 22g of agar, 2.2g of monopotassium phosphate and 1.5g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized composite CPDA culture medium, subpackaging the unsterilized composite CPDA culture medium into test tubes, and subpackaging each test tube for about 10mL; and (3) sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use.
(2) Culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong;
the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 28 ℃ when the mother culture medium is subjected to tissue culture, white villiated mycelium grows out after 4d of culture, and after the mycelium extends to the mother culture medium, the top end part of the mycelium is picked by a fungus grafting needle, and the mycelium is inoculated to a new mother culture medium;
the preparation method of the mother culture medium comprises the following steps: cleaning 240g of fresh potatoes, peeling, cutting up, adding 1.4L of water into 24g of pagodatree bark, boiling for 33min, adding water into the filtrate filtered by gauze to supplement 1L, adding 22g of glucose, 22g of agar, 2.2g of monopotassium phosphate and 1.3g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized composite CPDA culture medium, subpackaging the unsterilized composite CPDA culture medium into test tubes, and subpackaging each test tube for about 9mL; sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use;
(3) Preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room with the temperature of 28 ℃ for 30 days to obtain stock seeds of the locust Fan Shikong bacteria;
the stock culture medium comprises 80 parts of wood dust, 17 parts of bran, 5 parts of soybean powder, 1.5 parts of sucrose and 1.5 parts of gypsum powder, wherein the pH value of the stock culture medium is 6, and the water content of the stock culture medium is 60%;
(4) Preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at the temperature of 28 ℃ for 30 days to obtain cultivated species of the locust Fan Shikong bacteria;
the culture medium for the cultivated species comprises 80 parts of wood dust, 17 parts of bran, 5 parts of soybean powder, 1.5 parts of sucrose and 1.5 parts of gypsum powder, wherein the pH value of the stock culture medium is 6, and the water content is 60%;
ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 12 days, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, and harvesting when a large number of white spores are ejected from fruiting bodies and the fungus covers are not increased.
Examples
The mutagen dose was 500Gy, and the other protocols were the same as in example 1.
Examples
The mutagen dose was 1500Gy, and the other protocols were the same as in example 1.
The growth of the locust Fan Shikong strain obtained in examples 1 to 6 and the wild locust Fan Shikong strain in the control group CK were cultivated in a bag cultivation for two years under the same conditions as shown in Table 1.
Name of the name Whether or not to be able to people Work cultivation Growth status of mycelium Growth status of fruiting body Test fruiting body for continuous two-year cultivation Body per mu yield (jin/year)
Implementation of the embodiments Example 1 Is that The hypha is white, dense and strong, and has high growth speed and neatness Larger, the diameter is 13cm-15cm, the shape is regular in a sector shape, and the thickness of the fungus cover is 10mm-12mm 985
Implementation of the embodiments Example 2 Is that The hypha is white, dense and strong, and has high growth speed and neatness Larger, the diameter is 13cm-15cm, the shape is regular in a sector shape, and the thickness of the fungus cover is 10mm-12mm 1050
Implementation of the embodiments Example 3 Is that The hypha is white, dense and strong, and has high growth speed and neatness Larger, the diameter is 13cm-15cm, the shape is regular in a sector shape, and the thickness of the fungus cover is 10mm-12mm 993
Implementation of the embodiments Example 4 Is that The hypha is white, dense and strong, and has high growth speed and neatness Larger, the diameter is 13cm-15cm, the shape is regular in a sector shape, and the thickness of the fungus cover is 10mm-12mm 1120
Implementation of the embodiments Example 5 Is that The hypha is white, dense and strong, and has high growth speed and neatness Larger, the diameter is 13cm-15cm, the shape is regular in a sector shape, and the thickness of the fungus cover is 10mm-12mm 1066
