CN101138314B - Breeding method of oil-bearing crop with high content of carotene - Google Patents
Breeding method of oil-bearing crop with high content of carotene Download PDFInfo
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- CN101138314B CN101138314B CN2007100528417A CN200710052841A CN101138314B CN 101138314 B CN101138314 B CN 101138314B CN 2007100528417 A CN2007100528417 A CN 2007100528417A CN 200710052841 A CN200710052841 A CN 200710052841A CN 101138314 B CN101138314 B CN 101138314B
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Abstract
The present invention provides a cultivation method of oil plant containing rich vitamin A and relates to the oil plant breeding technology. The method is that the spectrophotometer is used for selecting large amounts of seed resource or the gene engineering technology is used for over-expressing the crucial genes of synthesizing the carotenoid; the basic germ plasm of seeds selected contain the carotenoid which is higher than 60Mug.gFW<-1>; the basic germ plasm with high carotenoid content is taken as the donor and the high-quality seeds are taken as the acceptor; the conventional breeding technology is used for hybridizing to obtain high-quality breeds with high carotenoid; the HPLC method is used for detecting the carotenoid content of the breeding seeds to control the content in the range of 60-300Mug.gFW<-1>. The selecting method of the present invention is simple with high efficiency, accurate results, low cost and reliable resources. The plant oil squeezed from the oil plant cultivated with the method of the present invention contains rich vitamin A and is characterized by high nutrition and anti-aging, disease resistant and deterioration resistant functions.
Description
Technical field
The present invention relates to a kind of oil crops new variety selective breeding technology field.
Background technology
Carotenoid be a class by isoprenoid deutero-pigment, extensively be present in plant and other biology.In plant, carotenoid is the important component part in the photosynthesizer, and auxiliary luminous energy absorption also has antioxygenation.Simultaneously, carotenoid is also most important for the mankind's nutrition and health.Existing studies show that, carotenoid can have the function of following several respects: 1. carotenoid is the precursor (Provitamin A, provitamin A) of vitamin A with beta carotene especially, and vitamin A is the micro-nutrient composition of needed by human; 2. have immunologic function, can improve the ability that the human immune system resists pathogen; 3. has antioxidant function, energy cancellation singlet oxygen and the detrimentally affect of removing interior free yl, thereby delaying human body caducity; 4. can prevent the generation of coronary heart disease and cancer, or delay the development of cancer; 5. promote to connect between cell seam and exchange (see the summary document, Han Yashan, the Research progress on Function of carotenoid, China Agricultural University's journal, 1999,4:64-70).
The mankind itself can not synthesize carotenoid, must absorb acquisition from the food that is rich in carotenoid, so that synthesize self required A1 vitamin group material (as xanthopsin, Vogan-Neu etc.).In the mankind's main food, only have the vegetables of minority, fruit (as: Radix Dauci Sativae) to be rich in carotenoid, then do not have in staple food crop (as paddy rice, wheat, corn) and the oil crops (as rape, soybean) or content seldom.The scarcity in carotenoid source makes healthy grave danger that faces of low developed area people (particularly children) in the world, endures the puzzlement of multiple diseases such as blind, diarrhoea, respiratory disease, children's measles, coronary heart disease and cancer to the fullest extent.Utilize the means of modern technology, by the kind genetic improvement, improve content of carotenoid in the human main food, ensure the supply of carotenoid, be expected to improve greatly people's nutrient health level, thereby have great economy and social effect (Naik P.S.et al., Genetic manpulation of carotenoid pathway in higher plants, Current Science, 2003,85:1423-1430).The scientist of the U.S. utilizes engineered method, has created the paddy rice (being commonly called as " gold rice ") of being rich in β-Hu Luobusu, has obtained the significant social effect.But aspect oil crops, application example is not arranged as yet.At this present situation, be necessary to cultivate a kind of novel oil crops kind that is rich in carotenoid, so that solve the insufficient problem of people's vitamin A intake.
Grease is the meeting slow oxidation in storage process, forms various oxide compounds, and causes spoiled by rancid oil or fat.Behind the Oxidation of Fat and Oils, nutritive substances such as VITAMIN wherein and indispensable fatty acid are destroyed, and the grease after the edible oxidation has detrimentally affect to HUMAN HEALTH.So most of edible oils, the fat that is used to toast and oils often need to add certain antioxidant and prevent its autoxidation.Years of researches show that carotenoid is a kind of effective natural antioxidants, and it is by cancellation
1O
2Reduce the generation of photosensitized oxidation thing.Studies show that of the anti-photosensitized oxidation of carotenoid oil-control, carotenoid has restraining effect to the photosensitized oxidation of soya-bean oil and rape oil, and antioxidant effect increases with the concentration rising of carotenoid, the anti-photosensitized oxidation effect of carotenoid mainly comes from the number of polyenoid chain, particularly conjugated double bond in the structure.
