CN1108373C - Isolated biological synthesis of elliptone - Google Patents
Isolated biological synthesis of elliptone Download PDFInfo
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- CN1108373C CN1108373C CN 00117506 CN00117506A CN1108373C CN 1108373 C CN1108373 C CN 1108373C CN 00117506 CN00117506 CN 00117506 CN 00117506 A CN00117506 A CN 00117506A CN 1108373 C CN1108373 C CN 1108373C
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- callus
- tubatoxin
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Abstract
The present invention relates to a biological technique for producing useful secondary metabolite by plant invitro tissue culture. The present invention comprises callus induction, callus growth culture, callus differentiation and synthesis, and rotenone extraction, and provides culture mediums, culture conditions (such as light intensity and temperatures) and culture periods suitable for the growth of invitro culture cells, and the synthesis and the accumulation of the rotenone. The present invention also provides an application of a rotenone biosynthesize precursor object in separation biosynthesis. The rotenone produced by the invitro culture can overcome the defect of a production method of farm living body cultivation, save the land consumption, shorten the production time, reduce the cost, be free from the limitations of external environmental conditions, and achieve the goal of continuous industrial production.
Description
The present invention relates to biotechnology, especially relate to the plant in vitro tissue and cultivate the useful secondary metabolite technology of producing.
Tubatoxin is famous vegetable source pesticide active ingredient, characteristics with efficient, low toxicity, low resistance and non-environmental-pollution can be used as the good sterilant of sanitary insect pests such as hundreds of insects on control fruit tree, vegetables, tealeaves, flowers and the food crop and mosquitos and flies.At present, the source of tubatoxin mainly obtains by the field cultivation derris root, and output is subjected to the restriction in region and season, and the content instability, and the production time is long, occupies cultivated land, and causes output relative problem such as excessive with production cost on the low side.
The object of the present invention is to provide a kind of stripped biosynthesizing production method of tubatoxin, utilization can produce the plant of tubatoxin, explant as the trifoliate jewelvine of pulse family, mountain green soy bean etc. carries out isolated culture biosynthesizing tubatoxin, overcome present tubatoxin and only obtain, occupy cultivated land, be subjected to deficiencies such as the big and resource-constrained of environmental influence by the field planting derris root.
The present invention includes that callus induction, callus growth are cultivated, the callus branch is combined to and the several programs of the extraction of tubatoxin, concrete performing step is as follows:
1. callus induction: get the plant that can produce tubatoxin, explant as the trifoliate jewelvine of pulse family, mountain green soy bean etc., behind sterilization, under aseptic condition, cut into fritter, place and be added with 6-BA 2.253 μ g/ml+2,4-D 2.210 μ g/ml, agar 0.8%, sucrose 30g/L is on the MS minimum medium of pH value 5.8, in illumination is light: dark=12: 12 hours, light intensity is about 2000 luxs, relative humidity 80 ± 5%, and isolated culture obtains callus lines in the culturing room that temperature is 28 ± 2.5 ℃.
2. callus growth is cultivated: with above-mentioned callus lines after cutting, in the MS substratum of access after improvement, in illumination is light: dark=12: 12 hours, light intensity is about 4200 luxs, relative humidity 80 ± 5%, cultivate under the condition of 30 ± 2.0 ℃ of temperature and ventilation and can obtain maximum biomass.The MS culture medium prescription of improvement is: trace element is consistent with the concentration and the MS substratum of macroelement, and organism is 2 times of concentration in the MS substratum, and sucrose is 50g/L, 0.8% agar, hormone combinations is 6-BA 0.225 μ g/ml+2,4-D 6.630 μ g/ml, and the pH value is 6.0.
3. differentiation of calli is synthetic cultivates: the callus after grown cultures is increased changes the callus branch over to and is combined to in the substratum, in illumination is light: dark=12: 12 hours, light intensity is about 1100 luxs, relative humidity 80 ± 5%, cultivate under the condition of 30 ± 2.0 ℃ of temperature and ventilation, make callus differentiate root and synthetic tubatoxin, after this change over to and continue under the dark condition to cultivate 5-10 days, make the biosynthetic tubatoxin accumulation of exsomatizing.
Be used for callus differentiation synthetic culture medium prescription, its each composition is identical with the modified MS medium of step 2, but hormone changes 0.1 μ g/ml NAA into, and the pH value changes 6.5 into.In order to promote the tubatoxin biosynthesizing of exsomatizing, in substratum, add 100-150 μ g/ml phenylalanine as the biosynthetic precursor compound of tubatoxin.
