CN114214208A

CN114214208A - High-selenium-resistance ganoderma lucidum mutant strain and application thereof

Info

Publication number: CN114214208A
Application number: CN202111524579.8A
Authority: CN
Inventors: 杨梦莲; 沈微; 单逸蓝; 陈献忠; 杨海泉; 夏媛媛; 陈磊
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-22
Anticipated expiration: 2041-12-14
Also published as: CN114214208B

Abstract

The invention relates to a high selenium-resistant ganoderma lucidum mutant strain and application thereof, belonging to the technical field of fungal breeding. The ganoderma lucidum sporocarp is collected in a selenium-rich area to obtain a mycelium culture, spores of the mycelium culture are collected and then subjected to mutation breeding, and enrichment culture is carried out in a high-selenium concentration culture medium to obtain the ganoderma lucidum mycelium with improved selenium resistance. The obtained Ganoderma lucidum mutant with improved selenium resistance is named as Ganoderma sp.ML2613, the mycelium culture of the mutant is preserved in China center for type culture Collection, and the preservation number is CCTCC NO: m20211217. The mutant Ganoderma sp.ml2613 can maintain normal growth in culture broth with selenium content of 1600mg/L compared to the original strain mycelium culture, while the original strain mycelium grows very slowly in the same fermentation broth. ML2613 is more suitable for culturing in high selenium content environment, has the potential of obtaining selenium-rich mycelium, and is also beneficial to field culture of Ganoderma lucidum in high selenium environment.

Description

High-selenium-resistance ganoderma lucidum mutant strain and application thereof

Technical Field

The invention relates to a high selenium-resistant ganoderma lucidum mutant strain and application thereof, in particular to a ganoderma lucidum mutant which is obtained by a mutation breeding method and can maintain a high growth speed under the condition of high selenium concentration and application thereof, and belongs to the technical field of fungal breeding.

Background

Ganoderma lucidum (Ganoderma lucidum) is a large fruiting body fungus which mainly saprophytic or parasitized on wood material or tree in nature, and belongs to Basidiomycota. Ganoderma lucidum and Ganoderma sinensis, Ganoderma Applanatum and other fungi belonging to the same genus as Ganoderma lucidum belong to medicinal or edible fungi with health promotion or treatment function in traditional Chinese medicine.

Selenium is a composition of various selenium-containing active proteins of a human body, the content of selenium element on the earth surface is low, the selenium obtained from normal food of the human body can not completely meet the requirements of the human body sometimes, and the selenium-rich food is an important way for helping people to supplement the selenium. Ganoderma lucidum has strong selenium enrichment capability, and Chinese science and technology workers have conducted more researches on adopting selenium enrichment of Ganoderma lucidum for many years. In the Lichuan city of Hubei province and Shitai county of Anhui province in China, small areas with high selenium content in soil exist, and lucid ganoderma growing in the small areas possibly have high selenium resistance and selenium enrichment capacity.

Disclosure of Invention

The invention aims to overcome the defects and provide a high selenium-resistant ganoderma lucidum mutant strain and application thereof.

The technical scheme of the invention is that a high selenium-resistant Ganoderma lucidum mutant strain ML2613 is preserved in China Center for Type Culture Collection (CCTCC) at Wuhan university, China, and is classified and named as Ganoderma lucidum ML2613Ganoderma sp.ML2613, and the preservation number is CCTCC NO: m20211217, preservation date 2021, 9 months and 26 days.

The ITS sequence of the high selenium-resistant ganoderma lucidum mutant strain ML2613 is shown in SEQ ID No. 1.

According to the preparation method of the high selenium-resistant ganoderma lucidum mutant strain ML2613, ganoderma lucidum in selenium-rich areas is adopted to carry out mycelium separation culture, selenium-resistant ganoderma lucidum is screened out, spore powder is collected after continuous culture, mutation is carried out on the spore powder, a mycelium colony is obtained through culture, a strain with the highest growth speed is selected, and finally the high selenium-resistant ganoderma lucidum mutant strain ML2613 is obtained.

Further, the mutagenesis method is specifically Co⁶⁰And (4) irradiating.

Further, the irradiation dose was controlled to 0.4 to 0.6 kGy.

