CN1240269C - Polygonatum cyrtonema Hua regeneration system establishment method - Google Patents
Polygonatum cyrtonema Hua regeneration system establishment method Download PDFInfo
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- CN1240269C CN1240269C CN 200410021659 CN200410021659A CN1240269C CN 1240269 C CN1240269 C CN 1240269C CN 200410021659 CN200410021659 CN 200410021659 CN 200410021659 A CN200410021659 A CN 200410021659A CN 1240269 C CN1240269 C CN 1240269C
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Abstract
The present invention discloses a method for establishing a regeneration system for polygonatum cyrtonema Hua, which comprises the steps of A, inducing explant calluses of the polygonatum cyrtonema Hua; B, inducing seedlings by inoculating the calluses to a differential medium; C, rooting by moving young seedlings which are not rooted in the step B to a rooting medium; D, firstly hardening and then cultivating the young seedlings which are rooted in the step B and the step C. The system for polygonatum cyrtonema Hua can be obtained only for 3 months to 4 months by using the method of the present invention, and the survival rate after transplantation is from 70% to 75%. When the rhizome of the polygonatum cyrtonema Hua is compared with medicinal polygonatum, the contents of mannose-binding lectin are similar, and the blood coagulation activities have no obvious differences.
Description
Technical field
The present invention relates to the method for a kind of a large amount of acquisition sealwort agglutinin II, especially a large amount of methods that obtain sealwort agglutinin II from capsule silk sealwort.
Background technology
Sealwort is the rhizome of liliaceous plant sealwort (Polygonatum sibiricum Redoute), capsule silk sealwort (Polygonatum cyrtonema Hua), hot river sealwort (Polygonatum macropodiumTurcz.), Yunnan sealwort (Polygonatum kingianum Coll.et Hemsl.), polygonatum cirrhifolium Royle (Polygonatum cirrhifolium (Wall.) Royle) etc.
[1]Sealwort be used as medicine cure the disease have existed since ancient times, Shennong's Herbal is classified it as top grade, sealwort is sweet, flavor is flat, return lung, spleen, kidney, warp, the effect that the tool Yin nourishing and lung moistening is invigorated the spleen and benefited qi is used for nourishing and fit keeping function and the disease for the treatment of deficiency of kidney essence, deficiency syndrome of the lung cough caused by dryness and taste void, is traditional Chinese medical science common drug.Modern pharmacology studies have shown that, multiple pharmacological effect such as sealwort has enhance immunity, reduces blood fat, blood sugar, delays senility, trophic nerve and function of detoxification, the sealwort The Chemical Constituents is shown sealwort contains sibiricoside, Yan acid, carbohydrate, quinones, amino acid and trace element.
In recent years, the research of Siberian solomonseal rhizome polysaccharide has been obtained some progress highly significant, the polysaccharide that extracts from capsule silk sealwort (providing group to cultivate the polysaccharide content of thing) has the activity that strengthens the rat immunity system
[2]Mannose binding lectin one sealwort agglutinin II (Polygonatumcyrtonema Hua.lectin II in its root-like stock, PCL II) has the activity of specific bond mannose, a member as the phytolectin superfamily has biologic activity widely, can be used as the damage by disease and insect etc. that retroviral effective inhibitor and being used to prevents crops
[3,4,5]
At present, sealwort mainly adopts the root-like stock breeding.Because the root-like stock reproduction rate is low, need 3-5 just can grow up to the plant of tool medical value, in addition, and the long-term large-scale unordered sealwort of excavating, China's sealwort has faced deficient danger.Though, U. S. application number provides the method for building up of a kind of new medium and a kind of Polygonatum plant polygonatum cirrhifolium Royle Regeneration in Vitro system for 20030100109 patent of invention, this method is cultivated the sealwort seed respectively on three kinds of medium, produce epicotyl, coleoptile, root and stop epicotylar dormancy, promote seed germination, but it is still very long that this method gained seedling will grow up to the required time limit of plant of tool medical value.
