CN116640673B - Low-temperature-resistant straw mushroom strain and preparation method thereof - Google Patents
Low-temperature-resistant straw mushroom strain and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 7
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- 230000000694 effects Effects 0.000 abstract description 22
- 102000006995 beta-Glucosidase Human genes 0.000 abstract description 20
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- 238000002474 experimental method Methods 0.000 abstract description 12
- 238000012216 screening Methods 0.000 abstract description 10
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- 235000002872 Volvariella volvacea V23 Nutrition 0.000 abstract description 6
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Abstract
The low temperature resistant straw mushroom strain has the preservation numbers of: cctccc No. M2023706. The preparation method of the strain comprises the steps of preparing the volvariella volvacea V23 strain spore liquid, and then adopting a normal-pressure room-temperature plasma mutagenesis technology to mutagenize the volvariella volvacea spore liquid; then, the induced strain is subjected to primary screening at 4 ℃; obtaining low temperature resistant mutant strains through antagonism, ITS sequencing and phylogenetic analysis classification and identification technology; screening the mutant strain with low beta-glucosidase activity under low temperature stress again by adopting the beta-glucosidase index; and combining fruiting and low temperature resistance experiments to verify the low temperature resistance of the mutant strain. The invention discovers that the mutant strain VG39 has certain low temperature stress resistance.
Description
Technical Field
The invention belongs to the field of foods, and relates to a breeding method of a straw mushroom strain, in particular to a low-temperature-resistant straw mushroom strain and a preparation method thereof.
Background
Straw mushroom (Volvariella volvacea) is called as Chinese mushroom, has delicious taste and has important medical care effects such as antioxidation, immunity regulation, anti-tumor and the like. The straw mushroom has short growth period (about 10 days), and is an edible mushroom with high economic benefit. The industrialized cultivation of straw mushrooms mainly uses straw and waste cotton as main matrixes, and is one of main varieties for degrading straw. In view of the annual production of crop straws of about 8 hundred million tons in China, the huge economic benefit of straw mushroom degradation straw will assist the development of resource saving and environment-friendly modern new agriculture.
The optimum temperature required for the growth and setting of the mycelium of straw mushrooms is (32+/-1 ℃), but at the conventional low temperature of 4 ℃, the mycelium and fruiting body soften, liquefy and even decay in a short time, which is known as 'low temperature autolysis'. The uniqueness of the straw mushroom provides a fresh-keeping problem, so that the shelf life of the straw mushroom is obviously shorter than that of other mushrooms. The low-temperature autolysis property of the straw mushroom variety greatly limits the cultivation and popularization of the straw mushroom variety, and indirectly influences the economic benefit of degrading straw. The cultivation of new varieties of low-temperature-resistant straw mushrooms is the most direct achievement transformation target for promoting the cultivation and popularization of straw mushrooms.
The normal pressure room temperature plasma (Atmospheric and room temperatureplasma, ARTP) is used for mutation breeding, and the permeability of a microbial cell membrane is changed by utilizing various electron flows generated by ARTP during discharge, so that genetic materials are damaged to induce the mutation. The technology is simple to operate, can operate in an environment of normal pressure and room temperature, has high safety, and simultaneously achieves better practical effect. Therefore, by mutagenesis of the volvariella volvacea spore liquid by the ARTP mutagenesis breeding technology, it is possible to screen low temperature resistant volvariella volvacea mutant strains.
Straw mushroom V23 is a main cultivation strain of straw mushroom which is screened by the south China academy of sciences of China in the middle 60s and is widely used at present. Straw mushroom V23 is deposited and provided by the edible fungus research institute of the Shanghai academy of agricultural sciences. The prior research on the prior art carries out ARTP mutagenesis on the volvariella volvacea V23 to obtain a new low temperature resistant mutagenesis strain, but the method for screening the strain with excellent low temperature resistance of the volvariella volvacea V23 is not researched. This is because the lack of the study of the low temperature resistant phenotype of the straw mushrooms limits the study of the low temperature resistant index of the straw mushrooms.
