CN105648030A - Method for judging degeneration of Phlebopus portentosus strains - Google Patents
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Abstract
The invention discloses a method for judging degeneration of Phlebopus portentosus strains. The activity of the Phlebopus portentosus strains is pre-judged through steps including colonial morphology observation, growth rate measurement, measurement of amylase secretion capacity of a colony, analysis of soluble proteins, analysis of esterase isozyme and the like, physiological and biochemical characteristics capable of characterizing that the Phlebopus portentosus strains degenerate with increase of subculture times are found out, the rapid and effective judging method is provided for identifying the activity of the strains and provides effective judgment basis for degeneration of the Phlebopus portentosus strains, the risks for misuse of degenerated strains are reduced greatly, and popularization of a Phlebopus portentosus cultivation technique is facilitated.
Description
Technical field
The invention belongs to agricultural technology field, be specifically related to the decision method of a kind of phlebopus portentosus decline.
Background technology
Phlebopus portentosus (Phlebopusportentosus) belongs to Boletales (Boletales) little Boletaceae (Boletinelaceae) net handle Boletus (Phlebopus), being edible fungi common on market in annual rainy season, Xishuangbanna of Yunnan province area, Phlebopus portentosus is also the currently the only bolete that can realize mushroom house annually cultivating. Obtaining female kind for the cultivation of Phlebopus portentosus mainly through wild strain or mushroom house cultivated strains separate tissue at present, mother carries out cultivation survey product and carrys out selection-breeding strain excellent after planting further expanding propagation. Under current cultivation mode, strain excellent will fail after the certain number of times of subculture, only by constantly separating, screen the continuity that strain excellent ensures to produce. In order to prevent not causing irremediable economic loss with decline bacterial strain, before entering production, effectively distinguish that bacterial strain vigor seems of crucial importance.
Summary of the invention
In view of this, it is an object of the invention to provide the decision method of a kind of phlebopus portentosus decline, by the method for the invention, in time phlebopus portentosus activity can be made anticipation, and then effectively prevent from cultivating the economic loss that decline bacterial strain causes.
The technical scheme that the present invention takes is as follows:
1, the decision method of phlebopus portentosus activity decay, comprises the steps:
(1) observe colonial morphology, measure growth rate: 30 DEG C of lucifuges of bacterial strain are cultivated, and measure colony radius respectively at least 5 different time points decussation methods, calculate average colony growth rate; When mycelium covers with culture dish, observe colony morphology characteristic simultaneously; Compared with bacterial strain first generation original seed, bacterium colony guttation is few, and sclerotium is little, average colony growth rate rate of change >=10%;
Described average colony growth rate rate of change=(| bacterial strain average colony growth rate-first generation original seed average colony growth rate |)/first generation original seed average colony growth rate �� 100%;
Described average colony growth rate is: with different time points for abscissa, and average colony radius corresponding to different time points is vertical coordinate, and the straight slope of acquisition is bacterial strain average colony growth rate;
(2) bacterium colony amylase secretion ability is measured: strain culturing obtained starch variable color circle by congo red method or Lugol's iodine solution method after 15 days, then decussation method is adopted to measure average colony diameter and average variable color loop diameter, and result data corresponding to bacterial strain first generation original seed compares, adopt the ratio that congo red method measures variable color loop diameter/colony diameter to reduce amplitude >=10%, adopt the ratio that Lugol's iodine solution method measures variable color loop diameter/colony diameter to reduce amplitude >=5%;
(3) soluble protein analysis: bacterial strain 150rpm, 30 DEG C of lucifuge shaken cultivation 7 days, filter, filtering residue cleans, dewater, liquid nitrogen grinding is to pulverizing, take the phosphate buffer mixing that ground product pH is 7.2,12000rpm is centrifuged 10min, Aspirate supernatant is soluble protein sample, denaturing polyacrylamide gel electrophoresis is adopted to carry out soluble protein analysis, compared with bacterial strain first generation original seed, the soluble protein band that relative molecular weight is 40.0kDa dies down, and the soluble protein band that relative molecular weight is 22.0kDa strengthens;
(4) Analysis of Esterase Isoenzyme: bacterial strain 150rpm, 30 DEG C of lucifuge shaken cultivation 7 days, filter, filtering residue cleans, and dewaters, and liquid nitrogen grinding is to pulverizing, take the phosphate buffer mixing that ground product pH is 5.6,12000rpm is centrifuged 10min, and Aspirate supernatant is esterase isozyme sample, uses native polyacrylamide gel electrophoresis to carry out Analysis of Esterase Isoenzyme, compared with bacterial strain first generation original seed, relative molecular weight is the esterase isozyme increased activity of 37.0kDa.