Further, the normal saline suspension of the spore powder obtained by mutagenesis is transferred into a PDA liquid culture medium with high selenium content for culture for 6-8 days.

Further, the high selenium content is 1800 mg/L.

The application of the strain is to cultivate the ganoderma lucidum in a high-selenium environment.

Further, the efficiency of obtaining selenium-rich mycelia is improved.

Furthermore, the growth speed is high in the environment with the selenium content of 1600 mg/L.

The invention has the beneficial effects that: compared with the mycelium culture of the original strain, the mutant Ganoderma sp.ML2613 of the strain obtained by screening can maintain normal growth in a culture solution with the selenium content of 1600mg/L, and the mycelium of the original strain grows extremely slowly in the same fermentation culture solution. ML2613 is more suitable for culturing in high selenium content environment, has the potential of obtaining selenium-rich mycelium, and is also beneficial to field culture of Ganoderma lucidum in high selenium environment.

Biological material sample preservation: a high selenium-resistant Ganoderma lucidum mutant strain ML2613 is preserved in China Center for Type Culture Collection (CCTCC) at Wuhan university, Wuhan, China, and is classified and named as Ganoderma lucidum ML2613Ganoderma sp.ML2613, and the preservation number is CCTCC NO: m20211217, preservation date 2021, 9 months and 26 days.

Drawings

FIG. 1ST308 shows the increase curve of the bacterial biomass in the fermentation medium.

FIG. 2 increase curves of bacterial mass in high selenium fermentation medium for ST308 and ML 2613.

Fig. 3 growth curve of Ganoderma sp.ml2613 in fermentation medium.

Fig. 4 growth curves of Ganoderma sp.ml2613 and Ganoderma sp.st308 in high selenium fermentation medium.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.

Example 1 preparation of highly selenium-resistant Ganoderma lucidum ML2613

(1) Collecting and separating culture of ganoderma lucidum sporocarp: in selenium-rich areas such as Lichuan county in Hubei province and Shitai county in Anhui province in China, Ganoderma lucidum growing on rotten wood, branches, tree roots and the like is searched, and the Ganoderma lucidum growing on the rotten tree roots is collected in an important manner. And (4) transporting the collected lucid ganoderma to a laboratory on the day of sampling to perform mycelium separation culture. The white mycorrhiza with long and thin root is first washed with tap water and then washed with great amount of sterile water. Soaking in 3 wt% hydrogen peroxide solution for about 5min, and washing with sterile water. And finally inoculating the washed ganoderma lucidum mycelia to a PDA culture medium for about 1 week.

If the culture has colored spores such as black, green and the like, the culture is judged to be the mold or the culture polluted by the mold and is not used any more; and if the mycelium is pure white, thick and extended to the periphery, the mycelium is primarily determined to be the mycelium of the ganoderma lucidum.

The above culture process can obtain more than 100 Ganoderma mycelia culture.

(2) Screening selenium-resistant ganoderma lucidum: the mycelium was inoculated into PDA solid medium containing selenium 3200mg/L, respectively. The results showed that 11 of these strains grew vigorously on the medium, while the other strains grew slowly or could not grow. The mycelium culture of the above 11 strains was inoculated with a high selenium fermentation medium containing 1400mg/L of selenium and growth was observed, showing that the strain originally numbered ST308 had a significantly faster growth rate. The strain ST308 is respectively preserved in an industrial microorganism resource information platform of the education department of the university of Jiangnan and a strain bank of Xianqin Biotech Co., Ltd, and the preservation numbers are CICICIM F7083 and XQ 5258.

(3) Mutagenesis of ganoderma lucidum and screening of high selenium-resistant ganoderma lucidum mutants:

a. collecting: the mycelium of the ST308 strain is inoculated to a wood chip culture medium. After about 5 months of culture, the part of the mycelium in the culture medium close to the periphery of the triangular flask shows the growth of the lucid ganoderma sporocarp, and the sporocarp is taken out to be inoculated into the wood chip culture medium without water. About 2 weeks of cultivation, some spore powder was observed on the aluminum foil around the partially cultured Ganoderma lucidum. Taking out the spore powder, collecting the spore powder in a sterile centrifuge tube, concentrating the spore powder collected for many times, and suspending the spore powder by using about 10mL of physiological saline;