Utilize method for tissue culture fast breeding nourish and generate and be and produce effective medicinal ingredient, become one of new direction of drug manufacture gradually.For capsule silk sealwort, yellow or vitrification phenomenon easily take place in tissue culture, up to the present the explant of also not seeing by inducing it produces callus, forms regenerating system through tissue differentiation again, and finds the report of mannose binding lectin in this regenerating system root-like stock.
Summary of the invention
Technical problem to be solved by this invention provides the method for a kind of a large amount of acquisition sealwort agglutinin II.This method can overcome the defective that capsule silk root of Rhizoma Polygonati shape stem breeding rate is low, growth year is long, and makes the protein component kind in the regenerating system similar to wild plant with content.
The present invention to the technical scheme that technical problem to be solved adopted is: the method for a kind of a large amount of acquisition sealwort agglutinin II is provided, the steps include:
Inducing of a, capsule silk sealwort (Polygonatum cyrtonema Hua) explant callus;
B, aforementioned callus is inoculated into induces living seedling in the differential medium;
C, the seedling that does not take root among the step b is moved to root induction in the root media;
D, with the seedling that takes root among step b and c elder generation hardening, cultivation then;
E, get the capsule silk sealwort regeneration root-like stock that above step obtains, extract preparation sealwort agglutinin II.
In step a, described capsule silk sealwort explant is its root, root-like stock or leaf; To be inoculated on the callus inducing medium after the capsule silk sealwort explant sterilization, inducing culture callus under the lucifuge condition, cultivation temperature is 25 ± 2 ℃, incubation time is 5-8 week, the inducing culture of described callus consists of: MS minimal medium, 0.5-2.0mg/L 6-BA, 0.5-4.0mg/L 2,4-D, 8g/L agar, 30g/L sucrose, pH5.8; Callus inducing medium preferred group of the present invention becomes: MS minimal medium, 1.0mg/L 6-BA, 2.0mg/L 2,4-D, 8g/L agar, 30g/L sucrose, pH5.8.
Consisting of of above-described MS minimal medium: macroelement (1.65g/L NH
4NO
3, 1.9g/L KNO
3, 0.44g/L CaCl
2.2H
2O, 0.37g/L MgSO
4.7H
2O, 0.17g/L KH
2PO
4), the trace element (37.25mg/L Na
2EDTA, 27.8mg/L FeSO
4.7H
2O, 0.83mg/L KI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
4.4H
2O, 8.6mg/L ZnSO
4.7H
2O, 0.25mg/LNa
2MoO
4.2H
2O, 0.025mg/L CuSO
4.5H
2O, 0.025mg/L CoCl
2.6H
2O), 2mg/L glycosides propylhomoserin, 100mg/L inositol, 0.1mg/L thiamine hydrochloride (B1), 0.5mg/L hydrochloric acid pyrrole diindyl alcohol (B6), 0.5mg/L nicotinic acid.
In step b, callus is inoculated into induces living seedling in the differential medium, cultivation temperature is 25 ± 2 ℃, light application time is 12-16 hour/day, intensity of illumination is 1200-2000Lx, and in incubation time 3-4 week, described differential medium consists of: MS minimal medium, 0.5-4.0mg/L 2,4-D, 0-2.0mg/L 6-BA, 0-0.6mg/L NAA, 8g/L agar, 30g/L sucrose, pH5.8; Differential medium preferred group of the present invention becomes: MS minimal medium, 2.0mg/L 2,4-D, 2.0mg/L6-BA, 0.3mg/L NAA, 8g/L agar, 30g/L sucrose, pH5.8, cultivation temperature is 25 ± 2 ℃, light application time is 12 hours/day, and intensity of illumination is 1200Lx, cultivates 2-3 week.
In step c, the seedling that does not take root is moved to the differentiation of inducing root in the root media, cultivation temperature is 25 ± 2 ℃, light application time is 12-16 hour/day, intensity of illumination is 1200-2000Lx, cultivate 3-4 week, described root media consists of: 1/2MS minimal medium, 0.3-0.5mg/LNAA, 8g/L agar, 30g/L sucrose, pH5.8; Root media preferred group of the present invention becomes: 1/2MS minimal medium, 0.5mg/L NAA, 8g/L agar, 30g/L sucrose, and pH5.8, light application time is 12 hours/day, intensity of illumination is 1200Lx, cultivates 3-4 week.