Preferably, step (1) used medium is M1 solid medium, (3) and in (4) used medium be M1 fluid medium.
Preferably, by weight, the one-tenth of described M1 fluid medium is grouped into:
Peeled potatoes 200.0 parts, glucose 20.0 parts, yeast extract 2.0 parts, MgSO4��7H2O1.0 part, KH2PO41.0 parts, 1000.0 parts of water; The basis of M1 solid medium liquid medium within adds 15.0 parts of agar powders.
Preferably, in step (2), Lugol's iodine solution method measures the bacterium culture medium of starch variable color loop diameter use is M1 starch solids culture medium, and by weight, the one-tenth of described M1 starch solids culture medium is grouped into:
Peeled potatoes 200.0 parts, glucose 10.0 parts, yeast extract 2.0 parts, MgSO4��7H2O1.0 part, KH2PO41.0 parts, 1000.0 parts of water, soluble starch 2.0 parts, agar powder 15.0 parts;
Congo red method measures the bacterium culture medium of starch variable color loop diameter use and adds 2 parts of Congo red on M1 starch solids medium base.
Preferably, bacterial strain first carries out actication of culture before cultivation, and described actication of culture condition is activated on M1 solid medium by bacterial strain, and 30 DEG C of lucifuges are cultivated 20 days.
Preferably, in step (3), in the concentration glue of denaturing polyacrylamide gel electrophoresis, acrylamide mass body volume concentrations is 5%, in separation gel, acrylamide mass body volume concentrations is 10%, and SDS mass body volume concentrations is 0.1%, and electrophoretic voltage is concentration glue 80V, separation gel 150V.
Preferably, in step (3), in the concentration glue of native polyacrylamide gel electrophoresis, acrylamide mass body volume concentrations is 5%, and in separation gel, acrylamide mass body volume concentrations is 8%, concentrates glue 80V, separation gel 150V.
Preferably, described step (1) is measured the time point of colony radius respectively at the 3rd of strain culturing the, 6,9,12,15 and 18 days.
The beneficial effects of the present invention is: the present invention is by analyzing Phlebopus portentosus colony growth speed under different subculture number, colony morphology characteristic, amylase activity, soluble protein pattern, the esterase isozyme and fruiting are surveyed and are produced all angles Changing Pattern, find can characterize phlebopus portentosus with subculture number increase occur decline physiological and biochemical property, identify for spawn activity and provide effective decision method, the strain decline that the method is Phlebopus portentosus provides effective judgment basis, greatly reduce the risk of misuse decline bacterial strain, be conducive to the popularization of Phlebopus portentosus cultivation technique.
Accompanying drawing explanation
In order to make the purpose of the present invention, technical scheme and beneficial effect clearly, the present invention provides drawings described below:
Fig. 1 bacterial strain colonial morphology figure.
Fig. 2 soluble protein gel electrophoresis collection of illustrative plates, M: pre-dyed albumen Marker, relative molecular weight size is 150,100,70,50,35,25,20kDa from top to bottom respectively.
Fig. 3 Esterase isoenzyme patterns, M: pre-dyed albumen Marker, relative molecular weight size is 100,70,50,35,25kDa from top to bottom respectively; Wherein, 11023,13013 loading protein contents are the half of A, B, C.
The figures above is original picture size.
Detailed description of the invention
Below the preferred embodiments of the present invention are described in detail. The experimental technique of unreceipted actual conditions in embodiment, generally conventionally condition or according to manufacturer it is proposed that condition.