b. mutagenesis: subjecting the suspended spore powder to Co⁶⁰Irradiation dose was controlled to about 0.5 kGy. Transferring the mutagenized spore suspension into a high-selenium PDA liquid culture medium with the selenium content of 1800mg/L for culture for 7 days; after dilution, the culture broth was plated with solid PDA medium containing 4000mg/L selenium, for a total of about 1000 plates. After about 1 month of culture, about 300 weakly growing mycelium colonies were formed on PDA medium in about 60 plates. The mycelia were transferred to a new high-selenium PDA plate with a selenium content of 4000mg/L, respectively. Approximately 120 mycelium colonies were obtained after approximately 5 days of culture. Inoculating the mycelium into a high-selenium PDA liquid culture medium with the selenium content of 1600mg/L, performing shake culture at 30 ℃ and 120r/min, and sampling at intervals of 24 hours from 48 hours to detect the dry weight of the mycelium. The results show that the growth rate of one strain with the number of ML2613 is obviously higher than that of other strains, and the strain is judged to be a selenium-resistant mutant strain.

(4) Identification and preservation of selenium-resistant mutant strains: and (3) culturing the selenium-resistant mutant ML2613 mycelium to extract chromosomes, and then analyzing ITS gene sequences. The result shows that the ITS sequence of ML2613 is completely consistent with the ITS gene sequence of the starting bacterium ST308, and the sequence is the same as SEQ ID NO. 1. According to the naming rule of fungi, the mycelium culture of the selenium-resistant mutant ML2613 is named as: ganoderma sp.ml2613, the mycelium culture of which has been deposited at the chinese type culture collection (university of wuhan, china) with the deposit number CCTCC NO: m20211217, preservation time 2021, 9 months and 26 days.

Materials and methods:

basal PDA culture medium: cleaning potato 400g, peeling, cutting into small pieces with thickness of about 3mm, adding water to 1L, heating and boiling for 10min, and filtering solid part with gauze. The filtrate was taken and water was added to 700 mL. Adding glucose according to the amount of 20g of final culture medium per liter to obtain a basic PDA liquid culture medium; and adding agar powder according to the amount of 15g/L of the final culture medium per liter to obtain the basic solid PDA culture medium. The culture medium is prepared by adopting a 1L triangular flask, 1L water is measured by using a measuring cylinder and added into the triangular flask before preparation, and a 1L volume water level line is accurately marked. Sterilizing the above components at 110 deg.C for 10 min.

In actual use, the sterilized basal culture medium is supplemented with sodium selenite aqueous solution and water according to the following preparation method of the high-selenium culture medium to prepare PDA culture media with different selenium contents. The culture medium only supplemented with water and not supplemented with sodium selenite solution is generally referred to as PDA culture medium or solid PDA culture medium.

High selenium culture medium: preparing 90g/L aqueous solution of sodium selenite pentahydrate, and filtering and sterilizing by using a bacterial filter to obtain the sodium selenite solution. When preparing the PDA culture medium or the fermentation culture medium, adding the sodium selenite solution with different volumes after the culture medium is sterilized to obtain the selenium-containing culture medium with corresponding concentration. The molecular weight of the sodium selenite pentahydrate is 262, the atomic weight of the selenium is 79, and the content of the selenium in the sodium selenite pentahydrate is 30%.

When 3200mg/L of the high selenium medium used in example 1 was prepared, 700mL of the previously sterilized medium was mixed with 118mL of the above sodium selenite solution, and 182mL of sterile water was added thereto to obtain 1L of the high selenium medium having a selenium content of 3200 mg/L. In practice, the amount of the final sterile water is based on the pre-marked 1L water line, since the sterilization process will cause the change of the volume of the culture medium. The high selenium culture medium with other concentration is prepared in the same way, for example, the high selenium culture medium with 2500mg/L is prepared by mixing 700mL of sterilized culture medium with 92mL of the above sodium selenite solution, and then adding about 108mL of water to obtain 1L of high selenium culture medium with 2500mg/L of selenium content.