Implementation of the embodiments Example 6 Is that The hypha is white, dense and strong, and has high growth speed and neatness Larger, the diameter is 13cm-15cm, the shape is regular in a sector shape, and the thickness of the fungus cover is 10mm-12mm 1043
Implementation of the embodiments Example 7 Is that The hypha is white, dense and strong, and has high growth speed and neatness Failure to form normal fruiting body, tumor formation 60
Implementation of the embodiments Example 8 Is that The hypha is white, dense and strong, and has high growth speed and neatness Failure to form normal fruiting body, tumor formation 75
Control Group CK Whether or not Mycelium-free (wild Robinia pseudoacacia Fan Shikong mycelium is milky white or off-white) Sparse and weak, slow growth speed and irregular shape No fruiting body (wild locust Fan Shikong fungus fruiting body is smaller, diameter 10cm-12cm, shape table) The surface is corrugated, the edge is irregular, and the thickness of the fungus cover is 6mm-8 mm) Unable to cultivate
The novel variety of the locust Fan Shikong bacterial fruiting body obtained by the method can be propagated when artificially cultivated, and after cultivation for 2 years under the same test condition, the higher quality of the locust Fan Shikong bacterial can be obtained in examples 1-6, wherein the best quality of the locust Fan Shikong bacterial obtained in example 4 under 1135Gy of irradiation dose is shown in FIG. 7 and the best quality of the novel variety of the locust Fan Shikong bacterial obtained in example 8 are shown in FIG. 8, the irradiation dose of 500Gy and 1500Gy cannot be obtained in FIG. 7, and the yield of the novel variety of the locust Fan Shikong bacterial obtained is higher as shown in FIG. 3 after continuous two years of tests, wherein the mycelium of the locust Fan Shikong bacterial under 1600 times of amplification of an optical microscope is shown in FIG. 4. The wild locust Fan Shikong bacteria are difficult to artificially cultivate, and the mycelium and fruiting body of the wild locust Fan Shikong bacteria have poor quality.
Although the present disclosure is disclosed above, the scope of the present disclosure is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the disclosure, and such changes and modifications would be within the scope of the disclosure.

Claims (9)

1. The mutagenesis method based on wild locust Fan Shikong bacteria is characterized by comprising the following steps:
dissolving spores of wild locust Fan Shikong bacteria in sterile physiological saline for dilution, and counting by a blood cell counting plate until the concentration of spores in the spore suspension is 10 7 personal/mL-10 8 Obtaining spore suspension by using each mL;
step two, preparing a mutagenesis medium: the mutagenesis medium consists of the following raw materials in parts by weight: 5-15 parts of wild locust Fan Shikong bacterial stock solution, 35-40 parts of corn starch, 15-25 parts of wheat bran, 8-12 parts of sucrose, 8-12 parts of glucose, 1.8-2.2 parts of monopotassium phosphate, 0.9-1.1 part of magnesium sulfate, 5-7 parts of yeast powder, 0.008-0.012 part of VB1, 16-20 parts of agar and 800-1200 parts of water, and controlling the ph value of the mutagenesis medium to be 6.3-6.5;
mixing the fruiting body of wild locust Fan Shikong bacteria of the same strain as the spores with water according to the mass ratio of 1 (5-10), boiling, and filtering to obtain a wild locust Fan Shikong bacteria stock solution;
step three, inoculating the spore suspension and hyphae of wild locust Fan Shikong bacteria of the same strain of spores in the step one into the same mutagenesis medium at the same time, wherein the volume ratio of the spore suspension to the mutagenesis medium is 1: (2-3), and then culturing at 26℃for 2d, and reutilizing 60 Co-gamma ray irradiation, wherein the irradiation dose is 1150Gy-1200Gy, the irradiation is performed three times, the time interval is 2 hours, and the irradiation time of the three times is sequentially 20s, 40s and 60s, so as to obtain the locust Fan Shikong bacteria after mutagenesis;
step four: culturing the obtained locust Fan Shikong strain after mutagenesis in the mutagenesis medium in the second step again, and continuously carrying out passage for at least 3 times to obtain the locust Fan Shikong strain after mutagenesis with stable heredity.