One, material and equipment
1.1 strains testeds
Phlebopus portentosus 11023 bacterial strain; 13013 bacterial strains; 11023 bacterial strain cultivated fruitbody tissue isolation strains 11023-1372 time subculture bacterial strains (hereinafter referred to as A), 5 subculture bacterial strains (hereinafter referred to as B), 8 subculture bacterial strains (hereinafter referred to as C).
1.2 reagent, instrument and equipment and culture medium
1.2.1 reagent
Glucose, yeast extract, Congo red, iodine, potassium iodide, acetic acid-1-naphthalene ester, acetic acid-2-naphthalene ester, Fast Blue RR salt, KH2PO4, MgSO4��7H2O is analytical pure, all purchased from Sangon Biotech (Shanghai) Co., Ltd.; Agar powder is Japan's import subpackage.
1.2.2 instrument and equipment
BIC-250 type growth cabinet, Shanghai Bo Xun company; DYCE-25E type P4 vertical electrophoresis apparatus, Beijing 61 company; SK-100B constant-temperature table, Shanghai Su Kun company.
1.2.2 culture medium
M1 culture medium: peeled potatoes 200.0g (liquor), glucose 20.0g, yeast extract 2.0g, MgSO4��7H2O1.0g, KH2PO41.0g, water 1000.0ml, M1 solid medium separately add 15.0g agar powder (screening of Phlebopus portentosus mother culture media, southwest agricultural journal, 2009, P209-211; The screening study of the carbon source of Phlebopus portentosus Mycelium culture base, nitrogenous source and inorganic salt, Yunnan Prov Agriculture University's journal, 2009, P145-149; The cultural method of phlebopus portentosus, CN101491195A).
M1 starch solids culture medium: peeled potatoes 200.0g (liquor), glucose 10.0g, yeast extract 2.0g, MgSO4��7H2O1.0g, KH2PO41.0g, water 1000ml, agar powder 15g, soluble starch 2g.
M1 starch Congo red solid medium: peeled potatoes 200.0g (liquor), glucose 10.0g, yeast extract 2.0g, MgSO4��7H2O1.0g, KH2PO41.0g, water 1000.0ml, agar powder 15.0g, soluble starch 2.0g, Congo red 2.0g.
Two, colony growth experiment
Test strain being activated on M1 solid medium, 30 DEG C of lucifuges are cultivated 20 days, take bacterium cake with the aseptic card punch that diameter is 1cm in the punching of eugonic mycelia leading edge, are inoculated in M1 culture medium central authorities, and 30 DEG C of lucifuges are cultivated. Each culture dish central authorities only one bacterium cake of inoculation, 5 parallel processing are at least done in every kind of process. Within 3rd, 6,9,12,15 and 18 days, utilize decussation method to measure colony diameter after cultivating, observe colony morphology characteristic (guttation situation, sclerotium situation). The SLOPE function in MicrosoftEXCEL is utilized to obtain colony growth speed (v): v=1/2*SLOPE ({ bacterium colony average diameter set }, { measuring time set }), wherein colony radius unit is mm, and measurement unit of time is d, then growth rate unit is mm/d. Such as, the t1 days measure colony diameter is d1, and the t2 days measure colony diameter is d2, and the t3 days measure colony diameter is d3, and the t4 days measure colony diameter is d4 ..., then v=1/2*SLOPE (d1, d2, d3, d4 ... }, t1, t2, t3, t4 ... }).Specifically for certain measurement result: t1=3, t2=6, t3=9, t4=12, t5=15; D1=10.41, d2=16.62, d3=23.93, d4=32.21, d5=41.39; Then v=1/2*SLOPE ({ 10.41,16.62,23.93,32.21,41.39}, { 3,6,9,12,15})=1/2*2.59=1.29 (mm/d). Arithmetic average �� the standard deviation of the colony growth speed of 5 times and above parallel laboratory test represents average growth rate, such as, in certain test of certain bacterial strain, 5 parallel laboratory test colony growth speed are respectively as follows: 2.31,2.94,2.81,2.90,3.15 (mm/d), then this empirical average growth rate=2.82 �� 0.28 (mm/d) of this bacterial strain.