4. the extraction of tubatoxin: adopt the organic solvent Soxhlet to extract or the dipping extraction.Isolated culture after with certain amount of organic solvent oven dry being ground carries out extracting, the extract of acquisition, after filtration, concentrate, with the refining tubatoxin that obtains of chromatography.
The major equipment that uses among the present invention has high-pressure sterilizing pot, aseptic technique platform.The biosynthetic rotenone content that exsomatizes can be analyzed with the HPLC method.
Utilize the technology of isolated culture to have special advantages and meaning: to produce the unfavorable factor that tubatoxin can avoid field cultivation to be brought by isolated culture with biosynthetic approach production tubatoxin, save the arable land, shorten the production time, reduce cost, and can not be subjected to the restriction of external environmental condition, reach the purpose of sustainable suitability for industrialized production.
Embodiment:
The healthy young leaflet tablet of getting trifoliate jewelvine is an explant, flooded 8-10 minute with 0.1% mercuric chloride solution, behind sterilization, under aseptic condition, cut into fritter, place and be added with 6-BA 2.253 μ g/ml+2,4-D 2.210 μ g/ml, agar 0.8%, sucrose 30g/L, on the MS minimum medium of pH value 5.8, in illumination is light: dark=12: 12 hours, light intensity is about 2000 luxs, relative humidity 80 ± 5%, in the culturing room that temperature is 28 ± 2.5 ℃ isolated culture 25-30 days, obtain that cell is loose, oyster white, well-grown callus lines.
Get above-mentioned callus lines, insert in the MS substratum of improvement after being cut into fritter, in illumination is light: dark=12: 12 hours, light intensity is about 4200 luxs, relative humidity 80 ± 5% was cultivated 30 days under the condition of 30 ± 2.0 ℃ of temperature and ventilation, can obtain maximum biomass, 7.03 times of average dry weight weightening finishes.The MS culture medium prescription of improvement is: trace element is consistent with the concentration and the MS substratum of macroelement, and organism is 2 times of concentration in the MS substratum, and sucrose is 50g/L, 0.8% agar, hormone combinations is 6-BA 0.225 μ g/ml+2,4-D 6.630 μ g/ml, and the pH value is 6.0.
Callus lines that will be after grown cultures increases, the callus branch of transferring is combined in the substratum of usefulness, its prescription: each composition is identical with above-mentioned modified MS medium, but hormone changes 0.1 μ g/mlNAA into, the pH value changes 6.5 into, and adds 100-150 μ g/ml phenylalanine as the biosynthetic precursor compound of tubatoxin.In illumination is light: dark=12: 12 hours, light intensity is about 1100 luxs, relative humidity 80 ± 5%, 30 ± 2.0 ℃ of temperature, with cultivation under the condition of ventilating 25 days, make callus differentiate root and synthetic tubatoxin, after this change over to and continue under the dark condition to cultivate 5-10 days, make the biosynthetic tubatoxin accumulation of exsomatizing.
After gained isolated culture drying grinds, put into container, add 10 times of quantitative analysis pure acetones, place then to flood on the shaking table and extracted 48 hours, filter out extract, through concentrating, get light brown solid, extraction yield is 3.96%.Extract is dissolved in methyl alcohol, with the reversed phase column chromatography method refining the tubatoxin product.HPLC measurement result, isolated culture tubatoxin productive rate reach every hectogram and cultivate produce tubatoxin 0.56 gram.
Callus was the lag-phase at preceding 5 days after being transferred on the growth medium, and the speed of growth is slow; It is slow vegetative period between the 5th day to the 10th day; Entered logarithmic phase on the 10th day, callus growth speed is very fast; To after 20 days, callus Growth speed begins to slow down, and after cell entered slow 30 days rise periods again, callus Growth almost stopped; Callus Growth enters stationary phase in the time of the 35th day.Callus synthesizes and the accumulation tubatoxin also presents certain rules, accumulation tubatoxin when callus begins from cultivating, from 5 days to 15 days, callus synthesizes and the accumulation tubatoxin presents the trend that increases progressively, when cultivating the 15th day, the rotenone content in the callus is the highest; The content of tubatoxin presents the trend of reduction after 15 days, almost detects the content less than tubatoxin when cultivating 35 days.