In step c, root media composition of the present invention also can be: 1/2MS minimal medium, 0.5mg/L6-BA, 8g/L agar, 30g/L sucrose, pH5.8; Cultivation temperature is 25 ± 2 ℃, and light application time is 12 hours/day, and intensity of illumination is 1200Lx, cultivates 3-4 week.
Consisting of of above-described 1/2MS minimal medium: macroelement (0.83g/L NH
4NO
3, 0.95g/L KNO
3, 0.22g/L CaCl
2.2H
2O, 0.185g/L MgSO
4.7H
2O, 0.085g/LKH
2PO
4), the trace element (37.25mg/L Na
2EDTA, 27.8mg/L FeSO
4.7H
2O, 0.83mg/LKI, 6.2mg/L H
3BO
3, 22.3mg/L MnSO
4.4H
2O, 8.6mg/L ZnSO
4.7H
2O, 0.25mg/LNa
2MoO
4.2H
2O, 0.025mg/L CuSO
4.5H
2O, 0.025mg/L CoCl
2.6H
2O), 2mg/L glycosides propylhomoserin, 100mg/L inositol, 0.1mg/L thiamine hydrochloride (B1), 0.5mg/L hydrochloric acid pyrrole diindyl alcohol (B6), 0.5mg/L nicotinic acid.
In steps d, described hardening and cultivation comprise: the test-tube plantlet that will take root moves under the natural daylight and cultivated 3-4 days, open bottle cap, hardening 2-3 days, and then taking-up seedling, cleaning agar, move into loose weight ratio and be in 1: 1 the peat soil and vermiculite, is cultivation under 27 ± 1 ℃, natural daylight in temperature.
Beneficial effect of the present invention is: the inventive method has higher differentiation rate, only need can obtain capsule silk sealwort regenerating system in 3-4 month, and the survival rate after it is transplanted reaches 70-75%, helps large-scale cultivation and use; And the protein component kind in its root-like stock is similar to wild plant with content, and particularly mannose binding lectin and wild plant content are close with the blood coagulation vigor, are the major protein in the root-like stock; In addition, the root-like stock of cultivation and wild type plesiomorphism (as shown in Figure 8), and growth is faster than wild type.
Description of drawings
The outer planting surface forms callus in Fig. 1 sealwort culture in vitro process of growth;
Callus expands in Fig. 2 sealwort culture in vitro process of growth;
Callus forms differentiation bud point in Fig. 3 sealwort culture in vitro process of growth;
Form indefinite bud in Fig. 4 sealwort culture in vitro process of growth;
Form the sealwort plant in Fig. 5 sealwort culture in vitro process of growth;
Form the root-like stock that expands in Fig. 6 sealwort culture in vitro process of growth;
The sealwort plant takes root in Fig. 7 sealwort culture in vitro process of growth;
The sealwort seedling of transplanting survival in Fig. 8 sealwort culture in vitro process of growth
The SDS-PAGE figure of Fig. 9 sealwort total protein and sealwort agglutinin I I;
Wherein, 1,2 be respectively wild and Regeneration in Vitro type root-like stock total protein; 3 is sealwort agglutinin II.
Embodiment
One, inducing of capsule silk sealwort explant callus of the present invention:
Embodiment 1
1, vegetable material
Capsule silk sealwort (Polygonatum cyrtonema Hua) is Liliaceae Polygonatum capsule silk sealwort.Material is taken from mountain area, Changning County, Sichuan Province.