Colonial morphology correlated results is in Table 1 and Fig. 1.
Table 1 colony growth experimental result
Different lower cases represent that the difference between process reaches pole significant level (p < 0.01), and guttation and sclerotium situation are that mycelia is close to covering with observed result during flat board.
By table 1 and Fig. 1 it can be seen that strain increasing along with subculture number, guttation and the Sclerotia forming ability of its bacterium colony all weaken. Cultivation strain excellent 11023,13013 average colony growth rate has pole significant difference with other 3 bacterial strains. Strains A, B colony growth speed pole significant difference are in bacterial strain C. Along with subculture number increases, colony growth speed rate of change >=10%. As compared with bacterial strain first generation original seed, go down to posterity the bacterial strain B after 5 times, and growth rate rate of change is (1.83-1.60)/1.83=12%.
Three, starch variable color circle experiment
Congo red method: by above-mentioned inoculation method by pure culture biscuits involvng inoculation in M1 starch Congo red solid medium, 30 DEG C of lucifuges are cultivated. Within 15th day, measure colony diameter and variable color loop diameter by decussation method. The mean+SD of data five and above parallel test represents.
Lugol's iodine solution method: by above-mentioned inoculation method by pure culture biscuits involvng inoculation in M1 starch solids culture medium, 30 DEG C of lucifuges are cultivated. Within 15th day, measure colony diameter by decussation method, then with Lugol's iodine solution dyeing, (5.0g iodine and 10.0g potassium iodide are dissolved in 85.0ml distilled water, the total concentration of iodine is 150mg/ml) it is sprayed on media surface with watering can, measure the diameter not being dyed to blue region. Data represent with the mean+SD of five and above parallel test.
Related experiment result is in Table 2.
Table 2 starch variable color circle experimental result
Different lower cases represent that the difference between process reaches significant level (p < 0.05).
Starch variable color circle experimental result is as shown in table 2, result shows that Phlebopus portentosus has secreted amylase, and along with subculture number increases, and the ratio of variable color loop diameter and colony diameter declines, namely bacterium colony secreting amylase ability of the same area reduces, and amylase secretion ability there occurs degeneration. Compared with first generation original seed, congo red method measures the ratio of variable color loop diameter/colony diameter and reduces amplitude >=10%, and Lugol's iodine solution method measures the ratio of variable color loop diameter/colony diameter and reduces amplitude >=5%.
Four, soluble protein analysis
It is that the aseptic card punch of 1cm takes bacterium cake four in the punching of eugonic mycelia leading edge with diameter, it is inoculated in equipped with in the 500ml triangular flask of 200mlM1 fluid medium, 150rpm, 30 DEG C of lucifuge shaken cultivation 7d, sterile gauze filters, distilled water cleans 3 times, squeezing with gauze and anhydrate point, liquid nitrogen grinding, to pulverizing, weighs 100mg abrasive material immediately and vibrates to the phosphate buffer that pH is 7.2 mixing, 12000rpm is centrifuged 10min, and careful Aspirate supernatant is protein sample.With modified form Bradford method determination of protein concentration test kit, protein sample is carried out quantitatively, by concentrating or diluting so that different samples (test strain and first generation original seed corresponding to this strain) protein concentration keeps consistent.
Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) is used to carry out soluble protein analysis: concentration gum concentration is 5%, resolving gel concentration is 10%, SDS concentration is 0.1%, the protein sample of test sample and this bacterial strain correspondence first generation original seed with same volume same protein concentration in same glue loading, applied sample amount is 20 �� l, and electrophoretic voltage is concentration glue 80V, separation gel 150V; Bromophenol blue migrates to and stops electrophoresis from about lower edge 1cm. Carry out coomassie brilliant blue staining according to a conventional method. According to gained electrophoresis pattern, calculating the relative mobility of variant protein band, computing formula is relative mobility (Rf)=target protein band migration distance/bromophenol blue migrate from. Relative molecular weight according to albumen Marker and the relative mobility of each band, Excel data fitting obtains relative mobility and protein relative molecular mass standard curve. Acquisition standard curve is: M=-44.540ln (Rf)+14.124,R2=0.998; LgM=-0.871Rf+ 2.093, R2=0.993. M relative molecular weight, unit kDa.