2. A method for cultivating the strain of acacia Fan Shikong, wherein the strain of acacia Fan Shikong is obtained by the mutagenesis method of claim 1, comprising the steps of:
culturing mother strain, aseptically inoculating the strain of Robinia pseudoacacia Fan Shikong after mutagenesis into mother strain culture medium for tissue culture, purifying for 6-8 days, and culturing to obtain pure mother strain of Robinia pseudoacacia Fan Shikong; the purification steps are as follows: after mutagenesis, the temperature of the locust Fan Shikong bacteria is kept at 24-28 ℃ when the mother culture medium is used for tissue culture, white villiated mycelium grows out after 3-4 d culture, after the mycelium extends to the mother culture medium, a fungus inoculating needle is used for picking the top part of the mycelium, and the mycelium is inoculated to a new mother culture medium;
the mother culture medium is a composite CPDA culture medium added with pagodatree bark juice and peptone;
preparing stock seeds, namely activating and propagating pure stock seeds of the locust Fan Shikong bacteria, then inoculating a stock seed culture medium, and culturing in a culture room at 25-28 ℃ for 25-30 d to obtain stock seeds of the locust Fan Shikong bacteria;
preparing cultivated species, namely inoculating the stock seeds of the locust Fan Shikong bacteria into a cultivated species culture medium, and culturing in a culture room at the temperature of 25-28 ℃ for 25-30 days to obtain a cultivated species of the locust Fan Shikong bacteria;
ear-growing management and harvesting, namely continuously culturing the cultivation seeds of the locust Fan Shikong bacteria for 10d-12d, then transferring the cultivation seeds to a fruiting room for fruiting, keeping ventilation and humidity during fruiting, and harvesting when a large number of white spores are ejected from fruiting bodies and the caps are not increased.
3. The cultivation method according to claim 2, wherein the composite CPDA medium is composed of the following raw materials in parts by weight: 200-250 parts of fresh potatoes, 20-25 parts of pagodatree bark, 1000-1500 parts of water, 18-22 parts of glucose, 18-22 parts of agar, 1.8-2.2 parts of monopotassium phosphate and 0.8-1.5 parts of magnesium sulfate.
4. A cultivation method according to claim 3, wherein said composite CPDA medium is prepared by the steps of: cleaning and peeling fresh potatoes, and cutting into pieces to obtain potato blocks; adding 200g-250g of potato blocks and 20g-25g of pagodatree bark into 1L-1.5L of water, boiling for 25min-35min, adding water into filtrate after gauze filtration to supplement 1L, adding 18g-22g of glucose, 18g-22g of agar, 1.8g-2.2g of monopotassium phosphate and 0.8g-1.5g of magnesium sulfate, heating and fully dissolving to obtain an unsterilized composite CPDA culture medium, subpackaging the unsterilized composite CPDA culture medium into test tubes, and subpackaging 5mL-10mL of each test tube; and (3) sterilizing the sub-packaged and unsterilized composite CPDA culture medium test tube for 20 minutes at 121 ℃, taking out the test tube, placing the test tube on an inclined plane, cooling and storing for later use.
5. The cultivation method according to claim 4, wherein: the temperature is kept at 24-28 ℃ during the culture period of the mother strain, the relative air humidity is controlled below 75%, and the mother strain is cultivated in a dark place.
6. The cultivation method according to claim 5, wherein said stock culture medium and cultivar culture medium each comprise: 75-80 parts of miscellaneous wood chips, 13-17 parts of bran, 4-5 parts of soybean meal, 1-1.5 parts of sucrose, 0.5-1.5 parts of gypsum powder, wherein the stock culture medium comprises pH value of 5-6 and water content of 55% -60%.
7. Sophora japonica Fan Shikong strainVanderbylia robiniophila) The method is characterized in that: the preservation number is CGMCC No.40140, the preservation is preserved in China general microbiological culture Collection center (China Committee for culture Collection), the preservation address is No. 1, no. 3, and the preservation time is 2022, 4 months and 24 days.
8. Use of the locust Fan Shikong bacteria obtained by the cultivation method according to any of claims 2 to 6, characterized in that: the fruiting body of the Robinia pseudoacacia Fan Shikong is used for preparing antitumor drugs.
9. The use of the fungus acacia Fan Shikong as defined in claim 8, wherein: the antitumor drugs include drugs for treating liver cancer, lung cancer, bone cancer, breast cancer and pancreatic cancer.
CN202210981857.0A 2022-08-16 2022-08-16 Mutagenesis and cultivation method of wild locust Fan Shikong bacteria, locust Fan Shikong bacteria and application Active CN115316196B (en)

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