2, callus inducing medium
Medium consists of:
(1) the MS minimal medium consists of: macroelement (1.65g/L NH
4NO
3, 1.9g/L KNO
3, 0.44g/L CaCl
2.2H
2O, 0.37g/L MgSO
4.7H
2O, 0.17g/L KH
2PO
4), the trace element (37.25mg/L Na
2EDTA, 27.8mg/L FeSO
4.7H
2O, 0.83mg/L KI, 6.2mg/LH
3BO
3, 22.3mg/L MnSO
4.4H
2O, 8.6mg/L ZnSO
4.7H
2O, 0.25mg/L Na
2MoO
4.2H
2O, 0.025mg/L CuSO
4.5H
2O, 0.025mg/L CoCl
2.6H
2O), 2mg/L glycosides propylhomoserin, 100mg/L inositol, 0.1mg/L thiamine hydrochloride (B1), 0.5mg/L hydrochloric acid pyrrole diindyl alcohol (B6), 0.5mg/L nicotinic acid.
(2) agar 8g/L, sucrose 30g/L.
(3) hormone composition and content see Table 1-1
(2.4-D 2,4-dichlorophenoxyacetic acid), 2,4 dichlorophenoxyacetic acid), 6-BA (N
6-Benzyladenine, N
6-benayl aminopurine), NAA (a-naphthaleneacetic acid, a-methyl) is the biochemical institute in a Chinese Academy of Sciences Shanghai product.
(4) pH is 5.8.
3, callus induces
Get the light yellow smart root-like stock of children, scrub clean gently with banister brush; Put into 75% ethanol again and soak 1min; Place 0.1% mercuric chloride solution (HgCl afterwards
2) middle immersion 8min sterilization, every 2min that crosses gently swings beaker; Use aseptic washing 5 ~ 6 times at last, each 2min.On aseptic paper, material is cut into 0.5cm * 0.5cm * 0.5cm size, is inoculated on the callus inducing medium.Placing temperature is that 25 ± 2 ℃, dark environment are cultivated 5-8 week down.
4, result
As shown in Figure 1, after two weeks of cultivation, the sealwort explant of inoculation begins to expand and there is yellow kick on the surface in dark surrounds, has obvious callus to generate about 4 weeks.By table 1-1 result as can be known, optimal medium consists of: MS minimal medium, 1.0mg/L 6-BA, 2.0mg/L2, and 4-D, 8g/L agar, 30g/L sucrose, pH5.8, effectively evoked callus forms.
The influence of the different medium of table 1-1 to inducing the sealwort callus to form
| | 2，4-D(mg/l) | 6-BA(mg/l) | The explant (individual) of inoculation | The callus (individual) that forms |
| 0.0 0.0 0.5 1.0 | 0.0 0.5 0.5 1.0 | 0 0 72 77 | 0 0 53 57 |
| 2.0 4.0 2.0 2.0 2.0 | 1.0 1.0 1.5 2.0 0.0 | 86 61 88 79 74 | 72 45 61 65 32 |
Two, the sealwort callus induces differentiation
1, material
The capsule silk sealwort callus that embodiment 1 is induced.
2, medium
The callus differential medium is: MS minimal medium, 0.50mg/L 2,4-D+0.5mg/L6-BA, 8g/L agar, 30g/L sucrose, pH5.8.
3, condition of culture
After 6 weeks, the callus that grows up among the embodiment 1 is directly changed in the differential medium, at 25 ± 2 ℃, illumination 13h/d cultivates 2-3 week under the condition of 2000Lx intensity of illumination.
Embodiment 3
Medium is: MS minimal medium, 1.0mg/L 2,4-D, A1.0mg/L 6-B, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 14h/d, and intensity of illumination is 1800Lx.
Other is identical with embodiment 2.
Embodiment 4
Medium is: MS minimal medium, 2.0mg/L 2,4-D, 1.0mg/L 6-BA, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 12h/d, and intensity of illumination is 1200Lx.
Other is identical with embodiment 2.
Embodiment 5
Medium is: MS minimal medium, 4.0mg/L 2,4-D, 1.0mg/L 6-BA, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 15h/d, and intensity of illumination is 1600Lx.
Other is identical with embodiment 2.
Embodiment 6
Medium is: MS minimal medium, 2.0mg/L 2,4-D, 1.0mg/L 6-BA, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 16h/d, and intensity of illumination is 1400Lx.
