CN104206139B - A kind of method improving content astaxanthin in astaxanthin-containing transgene tomato output and transgene tomato - Google Patents
A kind of method improving content astaxanthin in astaxanthin-containing transgene tomato output and transgene tomato Download PDFInfo
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Abstract
The invention provides a kind of method improving content astaxanthin in astaxanthin-containing transgene tomato output and transgene tomato, comprise sowing, apply fertilizer, shelter from heat or light, mark, Fruit post processing, freeze-drying, assay step, with the transgene tomato of astaxanthin-containing for research material, render transgenic tomato grows under full exposure, and at the clever solution of the tomato of Spraying in the flowering stage 0.02%.There is provided the tomato spirit solution spraying 0.02% to improve the method for the content astaxanthin of astaxanthin engineering tomato, and improve the method for astaxanthin engineering tomato yield with full exposure growth.It is simple that the present invention has method, and the advantages such as strong operability, the astaxanthin height accumulation technology and the industrialization that can be astaxanthin engineering tomato bring higher economic benefit.
Description
Technical field
The invention belongs to biological technical field, particularly, relate to a kind of method improving astaxanthin engineering tomato yield and content astaxanthin.
Background technology
Astaxanthin (Astaxanthin) is a kind of secondary carotenoid with superpower antioxidation activity; astaxanthin is also known as " ASX "; it is a kind of a kind of special Red carotenoids from finding from river crab shrimp shell, oyster, salmon and algae, fungi; there are protection blood vessel and eyes; radioresistance, anti-ageing, effect such as prevention senile dementia and cancer etc., be with a wide range of applications in fields such as medicine, food, cosmetics, feeding additive aquatic animals.Natural astaxanthin often synthesizes as secondary metabolite in vivo for some marine microorganism and a small amount of saccharomycete, but because extractive technique difficulty is high and be subject to the restriction such as algae kind or bacterial classification self problem, ubiquity astaxanthin yield is low, high in cost of production shortcoming, makes natural astaxanthin limits throughput.Chinese scholars has carried out much research to astaxanthin, mainly concentrates on its function and application prospect (Cao Xiuming etc., 2011; GoswamiG etc., 2010), biological accumulation (Geng Yuhuan etc., 2009; Li Xiaomeng etc., 2006) and extraction process (yellow virtuous just etc., 2009; Liu Hong is superfine, 2011) aspect such as.
At present, the production of astaxanthin technique possessed in the world mainly contains chemosynthesis and biotechnology is extracted, the astaxanthin of chemosynthesis is all much inferior compared with natural astaxanthin in structure, function, application and safety etc., therefore the extraction (Xiao Surong etc., 2011) that natural astaxanthin is generally tended in large-scale production is carried out.Haematococcus pluvialis cultivation, phaffia rhodozyma fermentation method, shrimp shell extraction all possesses the potentiality realizing natural astaxanthin industrialization, but because technical difficulty is high and by the restriction such as algae kind or bacterial classification self problem, ubiquity astaxanthin yield is low, high in cost of production shortcoming (Yan Yi etc., 2011), make natural astaxanthin limits throughput, price is high, and the price of its health products equals to gold especially, and most people are difficult to bear.Comparatively speaking, tomato is desirable astaxanthin processing factory, and the beta carotene in tomato and astaxanthin belong to carotenoid, and chemical constitution is similar.Therefore, Kunming Inst. of Botany, Chinese Academy of Sciences Huang Junchao researcher has made comprehensive comparative analysis (HuangJC etc. to the route of synthesis of micro-algae astaxanthin and the rate-limiting enzyme gene of catalysis committed step, 2012), solve the key issue of restriction higher plant synthesizing astaxanthin, and successfully develop the engineering New Tomato Variety of energy high-yield astaxanthin, its fruit can accumulate the astaxanthin of content close to haematococcus pluvialis.The more important thing is, tomato can be accomplished scale production as economic plants, and every mu of annual production can reach 20,000 jin, and farming is simple, cost is low, and thus the transgene tomato of astaxanthin-containing is desirable astaxanthin processing factory.There are no improving cultivation step in prior art, improve the report of the method for the content astaxanthin in the transgene tomato of astaxanthin-containing.