Our earlier studies showed that downregulation of β -glucosidase activity at 4 ℃ low temperature stress significantly improved the low temperature resistance of straw mushrooms, suggesting that β -glucosidase low activity could be an indicator of low temperature stress resistance of straw mushrooms (Gong M, et al, the promising application of a β -glucosidase inhibitor in the postharvest management of Volvariella volvacea. Postharvest Biology and Technology,2022,185,111784). Our recent studies showed that fruiting bodies sprayed with 100. Mu.M beta. -glucosidase inhibitor D-Glucono-1, 5-lactate solution (DG 100) during the small button stage of straw mushrooms could be kept fresh at low temperature of 4℃for 72 hours (Gong M, et al, the service/threonine protein kinase VSKM, 1 promotes cold stress resistance of the postharvest Volvariella volvacea by enhancing mitoribosome activity. Postharvest Biology and Technology (2023) 112465). In the low-temperature treatment for 24 hours, 48 hours and 72 hours, the beta-glucosidase activity of the fruiting body treated by DG100 is obviously lower than that of the fruiting body treated by water, and the low-activity beta-glucosidase is proved to be necessary for the low-temperature stress resistance of the straw mushrooms. In conclusion, the strain resistant to low-temperature mutagenesis can be bred by adopting the activity index of straw mushroom beta-glucosidase under low-temperature stress.
The research adopts a straw mushroom V23 strain as an initial strain for ARTP mutagenesis, and combines an ARTP mutagenesis technology to obtain a regenerated strain; the novel low-temperature resistant mutant strain is obtained through antagonism, ITS sequencing and phylogenetic tree classification and identification technology; and (3) carrying out re-screening research on the strain subjected to low temperature mutagenesis based on the activity index of the straw mushroom beta-glucosidase under low temperature stress, then carrying out fruiting experiments, collecting fruiting bodies, carrying out low temperature experiment verification, and providing a new technical support for breeding of the strain of the low temperature resistant straw mushroom.
Disclosure of Invention
The invention aims to provide a low-temperature-resistant straw mushroom strain and a preparation method thereof, and the low-temperature-resistant straw mushroom strain and the preparation method thereof aim to solve the technical problem that the straw mushroom strain in the prior art is poor in low-temperature resistance effect.
The invention provides a low temperature resistant straw mushroom strain, which has the preservation number of: CCTCC NO: M2023706, classified and named: volvariella volvacea.
The invention also provides a preparation method of the low-temperature-resistant straw mushroom strain, which comprises the following steps:
1) A step of preparing a spore suspension of a straw mushroom V23 strain, namely selecting fresh and elongated straw mushrooms, and processing the fresh and elongated straw mushrooms on an ultra-clean workbench: sterilizing the surface of a fungus cover by cotton containing 75% alcohol by mass percent, cutting off the fungus cover by a small knife, standing the straw mushroom by a toothpick, placing the straw mushroom in a sterile plate filled with sterile qualitative filter paper, placing the plate in a constant temperature incubator at 32 ℃ to enable fungus pleat spores to fall freely, and collecting the fungus pleat spores by using sterile paper; soaking the mixture containing spores in sterile water with a sterile knife to dilute the spores to 10 6 -10 8 Individual mL -1 ；
2) Performing mutagenesis on the volvariella volvacea V23 strain spore suspension by adopting a normal-pressure room-temperature plasma mutagenesis technology to obtain regenerated strains;
3) Preliminary screening is carried out on the regenerated bacterial strain by using the temperature of 4 ℃; the new low temperature resistant mutant strain is obtained through antagonism experiment of the low temperature resistant mutant strain, ITS sequencing and phylogenetic identification technology;
4) Screening the mutant strain with low beta-glucosidase activity under low temperature stress again by adopting the beta-glucosidase index; 5) And selecting the re-screened mutant strain for fruiting cultivation, collecting fruiting bodies and carrying out a low temperature resistance experiment to verify the low temperature resistance of the mutant strain.
Further, in step 1), the low temperature is 4 ℃.
Further, the step of adopting the normal pressure room temperature plasma mutagenesis technology to mutagenize the sporophytes of the volvariella volvacea V23 strain is as follows: mixing straw mushroom spore liquid and sterilized glycerol in a volume ratio of 1:1, then sucking 20 mu L of suspension liquid, mixing with sterilized glycerol in a volume ratio of 1:1, and setting parameters by a normal pressure room temperature plasma mutation breeding instrument: the output power is 125W, the radiation distance is 2mm, and the gas flow rate is 10L.min -1 The time gradients of treatment were 0s, 30s, 60s, 90s, 120s, 150s, three replicates per gradient, and the mutagenized colonies were re-inoculated into regeneration medium, medium: PDA+0.6mol.L -1 Mannitol, and then selecting a regeneration strain with vigorous metabolism.
The invention adopts spore preparation, ARTP mutagenesis technology, classification and identification experiment, beta-glucosidase screening, fruiting cultivation, low temperature resistant experiment and other technologies to select and breed the low temperature resistant strain of straw mushroom.