In the world, fundamental research for plant carotenoid pathways metabolism and gene regulatory network is more deep, the separated clone of gene (the Cunningham F.X.and Gantt E. that part is crucial, Genes andenzymes of carotenoid biosynthesis in plants.Annual Review of Plant Physiology andPlant Molecular Biology, 1998,49:557-583), by the genetic engineering technique means, key gene on the overexpression route of synthesis, can increase substantially content of carotenoid (Christine K, et al., Seed-specific overexpression of phytoene synthase:increase in carotenoids and othermetabolic effects, the plant Journal, 1999,20:401-412), but these researchs also rest on laboratory stage.At home, the this patent inventor has set up the fast survey technology (Gao Guizhen etc. of Semen Brassicae campestris carotenoid content, the research of the total quantity measuring method of Semen Brassicae campestris carotenoid, plant genetic gene journal, 2005,6:414-417), begin the screening of rape variety resource, but do not proposed clear and definite research purpose and the technical scheme of dealing with problems.
Summary of the invention
The objective of the invention is at above-mentioned present situation, aim to provide the method that a kind of cultivation is rich in carotenoid (provitamin A), is had the novel oil crops kind of anti-ageing, disease-resistant, anti-oxidant function, with the nutritive value and the medical care effect of further raising Vegetable oil lipoprotein.
The implementation of the object of the invention is, has the method for cultivation of the former oil crops of homovitamin A, and its concrete steps are as follows:
1) acquisition of basic germplasm, undertaken by following approach:
The oil crops germ plasm resource that China preserves is screened in a large number, and screening method adopts spectrophotometry, uses the spectrophotometric determination optical density value, the substitution formula
X-carotenoid content in the formula
The maximum absorbance value that A-measures at 445 ± 10nm place
The amount of the extracting solution that y-is used
The weight of g-analytic sample
Ask and calculate content of carotenoid X in the seed, select X and be higher than 60 μ ggW
-1Resource as the basic germplasm of high carotenoid breeding;
Spectrophotometry is, every part of resource takes by weighing the 1g seed respectively, fully dry, in mortar, grind, change triangular flask over to, add the 30ml extracting solution, extracting solution is the mixed solution of sherwood oil and acetone, the volume ratio of sherwood oil and acetone is 1: 1, vibration was extracted 6 hours under the lucifuge condition, and spectrophotometer is confirmed the existence of carotenoid in the interscan of 300-800nm wavelength region when observing three characteristic peaks, measure the optical density value at main peak 445 ± 10nm place, substitution formula compute classes carotene carotene content;
2) breed of variety, carry out in two kinds of situation:
1. 60 μ ggFW have been higher than for carotenoid content
-1, and other proterties meet the germ plasm resource of production requirement, direct utilization and extention;
2. 60 μ ggFW have been higher than for carotenoid content
-1, but other proterties does not meet the germ plasm resource of production requirement, then carries out genetic improvement, method is with improved seeds; Or the sterile line of cross-fertilize seed, maintenance line and recovery system; As receptor parent, high carotenoid basic germplasm is a donor parents, hybridizes transformation, high carotenoid content is carried out many for directive breeding, and the high carotenoid gene of enrichment obtains the kind or the hybrid parents of high carotenoid;
3) preparation of commodity kind:
1. for the kind of high carotenoid, expanding propagation under the isolation condition is prepared the oil crop seeds by using with high carotenoid;
2. for cross-fertilize seed:
Two-series hybrid: according to a conventional method, with sterile dual-purpose with recover system capable by 3: 1 than planting under the isolation condition, from dual-purpose system, pull out flowering period and can educate strain, account for 50%, gather in the crops seed from sterile strain during seed maturity, be the oil crop seeds by using with high carotenoid of two-series hybrid;
Three line hybrid seed according to a conventional method, is sowed in the isolation condition and to be planted sterile line and to recover system, row is than being 2: 1, and flowering period, supple-mentary pollination cut off the male parent row after the sterile line pollination, gather in the crops seed from sterile strain during seed maturity, be the oil crop seeds by using with high carotenoid of three line hybrid seed;
4) technical indicator: the technical indicator of the seed carotenoid content of seed selection kind is at 60-300 μ ggFW
-1Between, utilize the HPLC method accurately to measure or the spectrophotometry rapid determination, each crop seed carotenoid content of selection is in above-mentioned scope.