Fermentation medium: 40g of glucose, 0.2g of monopotassium phosphate, 1g of urea, 0.1g of anhydrous calcium chloride, 0.05g of anhydrous magnesium chloride, 10g of malt juice powder and 5g of yeast powder. Generally, 700mL of water is taken, the components are added, mixed and dissolved to obtain an initial culture medium, and after preparation, the initial culture medium is sterilized at 110 ℃ for 15min, and meanwhile, sterile water is prepared. When the culture medium is used, if a common selenium-free culture medium is prepared, water is directly supplemented to 1L, and the fermentation culture medium with the total volume of 1L is obtained. If the high-selenium fermentation culture medium is prepared, adding sodium selenite solution with corresponding concentration into the initial culture medium, and further supplementing water to 1L to obtain the high-selenium fermentation culture medium. The high-selenium fermentation medium generally refers to a medium prepared according to the method and having a selenium content of 1600 mg/L.

Wood chip culture medium: taking oak sawdust, drying in the sun, and crushing into small wood blocks with the size of 2 mm. Generally, 100mL of the small wood blocks and 10g of bran are put into a 250mL triangular flask, 100mL of water is added, an aluminum foil is covered on the small wood blocks and the bran, and a layer of cobblestone pebbles with the diameter of about 10mm is put on the aluminum foil. The triangular bottle is sealed by a sterile filter membrane and then sterilized to obtain the wood chip culture medium. In the inoculation process, a liquid transfer device is generally used for sucking 1mL of ganoderma lucidum mycelium culture solution, a small hole is formed in the center of an aluminum foil, and the culture solution is injected into lower-layer sawdust. Oak sawdust is collected by peasant households of Wuxi Nubia mountain.

Wood chip culture medium without water: preparing culture medium according to the above method, wherein the water addition amount is reduced from 100mL to 35mL, covering a layer of aluminum foil on the culture medium after preparation, and making a small hole with a diameter of about 20mm in the center of the aluminum foil for inoculating Ganoderma encarpium. The triangular bottle is sealed by a sterile filter membrane and then sterilized to obtain the water-deficient wood chip culture medium. In general, the fruit body grown in the wood chip culture medium is taken out together with the lower part of the fungal cord and a small amount of wood chips, and pricked into the small hole in the center of the aluminum foil.

Measurement of the Dry weight of the cells: the method 10 of the same document [ Shenmu, Wangbong, Zhujincun, etc. ] is a pullulanase chimera with improved performance and a Pichia pastoris mutant strain for highly producing the chimera, patent number is 201610306855.6.

The chromosome DNA extraction and ITS gene sequence analysis method comprises the following steps: the chromosome extraction method is the same as the literature (Zhouyinglingu, Shenwei, Lougangming, Wangzhenxiang, Zhugejian. a method for quickly extracting the chromosome DNA of fungi [ J ] microbiological report, 2004,31(4): 89-92); the method for further carrying out ITS gene amplification and sequence analysis after chromosome extraction is the same as the method related to ITS gene identification in the literature (Chenyuan source, Shenwei, Shigui Yang, Wangzhengxiang, comparison of two molecular markers in yeast molecular identification [ J ]. food industry science and technology, 2011,32(2): 175-.

The starting bacterium Ganoderma sp.ST308 used by the invention is preserved in an industrial microorganism resource platform of China colleges and universities in south Jiangnan university, and the strain number is as follows: CICIM F7083.

Example 2 stability of mutant strains Ganoderma sp.ML2613

The original strain Ganoderma lucidum ST308 and Ganoderma lucidum mutant ML2613 were inoculated into the fermentation medium of the material method of example 1, respectively, and cultured at 30 ℃ for 5 days. Transferring new fermentation medium according to 1/100 volume, and transferring new fermentation medium according to 1/100 inoculum size after culturing for 5 days. This was repeated 10 times.

The originally preserved starting strain ST308 and mutant strain ML2613, and the starting strain and the mutant strain (named as ST308b and ML2613b respectively for convenience of description) which are transferred by the shake flask are respectively inoculated with a fermentation medium and a high-selenium fermentation medium with the selenium content of 1600 mg/L.

The results show that the growth rates of the four bacteria in the fermentation medium without adding selenium are basically consistent, no obvious difference is seen, the time for achieving the highest bacterial quantity by shake flask fermentation after inoculation according to the inoculation amount of 1 percent is 4 days, and the highest bacterial quantities achieved are basically the same.