Other is identical with embodiment 2.
Embodiment 7
Medium is: MS minimal medium, 2.0mg/L 2,4-D, 2.0mg/L 6-BA, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 12h/d, and intensity of illumination is 1200Lx.
Other is identical with embodiment 2.
Embodiment 8
Medium is: MS minimal medium, 2.0mg/L 2,4-D, 0.4mg/L NAA, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 12h/d, and intensity of illumination is 1200Lx.
Other is identical with embodiment 2.
Embodiment 9
Medium is: MS minimal medium, 2.0mg/L 2,4-D, 0.5mg/L NAA, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 12h/d, and intensity of illumination is 1200Lx.
Other is identical with embodiment 2.
Embodiment 10
Medium is: MS minimal medium, 2.0mg/L 2,4-D, 0.6mg/L NAA, 8g/L agar, 30g/L sucrose, pH5.8.
Condition of culture is: temperature is 25 ± 2 ℃, illumination 12h/d, and intensity of illumination is 1200Lx.
Other is identical with embodiment 2.
The sealwort callus induction differentiated result of the foregoing description 2-10 sees Table 1-2:
Shown in accompanying drawing 2-6, be transferred to the callus of embodiment 1 gained on the differential medium after, callus further expands, color becomes green.From table 1-2 as can be known, differentiate budlet after two weeks, the differentiation rate of stem is the highest among the embodiment 4, can reach 83.7 ± 2.1%.
Differentiation effect the best of root and stem among the embodiment 7, average differentiation rate is 48.1 ± 3.1%.Along with the prolongation of incubation time, the stem continued growth also has a large amount of spherical expanding at base portion, and leaf launches until growing up to seedling.
Table 1-2 sealwort differentiation of calli result.
| Embodiment | The state of differentiation | Differentiation rate (%) | ||
| AS | AR | AS+ | ||
| Embodiment | ||||
| 2 embodiment 3 embodiment 4 embodiment 5 embodiment 6 embodiment 7 embodiment 8 embodiment 9 embodiment 10 | AS AS AS AS AS+AR AS+AR AR AR AR | 73.6 74.0 83.7 73.8 37.5 34.2 0.0 0.0 0.0 | 0.0 0.0 0.0 0.0 0.0 0.0 43.2 64.9 48.6 | 0.0 0.0 0.0 0.0 31.8 48.1 0.0 0.0 0.0 |
AS differentiates stem; AR differentiates root.
Differentiation rate=(the callus lines number of the number/inoculation of callus differentiation) * 100%
Three, root induction
Embodiment 11
1, material
The seedling that does not take root among the embodiment 2-10.
2, medium
Root media: 1/2MS minimal medium, 0.3mg/L NAA, 8g/L agar, 30g/L sucrose, pH5.8.
The 1/2MS minimal medium consists of: macroelement (0.83g/L NH
4NO
3, 0.95g/LKNO
3, 0.22g/L CaCl
2.2H
2O, 0.185g/L MgSO
4.7H
2O, 0.085g/L KH
2PO
4), the trace element (37.25mg/L Na
2EDTA, 27.8mg/L FeSO
4.7H
2O, 0.83mg/L KI, 6.2mg/LH
3BO
3, 22.3mg/L MnSO
4.4H
2O, 8.6mg/L ZnSO
4.7H
2O, 0.25mg/L Na
2MoO
4.2H
2O, 0.025mg/L CuSO
4.5H
2O, 0.025mg/L CoCl
2.6H
2O), 2mg/L glycosides propylhomoserin, 100mg/L inositol, 0.1mg/L thiamine hydrochloride (B1), 0.5mg/L hydrochloric acid pyrrole diindyl alcohol (B6), 0.5mg/L nicotinic acid.
3, condition of culture
Behind the seedling of turning out the sealwort explant, to root media, at 25 ± 2 ℃, illumination 14h/d cultivates 3-4 week under the condition of 2000Lx intensity of illumination with the sprigging of not taking root.