Experimental result is shown in Fig. 2. Result shows that bacterial strain C soluble protein is at relative mobility Rf=0.56, relative molecular weight be the protein band of 40.0kDa relative to strains A and B luminance-reduction, bacterial strain B, C soluble protein is at Rf=0.84 molecular weight is 22.0kDa, and band strengthens relative to strains A brightness.
Five, Analysis of Esterase Isoenzyme
It is that the aseptic card punch of 1cm takes bacterium cake four in the punching of eugonic mycelia leading edge with diameter, it is inoculated in equipped with in the 500ml triangular flask of 200mlM1 fluid medium, 150rpm, 30 DEG C of lucifuge shaken cultivation 7d, sterile gauze filters, distilled water cleans 3 times, squeezing with gauze and anhydrate point, liquid nitrogen grinding, to pulverizing, weighs 100mg abrasive material immediately and vibrates to the phosphate buffer that pH is 5.6 mixing, 12000rpm is centrifuged 10min, and careful Aspirate supernatant is sample. Use native polyacrylamide gel electrophoresis (Native-PAGE), concentration gum concentration is 5%, resolving gel concentration is 8%, the protein sample of test sample and this bacterial strain correspondence first generation original seed with same volume same protein concentration in same glue loading, applied sample amount is 20 �� l, concentration glue 80V, separation gel 150V, stop electrophoresis when bromophenol blue migrates to from end margin about 1cm. Electrophoresis terminates front half an hour carry out dyeing liquor, fixative and preservation liquid preparation (Guo Xiaoxia. the Phylogenetic Studies (Lepidoptera, butterfly suborder) [D] of butterfly esterase isozyme: Shaanxi Normal University, 2000). Glue is taken out after terminating by electrophoresis, removes concentration glue, after cleaning with distilled water, puts into dyeing liquor, and coloring case is observed in 37 DEG C of dyeing, stops dyeing when having clear band to occur. Taking out gel, distilled water rinsed clean is placed in fixative overnight, preserves in preservation liquid of taking pictures and put into for second day.
According to gained collection of illustrative plates, calculate relative mobility (Rf), the relative mobility computational methods that computational methods are analyzed with soluble protein. Relative molecular weight according to albumen Marker and the relative mobility of each band, Excel data fitting obtains relative mobility and protein relative molecular mass standard curve. Acquisition standard curve is: M=-69.164ln (Rf)+13.980, R2=0.998;LgM=-1.044Rf+ 2.307, R2=0.999. M is relative molecular weight, unit kDa.
Esterase isozyme electrophoresis result is as it is shown on figure 3, it is shown that tested strain occurs the esterase isozyme band of 4, wherein R altogetherf(corresponding relative molecular weight is=0.01,0.40 and 0.55 3 band: 332.0,77.0,55.0kDa) appearance in all bacterial strains, and Rf=0.80 (relative molecular weight is 37.0kDa) this isozyme adjusts band only to occur in A, B, C, and isozyme band darkens with subculture number increase, and enzymatic activity strengthens.
Six, cultivation fruiting
Method activated strains described in colony growth experiment, each bacterial strain buys the bacterium cake four that cut-off footpath is 1cm, it is inoculated in 300ml fluid medium and carries out seed culture medium fermentation, conventionally (Kai-PingJi after cultivating 7 days, YangCao, Chun-XiaZhangetal.CultivationofPhlebopusPortentosusinSou thernChina [J] .MycologicalProgress, 2011,10 (3): 293-300) connect cultigen and carry out mushroom producing culture. Inoculation work is completed by the consummate workman of same operation, each strain culturing 90 bags. Statistics pollution condition, adds up cultivating rate, single mushroom weight.
Fruiting result cultivated by table 3
Different lower cases represent that the difference between process reaches significant level (p < 0.05);-represent and do not test.