FIG. 1 is a graph showing the increase in the amount of bacteria in the fermentation medium in ST308, which is the average data of 3 replicates, with the error of data within 10% for three replicates. The same experiments are also carried out in ST308b, ML2613 and ML2613b, each strain is subjected to three parallel experiments, and the data error between the parallel experiments is within 10%. The average values of the cell amounts at the respective time points in the cell amount growth curves of the above four strains were compared, and the difference between the cell amounts at the respective time points was within 5%. The highest bacterial count was reached after 96hr fermentation. The highest bacterial loads were as follows: 19.1g/L ST308, 19.0g/L ST308b, 19.5 ML2613, and 19.2 ML2613 b.

In the high-selenium fermentation medium with the selenium content of 1600mg/L, the time for reaching the highest bacterial amount by ML2613 and ML2613b is 4 days, and the highest bacterial amounts reached by the ML2613 and the ML2613b are basically consistent and are not obviously different from the highest bacterial amounts reached by the ML2613 and the ML2613 in the selenium-free fermentation medium.

FIG. 2 is a plot of increase in the amount of bacteria in ML2613 and ST308 in a high selenium fermentation medium with a selenium content of 1600mg/L, the experimental data of the plot is the average data of 3 replicates, and the data error of three replicates is within 10%. As can be seen from FIG. 2, the time taken for ML2613 to reach the maximum bacterial load was 96hr, and the maximum bacterial load reached was 19.3g/L, which is consistent with the time taken for ML2613 to reach the maximum bacterial load in the selenium-free fermentation medium and the maximum bacterial load reached. However, the mass of the ML2613 in the early fermentation stage when the ML2613 is cultured in the high-selenium fermentation medium is obviously smaller than that in the selenium-free fermentation medium, the mass of the ML2613 in the early fermentation stage is close to that in the selenium-free fermentation medium when the ML2613 is fermented for 72 hours, and finally the ML2613 in the early fermentation stage reaches the same mass as that in the selenium-free fermentation medium at the end point of 96 hours. The same experiment was performed for ML2613b, and three parallel experiments were performed in the same manner, with data errors between the parallel experiments within 10%. The bacterial body quantity growth curve of ML2613b in the high selenium fermentation medium with the selenium content of 1600mg/L is consistent with the growth curve of ML2613 in figure 2, and the difference between the bacterial body quantities of ML2613 and ML2613b at each time point is within 5%. It can be seen that ML2613 and ML2613b grow substantially the same in a high selenium fermentation medium with a selenium content of 1600 mg/L.

The same experiment was carried out for ST308, and the growth of ST308 was extremely slow in the high-selenium fermentation medium with a selenium content of 1600mg/L, and the amount of the bacterial mass reached after 108 hours of culture was less than 1.2g, as shown in FIG. 2. The results of the test in ST308b were almost identical to those in ST308, and the amount of the cells reached less than 1.2g after 108 hours of culture.

The experiment shows that the ML2613 has no obvious degradation after multiple passages in the selenium-free culture medium, and is a selenium-resistant mutant strain with stable genetic performance.

Example 3 fermentation Performance of mutant strains Ganoderma sp.ML2613

Inoculating PDA liquid culture medium to mycelium culture of Ganoderma original strain ST308 and mutant strain ML2613, culturing for 5 days, inoculating fermentation culture medium according to 1/100 volume, and comparing fermentation performance. Wherein the experimental results of ML2613 are shown in FIG. 3. The experimental data of the graph are average data of 5 parallel experiments, and the data error of the parallel experiments is within 10%. The experimental result of the development ST308 is not obviously different from ML 2613.

Inoculating fermentation culture medium to mycelium culture of Ganoderma initial strain ST308 and mutant strain ML2613, culturing for 5 days, inoculating high selenium fermentation culture medium with selenium content of 1600mg/L according to volume of about 1/100, and comparing selenium-resistant fermentation performance.