Embodiment 12
1, medium
Root media: 1/2MS minimal medium, 0.5mg/L NAA, 8g/L agar, 30g/L sucrose, pH5.8.
2, condition of culture
Temperature is 25 ± 2 ℃, illumination 12h/d, and intensity of illumination is 1200Lx.
Other is with embodiment 11.
Embodiment 13
1, medium
Root media: 1/2MS minimal medium, 0.4mg/NAAL, 8g/L agar, 30g/L sucrose, pH5.8.
2, condition of culture
Temperature is 25 ± 2 ℃, illumination 116h/d, and intensity of illumination is 1500Lx.
Other is with embodiment 11.
Embodiment 14
1, medium
Root media: 1/2MS minimal medium, 0.5mg/L 6-BA, 8g/L agar, 30g/L sucrose, pH5.8.
2, condition of culture
Temperature is 25 ± 2 ℃, illumination 12h/d, and intensity of illumination is 1200Lx, cultivates 2-3 week.
Other is with embodiment 11.
The root induction of the foregoing description 11-14 the results are shown in Table 1-3:
As shown in Figure 7, the seedling that does not take root is transferred in the root media, after two weeks, seedling differentiates root, and the root induction rate of each embodiment sees Table 1-3.Sealwort cultured in vitro process of growth is seen Figure of description 1-8.
The situation that table 1-3 young shoot exsomatizes and takes root under different condition.
| Embodiment | SN | NSR | FR(％) |
| Embodiment 11 embodiment 12 embodiment 13 embodiment 14 | 20 20 20 20 | 6 13 9 3 | 30.0 65.0 45.0 15.0 |
The SN young shoot is counted the NSR young shoot of taking root and is counted the FR frequency of taking root
Embodiment 15
Rooting tube plantlet among the embodiment 2-14 moved to from glass greenhouse under the natural daylight cultivated 3 ~ 4 days; Open bottle cap afterwards, hardening three days; Take out seedling then, clean agar, move into loose peat soil: in the vermiculite (1: 1), under temperature is 27 ℃, natural daylight, cultivate.
Seedling percent after the transplanting reaches 70-75%, the root-like stock of cultivation and wild type plesiomorphism (as shown in Figure 8), and grow faster than wild type, but active ingredient is similar to wild type content.
Four, the evaluation and the assay of mannose binding lectin in the capsule silk sealwort regenerating system
Embodiment 16
1.1 the extraction of root-like stock total protein
Get the Regeneration in Vitro sealwort root-like stock of wild pharmaceutically useful sealwort and the foregoing description preparation respectively, add the Tris-HCl (50mM, pH6.8 contain 0.2M NaCl) of 2 times of volumes (W/V) after the liquid nitrogen grinding, 4 ℃ of standing over night.Homogenate filters the centrifugal 15min of back 3000g.Supernatant is filtered with filter paper (Whatman3MM), and-20 ℃ of preservations are stand-by.
1.2SDS-PAGE electrophoresis reactive analysis
Get wild type and Regeneration in Vitro type total protein extracting solution and from wild sealwort the sealwort agglutinin I I sample of purifying (purification process is seen document
[6]) carry out discontinuous SDS-PAGE electrophoresis
[7](Tris-HCl, pH of buffer 7.2 concentrate glue 5%, separation gel 10%), silver dyes the observation electrophoresis result.
1.3 the rabbit erythrocyte agglutination activity is analyzed
Get above-mentioned two kinds of extracts and sealwort agglutinin II 25ul (concentration is identical) respectively, on 96 holes trace serum dilution plate, carry out serial doubling dilution
[8], in every hole, add 25ul rabbit erythrocyte (the rabbit erythrocyte washs 3 times with physiological saline, and is mixed with 2% cell suspension with same solution) again.Behind the concussion mixing, room temperature is placed 2h, test under microscope aggegation result.