Cultivation fruiting result is as shown in table 3, and along with subculture number increases, average single mushroom heavily reduces, and standard deviation increases. Along with subculture number increases, anti-hybrid ability weakens gradually, and pollution rate increases. As compared to strain excellent 11023,13013, A, B, C mono-mushroom weight standard deviation is bigger than normal, and fruiting is irregular, and mushroom type is inconsistent, and anti-hybrid ability is weak.
Summary research finds, along with the increase of subculture number, Phlebopus portentosus mycelium at colony morphology characteristic, colony growth speed, exoenzyme, soluble protein, esterase isozyme and change in various degree all occurs on Fruiting Characters. Amylase is that under Phlebopus portentosus artificial cultivation condition, substrate utilizes relevant topmost exoenzyme, and therefore monitoring diastatic activity performance characterizes spawn activity preferably. The change of soluble protein and the esterase isozyme is that a kind of directly perceived of the difference of gene expression dose embodies. Phlebopus portentosus increases along with subculture number, and some protein band or esterase isozyme bar are with expressing the trend raised, and some expression then reduces. Protein is the product of gene expression, its kind and expression along with incubation time, kinds of culture medium, condition of culture, sampling point change difference to some extent, therefore in testing, condition needs to keep consistent. Along with the increase generation strain degeneration of strain subculture number, final consequence is exactly that yield falls sharply, and between fruiting individuality, variation increases, and fruiting is irregular, directly contributes economic loss. Therefore, in Bacterial Strains Managing process, the above-mentioned physio-biochemical characteristics of periodic monitoring can as the effective means of a strain degeneration assessment.
What finally illustrate is, preferred embodiment above is only in order to illustrate technical scheme and unrestricted, although the present invention being described in detail by above preferred embodiment, but skilled artisan would appreciate that, in the form and details it can be made various change, without departing from claims of the present invention limited range.
Claims (8)
1. the decision method of phlebopus portentosus activity decay, it is characterised in that comprise the steps:
(1) observe colonial morphology, measure growth rate: 30 DEG C of lucifuges of bacterial strain are cultivated, and measure colony radius respectively at least 5 different time points decussation methods, calculate average colony growth rate;When mycelium covers with culture dish, observe colony morphology characteristic simultaneously; Compared with bacterial strain first generation original seed, bacterium colony guttation is few, and sclerotium is little, average colony growth rate rate of change >=10%;
Described average colony growth rate rate of change=(| bacterial strain average colony growth rate-first generation original seed average colony growth rate |)/first generation original seed average colony growth rate �� 100%;
Described average colony growth rate is: with different time points for abscissa, and average colony radius corresponding to different time points is vertical coordinate, and the straight slope of acquisition is bacterial strain average colony growth rate;
(2) bacterium colony amylase secretion ability is measured: strain culturing obtained starch variable color circle by congo red method or Lugol's iodine solution method after 15 days, then decussation method is adopted to measure average colony diameter and average variable color loop diameter, and result data corresponding to bacterial strain first generation original seed compares, adopt the ratio that congo red method measures variable color loop diameter/colony diameter to reduce amplitude >=10%, adopt the ratio that Lugol's iodine solution method measures variable color loop diameter/colony diameter to reduce amplitude >=5%;
(3) soluble protein analysis: bacterial strain 150rpm, 30 DEG C of lucifuge shaken cultivation 7 days, filter, filtering residue cleans, dewater, liquid nitrogen grinding is to pulverizing, take the phosphate buffer mixing that ground product pH is 7.2,12000rpm is centrifuged 10min, Aspirate supernatant is soluble protein sample, denaturing polyacrylamide gel electrophoresis is adopted to carry out soluble protein analysis, compared with bacterial strain first generation original seed, the soluble protein band that relative molecular weight is 40.0kDa dies down, and the soluble protein band that relative molecular weight is 22.0kDa strengthens;
(4) Analysis of Esterase Isoenzyme: bacterial strain 150rpm, 30 DEG C of lucifuge shaken cultivation 7 days, filter, filtering residue cleans, and dewaters, and liquid nitrogen grinding is to pulverizing, take the phosphate buffer mixing that ground product pH is 5.6,12000rpm is centrifuged 10min, and Aspirate supernatant is esterase isozyme sample, uses native polyacrylamide gel electrophoresis to carry out Analysis of Esterase Isoenzyme, compared with bacterial strain first generation original seed, relative molecular weight is the esterase isozyme increased activity of 37.0kDa.