The experimental results are shown in fig. 4, each point in the graph is the average data of 5 parallel experiments, and the data error of the parallel experiments is within 10%. As can be seen from fig. 4, the growth rate of ML2613 was significantly higher in the high selenium fermentation medium than ST 308. ML2613 can reach the highest thalli volume when culturing for 4 days, there is about 19.1g dry thalli per liter of fermentation liquor, and the starting bacterium ST308 only has about 1g dry thalli under the same fermentation condition and the same fermentation time. If the cultivation is continued, the bacterial count of ST308 can be increased to a maximum value of about 5g at about 15 days. It can be seen that the growth of the starting bacterium ST308 in the high-selenium culture medium is obviously inhibited, the growth of ML2613 is not obviously inhibited, the growth speed is obviously higher than that of the starting bacterium ST308, and the final bacterium amount is also obviously higher than that of the starting bacterium.

ML2613 is more suitable for culturing under the high selenium culture condition, is more suitable for obtaining the mutant strain with stable performance of the selenium-rich thallus.

Sequence listing

<110> university of south of the Yangtze river

<120> high selenium-resistant ganoderma lucidum mutant strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 428

<212> DNA

<213> ITS gene sequence of Ganoderma sp. ML2613 (2 Ambystoma laterale x Ambystoma jeffersonanium)

<400> 1

ttttgaaagt ggagtctgct gctggtgtgg aaacatgcat gtgcacggct ctgtgctcaa 60

gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat 120

catcgaatct ttgaacgcac cttgtgctcc ttggtattcc gaggagcatg cctgtttgag 180

tgtcatgaaa tcttcaacct acaagcttct gtggtttgta ggcttagact tggaggcttg 240

tcggccgtca tcggtcggct cctcttaaat gcattagctt ggttccttgc ggatcggctc 300

tcggtgtgat aatgtctacg ccgtgaccgt gaagcgtttg gcaagcttct aaccgtctta 360

taagacagct ttatgacctc tgacctcaaa tcaggtagga ctacccgctg aacttaagca 420

tatcaata 428

Claims

1. A high selenium-resistant Ganoderma lucidum mutant strain ML2613 is preserved in China Center for Type Culture Collection (CCTCC) at Wuhan university, Wuhan, China, and is classified and named as Ganoderma lucidum ML2613Ganoderma sp.ML2613, and the preservation number is CCTCC NO: m20211217, preservation date 2021, 9 months and 26 days.

2. The selenium-tolerant ganoderma lucidum mutant strain ML2613 as claimed in claim 1, wherein: the ITS sequence is shown in SEQ ID No. 1.

3. The method for preparing the selenium-resistant ganoderma lucidum mutant strain ML2613 as claimed in claim 1, is characterized in that: the method comprises the steps of performing mycelium separation culture on ganoderma lucidum in selenium-rich areas, screening selenium-resistant ganoderma lucidum, continuously performing culture, collecting spore powder, performing mutagenesis on the spore powder, obtaining mycelium colonies through culture, selecting a strain with the highest growth speed, and finally obtaining a high-selenium-resistant ganoderma lucidum mutant strain ML 2613.

4. The method for preparing the selenium-resistant ganoderma lucidum mutant strain ML2613 as claimed in claim 3, wherein the method comprises the following steps: the mutagenesis method is specifically Co⁶⁰And (4) irradiating.

5. The method for preparing the selenium-resistant ganoderma lucidum mutant strain ML2613 as claimed in claim 4, wherein the method comprises the following steps: the irradiation dose is controlled to be 0.4-0.6 kGy.

6. The method for preparing the selenium-resistant ganoderma lucidum mutant strain ML2613 as claimed in claim 5, wherein the method comprises the following steps: and transferring the physiological saline suspension of the spore powder obtained by mutagenesis into a PDA liquid culture medium with high selenium content for culture for 6-8 days.

7. The method for preparing the selenium-resistant ganoderma lucidum mutant strain ML2613 as claimed in claim 6, wherein the method comprises the following steps: the high selenium content is 1800 mg/L.

8. Use of the strain according to claim 1, characterized in that: the method is applied to the cultivation of the lucid ganoderma in the high-selenium environment.

9. Use of a strain according to claim 8, characterized in that: improving the efficiency of obtaining the selenium-rich mycelium.

10. Use of a strain according to claim 8, characterized in that: the growth speed is high in the environment with the selenium content of 1600 mg/L.