Summary of the invention
The present invention is intended to solve the above-mentioned problems in the prior art, provides a kind of method improving content astaxanthin in astaxanthin-containing transgene tomato output and tomato.The present invention with the transgene tomato of astaxanthin-containing for research material, carry out different cultivation experiments process, there is provided the tomato spirit solution spraying 0.02% to improve the method for the content astaxanthin of astaxanthin-containing transgene tomato, and improve the method for astaxanthin-containing engineering tomato yield with full exposure growth.It is simple that the present invention has method, and the advantages such as strong operability, the astaxanthin height accumulation technology and the industrialization that can be astaxanthin engineering tomato bring higher economic benefit.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of method improving content astaxanthin in astaxanthin-containing transgene tomato output and transgene tomato, comprise sowing, apply fertilizer, shelter from heat or light, mark, Fruit post processing, freeze-drying, assay step, it is characterized in that render transgenic tomato grows under full exposure, and at the clever solution of the tomato of Spraying in the flowering stage 0.02%.
Method is first by transgene tomato planting seed particularly, and after 2 months by little transplantation of seedlings in the ground, according to the form below sprays tomato spirit to tomato, process of sheltering from heat or light;
| No. 1 | No. 2 | No. 3 | No. 4 | |
| Spray tomato spirit solution | Do not spray | 0.02％ | Do not spray | 0.02％ |
| Shade net | Nothing | Nothing | Obscurity 80% | Obscurity 80% |
| Training measure | Two bar training | Two bar training | Two bar training | Two bar training |
Then, the 20-50 strain tomato of each process of random selecting, mark, treat that tomato enters fruiting period, each fruit on the every strain tomato marked also is marked, to obtain every strain gross yield, finally calculate average single plant yield by the total amount of the 20-50 strain of each process, and using the ripening fruits of plucking different disposal in batches as the material comparing content astaxanthin;
Fruit post processing: each 3-5 of ripening fruits that at every turn wins different disposal, repeat 3 times, fruit is cleaned, goes carefully, put into freezing more than 48 hours of the refrigerator-freezer that temperature is-20 DEG C, fruit freezes in fact, fruit is taken out and naturally thaws under the condition of lucifuge, in this cryogenic freezing process, cell sap can break through cell wall because of expanding, naturally flow out in the process of thawing, slough the moisture of fresh fruit 65-70%, then use the broken pulp of agitator, and use centrifuge, slough the moisture of material 60-65%;
Freeze-drying: use the material of freeze-drying machine to above-mentioned dehydration processing to carry out freeze drying, freeze-drying is set to: pre-freeze, to less than-25 DEG C, vacuumizes, opens heating (when vacuum reaches 0.03MPa, be set as 0 DEG C, can be progressive after 12 hours be transferred to 30 DEG C);
Content astaxanthin measures: by sample comminution good for freeze drying, pack, sealing, carry out content astaxanthin mensuration, measure and adopt high performance liquid chromatography to be separated, its liquid-phase condition: chromatographic column is WatersSpherisorbOSD2 (5 μm, 4.6,250mm), liquid phase model is Waters (Milford, MA, USA), sample size is 20 μ l; Mobile phase A is acetonitrile/methanol/Tris-HCL (pH8.0) mutually, and volume ratio is 84:2:14, B phase is methanol/ethyl acetate, and volume ratio is 68:32; With the flow velocity linear gradient elution pigment of 1.2mL/min, B phase from 100%A phase to 100%, 15 minutes each gradient used times, subsequently with B phase wash-out 10 minutes, each component is identified by the spectral absorption of each color component of comparison and standard items and relative retention time, be translated into concentration value by the integrated value of peak area and standard items being contrasted thus carry out quantitatively to each component, standard items are bought from sigma company and Wako company.