The content of product tubatoxin is measured with the HPLC method, the instrument title: U.S. Hewlett-Packard 1100 high performance liquid chromatographs (configuration: quaternary gradient pump, UV, visible light optical wavelength detector, manual injector, Hewlett-Packard's chem workstation); Pillar: HEWLWTT PACKARD (ODS HYPERSIL, 5 μ m, 125 * 4mm) condition determinations: detect wavelength: λ=295nm; Moving phase: 75: 25=methyl alcohol: water; Flow velocity: 1ml/min; Detection sensitivity: AUFS=0.001; Column temperature: normal temperature; Sample size: 20 μ l.The standard specimen retention time is 5.661 minutes.According to the absorption peak of the tubatoxin of concentration known standard specimen ratio and the relation that the ratio of their content separately is directly proportional, can draw the rotenone content of sample with the tubatoxin absorption peak area of institute test specimens.
Test with acetone extract, the biosynthetic tubatoxin that find to exsomatize is suitable with the tubatoxin of field planting acquisition to the biological activity of cabbage caterpillar, and it is to concentration (AFC in 24 hours the food refusal of small white 5 instar larvaes
50) be 2.375 μ g/ml, 24 hours median lethal dosage (LD
50) be 0.283 μ g/ head, 24 hours restraining effect 50% effective concentration (EC to body weight gain
50) be 2.050 μ g/ml; 48 hours EC
50Be 1.170 μ g/ml, egg hatching rate is 14.56%, and adult eclosion rate is 22.22%, and the larvae pupation abnormal rate is 87.50%.Indoor pot experiment is the result show, is under 40 μ g/ml samples are handled, can prevent and treat well causing harm of cabbage caterpillar in tubatoxin concentration.
Claims (3)
1. isolated biological synthesis of elliptone is characterized in that the tubatoxin biosynthesizing of exsomatizing, and comprises that callus induction, callus growth are cultivated, differentiation of calli is synthetic and the extraction of tubatoxin;
(1) callus induction: get the plant that can produce tubatoxin, explant as the trifoliate jewelvine of pulse family, mountain green soy bean etc., behind sterilization, under aseptic condition, cut into fritter, place and be added with 6-BA 2.253 μ g/ml+2,4-D 2.210 μ g/ml, agar 0.8%, sucrose 30g/L is on the MS minimum medium of pH value 5.8, in illumination is light: dark=12: 12 hours, light intensity is about 2000 luxs, relative humidity 80 ± 5%, the callus lines that isolated culture obtains in the culturing room that temperature is 28 ± 2.5 ℃;
(2) callus growth is cultivated: after the callus lines cutting, inserting in the MS substratum after improvement, is light in illumination: dark=12: 12 hours, light intensity is about 4200 luxs, relative humidity 80 ± 5% is cultivated under the condition of 30 ± 2.0 ℃ of temperature and ventilation and can be obtained maximum biomass; The MS culture medium prescription of improvement is: trace element is consistent with the concentration and the MS substratum of macroelement, and organism is 2 times of concentration in the MS substratum, and sucrose is 50g/L, 0.8% agar, hormone combinations is 6-BA 0.225 μ g/ml+2,4-D 6.630 μ g/ml, and the pH value is 6.0;
(3) the callus branch is combined to cultivation: will be by callus lines, inserting the callus branch is combined to in the substratum, in illumination is light: dark=12: 12 hours, light intensity is about 1100 luxs, relative humidity 80 ± 5%, 30 ± 2.0 ℃ of temperature, with the ventilation condition under cultivate, make callus differentiate root and synthetic tubatoxin, after this change over to and continue under the dark condition to cultivate, make the biosynthetic tubatoxin accumulation of exsomatizing; Be used for callus differentiation synthetic culture medium prescription, its each composition is identical with the modified MS medium of (2), but hormone changes 0.1 μ g/ml NAA into, and the pH value changes 6.5 into;
(4) extraction of stripped biosynthetic tubatoxin: adopt the organic solvent Soxhlet to extract or the dipping extraction, isolated culture after with certain amount of organic solvent oven dry being ground carries out extracting, the extract that obtains, after filtration, concentrate, with the refining tubatoxin that obtains of chromatography.
2. according to the said isolated biological synthesis of elliptone of claim 1, it is characterized in that in the callus branch is combined to the substratum of cultivation, adding tubatoxin biosynthesizing precursor compound phenylalanine, its add-on is 100-150 μ g/ml.
3. according to the said isolated biological synthesis of elliptone of claim 1, it is characterized in that callus differentiates root after, change over to and continue under the dark condition to cultivate 5-10 days.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 00117506 CN1108373C (en) | 2000-10-18 | 2000-10-18 | Isolated biological synthesis of elliptone |
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| CN 00117506 CN1108373C (en) | 2000-10-18 | 2000-10-18 | Isolated biological synthesis of elliptone |
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| CN1290745A CN1290745A (en) | 2001-04-11 |
| CN1108373C true CN1108373C (en) | 2003-05-14 |
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