2.1SDS-PAGE electrophoresis qualification result
The SDS of the root-like stock total protein extract of wilder root-like stock and the Regeneration in Vitro electrophoresis pattern (accompanying drawing 9) that dissociates, sealwort agglutinin II occupies bigger ratio (accounting for 20%), is the major protein colored zone; The albumen kind is similar with content in Regeneration in Vitro type and the wild type root-like stock total protein, and especially mannose binding lectin content does not have significant difference, and is key component in the total protein.
2.2 the measurement result of sealwort agglutinin blood coagulation activity
The blood coagulation activity measurement result of root-like stock total protein extract sees Table 1-4, and sealwort agglutinin II still can the aggegation rabbit erythrocyte when 0.25 μ g/ml; The rhizomatic total protein extract of wild sealwort has activity when 1.95 μ g/ml, and the total protein of extracting still has faint activity in the Regeneration in Vitro type root-like stock when 1.95 μ g/ml, and has obvious activity when 3.9 μ g/ml.The minimum aggegation concentration of mannose binding lectin differs less than an order of magnitude in wild type and the Regeneration in Vitro type root-like stock, the content that can judge mannose binding lectin in the sealwort root-like stock of wild type and Regeneration in Vitro is close, is the major protein in the root-like stock.The conclusion that is drawn with the SDS-PAGE electrophoresis result is consistent.
Wild and the Regeneration in Vitro type root-like stock total protein extract rabbit haemagglutination activity of table 1-4
| Protein concentration (ug/ml) | ||||||||||||
| Sample | 1000 | 500 | 250 | 125 | 62.5 | 31.25 | 15.6 | 7.8 | 3.9 | 1.95 | 0.98 | 0.49 |
| Wild type regeneration type sealwort agglutinin II | + + + | + + + | + + + | + + + | + + + | + + + | + + + | + + + | + + + | + +- + | - - + | - - + |
+: blood coagulation-: not blood coagulation
Obviously, according to foregoing of the present invention, ordinary skill knowledge and customary means according to this area, do not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways, all technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
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2, Huang Taikang work, " conventional Chinese medicine composition and pharmacology handbook, Beijing: Chinese Medicine science and technology publishing house, 2000,1590-1596
3、Blazarini J，Neyts J，Schols D，Hosoya M，Van Damme E J M，Peumans W，and De Clercq E，The mannose-specific plant lectinsfrom Cymbidium hybrid and Epipactis helleborine andthe(N-Acetylgucosamine)
n-specific plant lectin from Urticadioica are potent and selective inhibitors of humanimmunodeficiency virus and cytomegalovirus replication in vitro.Antiviral Res，1992，18：191-207
4、Powell K S，Gatehouse A M R，Hilder V A，Van Damme E J M，PeumansW J，
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Claims (7)
1. an a large amount of method that obtains sealwort agglutinin II is characterized in that: the steps include:
Inducing of a, capsule silk sealwort explant callus;
B, aforementioned callus is inoculated into consists of: MS minimal medium, 0.5-4.0mg/L2,4-D, 0-2.0mg/L 6-BA, 0-0.6mg/L NAA, 8g/L agar, 30g/L sucrose are induced living seedling in the differential medium of PH5.8; Cultivation temperature is 25 ± 2 ℃, and light application time is 12-16 hour/day, and intensity of illumination is 1200-2000Lx, cultivates 3-4 week;
C, the seedling that does not take root among the step b moved to consist of: 1/2MS minimal medium, 0.3-0.5mg/L NAA, 8g/L agar, 30g/L sucrose, root induction in the root media of PH5.8, cultivation temperature is 25 ± 2 ℃, light application time is 12-16 hour/day, intensity of illumination is 1200-2000Lx, cultivates 3-4 week;
D, with the seedling that takes root among step b and c elder generation hardening, cultivation then;
E, get the capsule silk sealwort regeneration root-like stock that above step obtains, extract preparation sealwort agglutinin II.
2. the method for acquisition sealwort agglutinin II according to claim 1 is characterized in that the capsule silk sealwort explant described in the step a is its root, root-like stock or leaf.