According to described cultivation method, wherein said transgene tomato to be preserving number the be transgene tomato of No. CGMCCNo9223.
The present invention with the transgene tomato of astaxanthin-containing for research material, carry out different cultivation experiments process, there is provided the tomato spirit solution spraying 0.02% to improve the method for the content astaxanthin of astaxanthin-containing transgene tomato, and improve the method for astaxanthin-containing engineering tomato yield with full exposure growth.It is simple that the present invention has method, and the advantages such as strong operability, the astaxanthin height accumulation technology and the industrialization that can be astaxanthin engineering tomato bring higher economic benefit.
Embodiment
Further illustrate essentiality content of the present invention by specific embodiments of the invention below, but do not limit the present invention with this.
Embodiment 1
1. materials and methods
1.1 research material
By (HuangJC etc. such as Huang Junchao, 2013) can the ketonize enzyme of astaxanthin and '-hydroxylase gene be become to proceed in wild-type tomatoes plant (cv.UC82B) with luteole by beta carotene in catalysis plant cell with being separated to screening in green alga by gene engineering method, the transgene tomato that obtains (on May 14th, 2014 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is: No. CGMCCNo9223, Classification And Nomenclature: red No. 1 transgene tomato of shrimp.Depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Phone: 010-64807355; Fax: 010-64807288.) planting seed is to little basin, latter two month of emerging transplants in the ground.Beat the cultivation steps such as bud, training, the extermination of disease and insect pest in guarantee consistent, and liquid manure abundance and under basically identical prerequisite, experimentally design, tomato spirit is sprayed to tomato, the different experiments process of sheltering from heat or light.
30 strain tomatoes of each process of random selecting, carry out mark.Treat that tomato enters fruiting period, each fruit on the every strain tomato marked also is marked, to obtain every strain gross yield, finally calculate average single plant yield by the total amount of 30 strains of each process.And with the ripening fruits of plucking different disposal in batches for comparing the research material of content astaxanthin.
1.2 research method
1.2.1 test arrangement is in greenhouse, designs four different experiments process (table 1).
The process of table 1 different experiments
| No. 1 | No. 2 | No. 3 | No. 4 | |
| Spray tomato spirit solution | Do not spray | 0.02％ | Do not spray | 0.02％ |
| Shade net | Nothing | Nothing | Obscurity 80% | Obscurity 80% |
| Training measure | Two bar training | Two bar training | Two bar training | Two bar training |
1.2.2 Fruit post processing: each 3-5 of ripening fruits that at every turn wins different disposal, repeat 3 times.Fruit is cleaned, stalk, put into freezing more than 48 hours of the refrigerator-freezer that temperature is-20 DEG C.Fruit freezes in fact, is taken out by fruit and naturally thaws under the condition of lucifuge.In this cryogenic freezing process, cell sap can break through cell wall because of expanding, and naturally flows out, slough the moisture of fresh fruit 65-70% in the process of thawing.Then use the broken pulp (so that post-production) of agitator, and use centrifuge, slough the moisture of material 60-65%.
1.2.3 freeze-drying: use the material of freeze-drying machine to above-mentioned dehydration processing to carry out freeze drying.Freeze-drying is set to: pre-freeze is to less than-25 DEG C; Vacuumize; Open heating (when vacuum reaches 0.03MPa, be set as 0 DEG C, can be progressive after 12 hours be transferred to 30 DEG C).