2. the decision method of phlebopus portentosus activity decay according to claim 1, it is characterized in that, step (1) used medium is M1 solid medium, (3) and in (4) used medium be M1 fluid medium.
3. the decision method of phlebopus portentosus activity decay according to claim 2, it is characterised in that by weight, the one-tenth of described M1 fluid medium is grouped into:
Peeled potatoes 200.0 parts, glucose 20.0 parts, yeast extract 2.0 parts, MgSO4��7H2O1.0 part, KH2PO41.0 parts, 1000.0 parts of water; M1 solid medium separately adds 15.0 parts of agar powders.
4. the decision method of phlebopus portentosus activity decay according to claim 2, it is characterized in that, in step (2), Lugol's iodine solution method measures the bacterium culture medium of starch variable color loop diameter use is M1 starch solids culture medium, by weight, the one-tenth of described M1 starch solids culture medium is grouped into:
Peeled potatoes 200.0 parts, glucose 10.0 parts, yeast extract 2.0 parts, MgSO4��7H2O1.0 part, KH2PO41.0 parts, 1000.0 parts of water, soluble starch 2.0 parts, agar powder 15.0 parts;
It is that M1 starch solids culture medium separately adds 2 parts of Congo red that congo red method measures the bacterium culture medium of starch variable color loop diameter use.
5. the decision method of phlebopus portentosus activity decay according to claim 1, it is characterised in that bacterial strain first carries out actication of culture before cultivation, described actication of culture condition is activated on M1 solid medium by bacterial strain, and 30 DEG C of lucifuges are cultivated 20 days.
6. the decision method of phlebopus portentosus activity decay according to claim 1, it is characterized in that, in step (3), in the concentration glue of denaturing polyacrylamide gel electrophoresis, acrylamide mass body volume concentrations is 5%, in separation gel, acrylamide mass body volume concentrations is 10%, SDS mass body volume concentrations is 0.1%, and electrophoretic voltage is concentration glue 80V, separation gel 150V.
7. the decision method of phlebopus portentosus activity decay according to claim 1, it is characterized in that, in step (3), in the concentration glue of native polyacrylamide gel electrophoresis, acrylamide mass body volume concentrations is 5%, in separation gel, acrylamide mass body volume concentrations is 8%, concentration glue 80V, separation gel 150V.
8. the decision method of phlebopus portentosus activity decay according to claim 1, it is characterised in that measure the time point of colony radius in described step (1) respectively at the 3rd of strain culturing the, 6,9,12,15 and 18 days.
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| CN111088321A (en) * | 2020-01-04 | 2020-05-01 | 福建农林大学 | Method for rapidly judging hypsizigus marmoreus strain decline based on morphological characteristics and physiological and biochemical indexes and application thereof |
| CN111118105A (en) * | 2020-01-04 | 2020-05-08 | 福建农林大学 | Method for rapidly judging degradation of pleurotus eryngii strain based on hypha characteristics and biochemical indexes and application of method |
| CN117402743A (en) * | 2023-08-17 | 2024-01-16 | 景洪宏臻农业科技有限公司 | Phlebopus portentosus strain HZ2302 and application thereof |
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| CN111118105A (en) * | 2020-01-04 | 2020-05-08 | 福建农林大学 | Method for rapidly judging degradation of pleurotus eryngii strain based on hypha characteristics and biochemical indexes and application of method |
| CN117402743A (en) * | 2023-08-17 | 2024-01-16 | 景洪宏臻农业科技有限公司 | Phlebopus portentosus strain HZ2302 and application thereof |
| CN117402743B (en) * | 2023-08-17 | 2024-04-05 | 景洪宏臻农业科技有限公司 | Phlebopus portentosus strain HZ2302 and application thereof |
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