In particular, straw mushroom V23 is a commercially available product and will not be described in detail herein.
The VG39 strain of the invention is preserved in China center for type culture Collection (CHINA CENTER FOR TYPE CULTURE COLLECTION), and the preservation number is: CCTCC NO: M2023706, the preservation date is 2023, 05 and 08, and the classification name is: volvariella volvacea, china center for type culture collection: the preservation date of the Wuhan district No. 299 of Wuchang district of Hubei province is 2023, 05 and 08.
Compared with the prior art, the invention has the advantages that the technical effect is positive and obvious, and the invention has the following advantages:
(1) The re-screening of the low temperature resistant mutant strain is carried out by better utilizing the activity index of beta-glucosidase under low temperature stress.
(2) Obtaining a low-temperature-resistant straw mushroom mutant strain.
Drawings
FIG. 1 photographs of 32℃plate growth of low temperature resistant mutant VG 39.
FIG. 2 shows the antagonistic comparison of the low temperature resistant mutant strain VG39 with the starting strain V23. CK represents V23.
FIG. 3 shows the ITS4 phylogenetic analysis of low temperature resistant mutant strains. Phylogenetic analysis was performed using the ITS4 gene sequences of a total of 15 volvariella volvacea including volvariella volvacea starting strain V23 and 14 mutant strains.
FIG. 4 shows a comparison of beta-glucosidase activity of straw mushroom mycelia under low temperature stress of 4℃and h represents hours.
Fig. 5, fig. 39, egg-shaped fruiting body photo.
FIG. 6 shows the phenotype of Volvariella volvacea VG39 fruiting body at 4℃under low temperature stress, h representing hours.
FIG. 7 shows the phenotype of Volvariella volvacea V23 fruiting body at 4℃under low temperature stress, h represents hours.
Detailed Description
Example 1
(1) ARTP obtaining regenerated Strain
And (3) mutagenesis of the straw mushroom spores by utilizing an ARTP technology. Fresh, elongated straw mushrooms were selected and treated on an ultra clean bench: sterilizing the surface of the fungus cover by 75% alcohol cotton, cutting off the fungus cover by a small knife, standing the straw mushroom by a toothpick, placing the straw mushroom in a sterile plate filled with sterile qualitative filter paper, placing the plate in a constant temperature incubator at 32 ℃ to enable the fungus pleat spores to fall freely and collect; cutting sterile paper containing spores with a sterile knife, soaking in sterile water to dilute spore concentration to 10 6 -10 8 Individual mL -1 。
(2) Screening of regenerated strains at 4 DEG C
The selected regenerated strain with vigorous metabolism is inoculated in a PDA flat plate, firstly placed in a 32 ℃ incubator for 1d, then placed in a 4 ℃ refrigerator for culture, the morphology and low-temperature lethal activity are analyzed, and then a low-temperature resistant mutant strain VG39 (CCTCC NO: M2023706) is screened out (figure 1).
(3) Regenerated strain antagonism experiment
Analysis of the results of the antagonism experiments showed that the VG39 mutant strain antagonized from the original strain V23, producing antagonistic lines, which clearly distinguished from each other (FIG. 2). The antagonistic experimental results confirm that the mutant strain VG39 and the original strain V23 belong to different strains from the morphological level.
(4) ITS phylogenetic analysis of mutagenized strains
Phylogenetic analysis was performed using the ITS4 gene sequences of a total of 15 volvariella volvacea including volvariella volvacea starting strain V23 and 14 mutant strains. Sequence alignment was performed using Clustalw2, default parameters. And constructing a phyML LG model by using a maximum likelihood method. The fast likelihood method aLRT SH-like is used as a branch support for the tree. The results of the phylogenetic tree showed that the branching support of each cluster had a very high confidence (100%) with the mutant strain VG39 being located in a different cluster than the starting strain V23, with a large genetic gap. The above results from the systematic taxonomic status confirm that the mutant strain VG39 and the starting strain V23 belong to different strains (FIG. 3). The sequence of VG39_ITS4 is shown in SEQ ID NO. 1.