1.2.4 content astaxanthin measures: by sample comminution good for freeze drying, pack, sealing.And entrust Kunming Institute of Zoology Institute of Analysis of the Chinese Academy of Sciences to carry out content astaxanthin mensuration.Measure and adopt high performance liquid chromatography to be separated, its liquid-phase condition: chromatographic column is WatersSpherisorbOSD2 (5 μm, 4.6,250mm), and liquid phase model is Waters (Milford, MA, USA), and sample size is 20 μ l.Mobile phase A is acetonitrile/methanol/Tris-HCL (pH8.0) mutually, and volume ratio is 84:2:14, B phase is methanol/ethyl acetate, and volume ratio is 68:32.With the flow velocity linear gradient elution pigment of 1.2mL/min, the B phase from 100%A phase to 100%, 15 minutes each gradient used times, subsequently with B phase wash-out 10 minutes.Each component is identified by the spectral absorption of each color component of comparison and standard items and relative retention time.Be translated into concentration value by the integrated value of peak area and standard items are contrasted thus each component is carried out quantitatively.Standard items are bought from sigma company and Wako company.
1.2.5 data analysis: all data are all that Using statistics software SPSS16.0forWindows (SPSSInc., Chicago, USA) analyzes, between different disposal, the comparison in difference of (interior) adopts one-way analysis of variance and LSD inspection.All statistical chart SigmaPlot10.0forwindows (SystatSoftwareInc., city, USA) are drawn.
2. result and discussion
The average single plant yield (table 2) of 2.1 different experiments process
The average single plant yield of table 2 different experiments process
| No. 1 | No. 2 | No. 3 | No. 4 | |
| Average single plant yield (kg) | 10.99±0.15a | 11.20±0.51a | 2.90±0.13b | 4.37±0.53c |
In table, data are mean value ± standard error (n=30), are shown with significant difference (P≤0.05) with alphabet different in a line.
Experimental result shows, 80% shelters from heat or light can have a strong impact on the output of astaxanthin-containing transgene tomato, the average yield of the transgene tomato of growth under full exposure is 10.99 kilograms/strain, and growth is 2.9 kilograms/strain (table 2) at the average yield of 80% transgene tomato sheltered from heat or light under condition.Tomato is a kind of sun plant, has higher photosynthesis potentiality and higher light saturation point, and the tomato leaf grown under high light has higher photosynthetic capacity than the tomato leaf of growth under low light environment.Due to the output of tomato and the photosynthesis of tomato plant closely related, the tomato of growth under high light can synthesize more carbohydrate, for the g and D of tamato fruit, so 80% main cause of sheltering from heat or light reduction transgene tomato output limits its photosynthetic capacity because shelter from heat or light, the g and D of tamato fruit is restricted.
Spray tomato spirit not affect the output of the astaxanthin-containing transgene tomato grown under full exposure, but 80% output of sheltering from heat or light astaxanthin-containing transgene tomato can be improved.After spraying tomato spirit, 80% output of sheltering from heat or light astaxanthin-containing transgene tomato is increased to 4.37 kilograms/strain (table 2) by 2.9 kilograms/strain.Tomato spirit (p-chlorophenoxyacetic acid) is a kind of phenol plant growth regulator, and it can prevent fallen flowers, shedding.The tomato spirit solution spraying 0.02% can improve percentage of fertile fruit, and then increases by 80% output of sheltering from heat or light astaxanthin-containing transgene tomato.
The content astaxanthin of 2.2 different experiments process
This experiment be investigated spray 0.02% tomato spirit solution on the impact of the content astaxanthin in astaxanthin-containing transgene tomato, after spraying tomato spirit, under full exposure condition, be all greatly improved (table 3) with 80% content astaxanthin sheltered from heat or light in the astaxanthin-containing transgene tomato of lower growth.Tamato fruit is after full maturity, and three Carotenoids (lycopene, beta carotene and xanthophyll) content reaches peak (journey Sun Liang etc., 2007).Because tomato spirit has dematuration, spray the content that tomato spirit may improve carotenoid in tomato, and carotenoid is the precursor of chemical activators, can as a kind of method of the content astaxanthin improved in astaxanthin-containing transgene tomato so spray tomato spirit.
The content astaxanthin of table 3 different experiments process
In table, data are mean value ± standard error (n=3).