3. the method for acquisition sealwort agglutinin II according to claim 1, it is characterized in that: the inducing of step a described capsule silk sealwort explant callus, use consists of MS minimal medium, 0.5-2.0mg/L 6-BA, 0.5-4.0mg/L 2,4-D, 8g/L agar, 30g/L sucrose, the callus inducing medium of PH5.8; Cultivation temperature is 25 ± 2 ℃, and lucifuge is cultivated, incubation time 5-8 week.
4, the method for acquisition sealwort agglutinin II according to claim 3, it is characterized in that: in step a, described callus inducing medium consists of: MS minimal medium, 1.0mg/L 6-BA, 2.0mg/L 2,4-D, 8g/L agar, 30g/L sucrose, PH5.8.
5, the method for acquisition sealwort agglutinin II according to claim 1, it is characterized in that: the differential medium described in the step b consists of: MS minimal medium, 2.0mg/L2,4-D, 2.0mg/L 6-BA, 0.3mg/L NAA,, 8g/L agar, 30g/L sucrose, PH5.8; Cultivation temperature is 25 ± 2 ℃, and light application time is 12 hours/day, and intensity of illumination is 1200Lx, cultivates 3-4 week.
6, the method for acquisition sealwort agglutinin II according to claim 1, it is characterized in that: the root media described in the step c consists of: 1/2MS minimal medium, 0.5mg/LNAA, 8g/L agar, 30g/L sucrose, root induction in the root media of PH5.8, cultivation temperature is 25 ± 2 ℃, light application time is 12 hours/day, intensity of illumination is 1200Lx, cultivates 3-4 week.
7, the method for acquisition sealwort agglutinin II according to claim 1, it is characterized in that: hardening described in the steps d and cultivation comprise: the test-tube plantlet that will take root moves under the natural daylight and cultivated 3-4 days, opened the bottle cap hardening 2-3 days, and then taking-up seedling, clean agar, moving into loose weight ratio and be in 1: 1 the peat soil and vermiculite, is cultivation under 27 ± 1 ℃, natural daylight in temperature.
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| CN102550413B (en) * | 2011-12-26 | 2013-04-10 | 福建省三明市农业科学研究所 | Rapid propagation method for tube seedlings of high-grade polygonatum cyrtonema hua |
| CN102550409A (en) * | 2011-12-29 | 2012-07-11 | 重庆市秀山红星中药材开发有限公司 | Method of tissue culture propagation for rhizome polygonati |
| CN102630562B (en) * | 2012-03-28 | 2015-07-01 | 青阳县九华黄精产业化协会 | Rapid propagation method of Jiuhua Polygonatum sibiricum |
| CN102771395B (en) * | 2012-08-13 | 2014-04-16 | 安徽农业大学 | Tissue culture method for Polygonaturm sibiricum Redoute |
| CN103641543A (en) * | 2013-11-23 | 2014-03-19 | 大连大学 | Culture medium for prevention of astragalus mongolicus test-tube sprout etiolation |
| CN104396493A (en) * | 2014-11-03 | 2015-03-11 | 贵州省植物园 | Sexual propagation method for polygonatum zanlanscianense |
| CN105165321A (en) * | 2015-08-11 | 2015-12-23 | 玉龙县凤翔旺生态养殖有限责任公司 | Polygonatum kingianum cultivation method |
| CN106717690A (en) * | 2015-11-20 | 2017-05-31 | 梁成湖 | A kind of cultural method for being enriched with organic chrome yellow essence |
| CN106134996B (en) * | 2016-06-30 | 2018-06-29 | 恩施州源惠科技开发有限公司 | A kind of method of sealwort once-seedling forming |
| CN108812321B (en) * | 2018-07-09 | 2021-09-14 | 重庆市药物种植研究所 | Tissue culture rapid propagation method of polygonatum kingianum |
| CN109169286B (en) * | 2018-10-17 | 2021-06-18 | 重庆市药物种植研究所 | Polygonatum cyrtonema tissue culture method |
| CN111183902B (en) * | 2020-02-26 | 2022-03-08 | 四川农业大学 | Tissue culture method for polygonatum sibiricum |
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