>VG39_ITS4
CCTTGGATTTGAGGTCAGGGTGTCAAGGGGTGAGGCCGGGGTGTGTCGGCCTGTCCGGCTCGAGGGGCCGGACGGTTCGAAGCCGAGCCACGCTGAGTGCGGCCGCGGGGGGGCGTAGATGGACTATCAACGCCACGAGGCCGACTGCGACTGGGCCCTGCTGATGCCTTTGAGGAGAGCGGATCGCGAAGGCTAGCGACCCGCACAGCTCCCAACTCCAAGCCCCCAAAAGCCCGGGGAGAAGCCGGGCTGGGCTTGAGGATTCGATGACACTCAAACAGGCATGCTCTTCGGAATGGCCAAAGAGCGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGTTGAAAGTTGTATTGGTATGTCTATGGGGCCGACGAGGGGTCCCCGGTCGTACAAGAACACTCTGCAACACGTTTTTCTCAAGGGTGTACAAAAGATCTCGTAGAGCCGAGGGAGCCGAACGAGGCGGCTTCACGGCCTACAGAAGGTGCACAGGTGGGGACGTGGAGAATGGAAGGCGTCGGGGAGGGCGTGCACCTGCTCCGAGGAGCCAGCAATCAGCCCGACCCGCGTTCGATTCTGTAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTACATTTTTTTACCTTCCAATAGTT。
(5) Trend of activity change of beta-glucosidase under 4 ℃ low temperature stress treatment of straw mushroom strain
The β -glucosidase activity of V23 was significantly higher (P < 0.01) than that of the mutant strain VG39 (fig. 4) at both 0h and 16h of low temperature stress treatment. Compared with V23, VG39 obviously inhibits the activity of beta-glucosidase, which is consistent with the result reported in the literature that low-activity beta-glucosidase helps to improve the low temperature stress resistance of straw mushrooms.
(6) Volvariella volvacea VG39 cultivation experiment
And (3) fruiting cultivation is carried out by adopting VG39, wherein the VG39 shows primordia on the 7 th day of sowing, the straw mushroom original strain V23 shows primordia on the 9 th day after sowing, and the time for showing the primordia by the VG39 is earlier than that of V23. The entire fruiting body of strain VG39 became grey black (FIG. 5). The bioconversion rate of VG39 is inversely proportional to fruiting temperature, the fruiting efficiency of VG39 is optimal at 28 ℃, the bioconversion rate is 11.489%, and the bioconversion rate is better than 30 ℃ and 32 ℃.
(7) Volvariella volvacea VG39 low temperature resistance verification test
Collecting VG39 fruiting body for low temperature resistance experiment, and showing that the VG39 fruiting body keeps complete mushroom shape and dry surface in 24 hours of low temperature treatment; the cut-out view shows that the fruiting body has almost no browning, and maintains a relatively fresh state (FIG. 6).
The results show that: the low beta glucosidase activity can be used as a screening index of the low temperature resistant strain of the straw mushroom; in antagonism experiments, RAPD and classification and identification of phylogenetic tree, the low temperature resistant mutant strain VG39 and the original strain V23 belong to different strains; the beta-glucosidase activity of VG39 is low; the low temperature resistance test proves that the VG39 fruiting body has certain low temperature stress resistance. In conclusion, VG39 belongs to a new low temperature resistant strain.
VG39 strain is deposited in China center for type culture Collection, with accession number: CCTCC NO: M2023706, classified and named: volvariella volvacea.
Comparative example 1
(1) Antagonistic lines were present for both the starting strain V23 and the mutant strain VG39 (FIG. 2), suggesting that V23 and the mutant strain belong to different strains.
(2) ITS phylogenetic analysis showed that the original strain V23 was located in a different cluster than the mutant strain VG39, confirming that the mutant strain VG39 belongs to the new strain (FIG. 3).
(3) The beta-glucosidase activity of V23 was significantly higher than that of the mutant strain VG39 at low temperature stress treatment of 4 ℃ for 0h and 16h (fig. 4).
(4) The low temperature stress resistance test proves that the V23 fruiting body subjected to water treatment has atrophy and collapse of mushroom shape in 24-hour low-temperature fresh-keeping; the cut-out view shows that the fruiting body part has brown stain phenomenon. In the low-temperature preservation for 48 hours, the mushroom shape is seriously deformed and collapses; the cut-away view shows that the fruiting body has been fully browned and dissolved (figure 7).
It will be apparent to those skilled in the art from this disclosure that various other modifications and variations can be made in the light of the above teachings, and all such modifications and variations are intended to be within the scope of the appended claims.
Claims (1)
1. A low temperature resistant straw mushroom strain, which is characterized in that the strain is preserved in China center for type culture Collection, and has the preservation number of: CCTCC NO: M2023706, classified and named: straw mushroom VG39 (Volvariella volvaceaV G39) with a date of storage of 2023, 05 and 08.
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