3. best-of-breed technology scheme
According to above-mentioned experimental result, astaxanthin-containing transgene tomato grows in full exposure, and can improve tomato yield and content astaxanthin at the tomato spirit solution of Spraying in the flowering stage 0.02%, these two kinds of conditions is combined the astaxanthin that can obtain and contain most volume.
Claims (1)
1. one kind is improved the method for content astaxanthin in astaxanthin-containing transgene tomato output and transgene tomato, comprise sowing, apply fertilizer, shelter from heat or light, mark, Fruit post processing, freeze-drying, assay step, it is characterized in that render transgenic tomato grows under full exposure, and at the clever solution of the tomato of Spraying in the flowering stage 0.02%, first by transgene tomato planting seed, after 2 months by little transplantation of seedlings in the ground, according to the form below tomato spirit is sprayed to tomato, process of sheltering from heat or light;
Then, the 20-50 strain tomato of each process of random selecting, mark, treat that tomato enters fruiting period, each fruit on the every strain tomato marked also is marked, to obtain every strain gross yield, finally calculate average single plant yield by the total amount of the 20-50 strain of each process, and using the ripening fruits of plucking different disposal in batches as the material comparing content astaxanthin;
Fruit post processing: each 3-5 of ripening fruits that at every turn wins different disposal, repeat 3 times, by fruit cleaning, stalk, put into freezing more than 48 hours of the refrigerator-freezer that temperature is-20 DEG C, fruit freezes in fact, fruit is taken out and naturally thaws under the condition of lucifuge, in this cryogenic freezing process, cell sap can break through cell wall because of expanding, naturally flow out in the process of thawing, slough the moisture of fresh fruit 65-70%, then use the broken pulp of agitator, and use centrifuge, slough the moisture of material 60-65%;
Freeze-drying: use the material of freeze-drying machine to above-mentioned dehydration processing to carry out freeze drying, freeze-drying is set to: pre-freeze, to less than-25 DEG C, vacuumizes, opens heating, when vacuum reaches 0.03MPa, be set as 0 DEG C, progressively after 12 hours is transferred to 30 DEG C;
Content astaxanthin measures: by sample comminution good for freeze drying, pack, sealing, carry out content astaxanthin mensuration, measure and adopt high performance liquid chromatography to be separated, its liquid-phase condition: chromatographic column is WatersSpherisorbOSD25 μm, 4.6,250mm, liquid phase model is WatersMilford, MA, USA, sample size is 20 μ l; Mobile phase A is acetonitrile/methanol/Tris-HCLpH8.0 mutually, and volume ratio is 84:2:14, B phase is methanol/ethyl acetate, and volume ratio is 68:32; With the flow velocity linear gradient elution pigment of 1.2mL/min, B phase from 100%A phase to 100%, 15 minutes each gradient used times, subsequently with B phase wash-out 10 minutes, each component is identified by the spectral absorption of each color component of comparison and standard items and relative retention time, be translated into concentration value by the integrated value of peak area and standard items being contrasted thus carry out quantitatively to each component, standard items are bought from sigma company and Wako company; Described transgene tomato to be preserving number the be transgene tomato of No. CGMCCNo9223.
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| CN106106122B (en) * | 2016-01-04 | 2018-03-06 | 中国科学院昆明植物研究所 | Leaf produces the breeding method of astaxanthin tomato variety |
| CN106106121B (en) * | 2016-01-04 | 2017-11-21 | 中国科学院昆明植物研究所 | A kind of breeding method of leaf production astaxanthin tomato variety |
| CN107235881B (en) * | 2017-06-15 | 2019-06-11 | 中国科学院昆明植物研究所 | The supercritical carbon dioxide extraction method of astaxanthin in transgenic engineering tamato fruit |
| CN114885635A (en) * | 2022-03-31 | 2022-08-12 | 西北农林科技大学 | Method for accumulating carotenoids in seedling stage of orange Chinese cabbage |
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| CN102888425B (en) * | 2012-07-02 | 2015-02-25 | 中国科学院昆明植物研究所 | Method for producing astaxanthin by using transgenic plant |
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