Advantage of the present invention and positively effect are as follows:
1, utilize content of carotenoid in the spectrophotometry Semen Brassicae campestris, method is simple efficiently, the result is accurate, with low cost, can realize extensive screening assay;
2, be used in combination the accurate assay method of spectrophotometer rapid method and HPLC, the high carotenoid germ plasm resource that screening obtains has higher reliability;
3, in the process of cultivating high carotenoid rape new variety, content of carotenoid is controlled at 60-300 μ ggFW
-1Scope within, make to prevent too high some negative impacts that bring of carotenoid content again by the physiologically active can give full play to carotenoid, as rate of emergence reduction, dormancy time prolongation etc.;
4, the excellent gene transformation of high carotenoid is cultivated high carotenoid oil vegetable kind in good rape variety, with this rape is the vegetables oil rich vitamin A that raw material squeezes, have multiple efficacies such as high nutrition, anti-aging, disease-resistant, anti-oxidant and anti-bad change, can satisfy people's high-quality life requirement, its economic use value is very big.
Description of drawings
Fig. 1 is a breed breeding programchart of the present invention
Fig. 2 is that spectrophotometry scanning obtains the carotenoid typical absorption spectrogram in the Semen Brassicae campestris
Fig. 3 is that spectrophotometer scans the carotenoid typical absorption spectrum in the soybean seeds that obtains
Embodiment
Method of the present invention is: 1) screen germ plasm resource in a large number with spectrophotometry, perhaps utilize genetic engineering technique overexpression carotenoid to synthesize key gene, obtain the seed carotenoid content and be higher than 60 μ ggFW
-1Basic germplasm; 2) be donor with high carotenoid basic germplasm, improved seeds are acceptor, utilize traditional breeding method to hybridize transformation, obtain high carotenoid improved seeds; 3) utilize the HPLC method to measure the seed carotenoid content of seed selection kind, it is controlled at 60-300 μ ggFW
-1In the scope.
Oil crops have rape, soybean, peanut, sesame, Sunflower Receptacle, safflower, flax and corn.
In order to understand the present invention better, with the rape embodiment below, further illustrate content of the present invention:
With reference to Fig. 1, concrete steps of the present invention are:
1) by rapid determination to the Semen Brassicae campestris carotenoid content, screening rape germ plasm resource
Every part of resource takes by weighing the 1g seed respectively, fully dry, in mortar, grind, change triangular flask over to, add the 30ml extracting solution, extracting solution is that volume ratio is a sherwood oil: acetone mixes=1: 1 mixed solution, vibration was extracted 6 hours under the lucifuge condition, and spectrophotometer is confirmed the existence of carotenoid in the interscan of 300-800nm wavelength region when observing three characteristic peaks, measure main peak (445 ± 10nm) optical density value, substitution formula
X-carotenoid content in the formula
The maximum absorbance value that A-measures at 445 ± 10nm place
The amount of the extracting solution that y-is used
The weight of g-analytic sample
Ask and calculate content of carotenoid X in the seed, select X and be higher than 80 μ ggFW
-1Seed make basic germplasm,
The HPLC method is, every part of resource takes by weighing 100mg seed (about 25 seeds) respectively, add the 3ml extracting solution, extracting solution is that volume ratio is a normal hexane: acetone: the mixed solution of ethanol=50: 25: 25, residue repeatedly extracting of 2ml extracting solution, till leach liquor is colourless, merge leach liquor, centrifugation 3min, getting supernatant liquor transfers in the Glass tubing, nitrogen dries up, with 3ml normal hexane and 1ml dissolve with methanol settling, add the saturated NaCl solution of 1ml again, abundant mixing, centrifugal 3min, supernatant liquor is transferred in the new Glass tubing, the liquid of bottom with the extracting of 2ml normal hexane repeatedly, till supernatant liquor is colourless, merge supernatant liquor, nitrogen dries up, settling 2ml acetonitrile, methylene dichloride, the methyl alcohol mixed liquor dissolving, mixeding liquid volume is than being acetonitrile: methylene dichloride: methyl alcohol=50: 40: 10, centrifugal 3min gets supernatant liquor by 0.45 μ membrane filtration, and the filtrate sealing is preserved, with the anti-phase C-18 post of Spherisorb ODS2 is separator column, two probes carry out determination and analysis, and moving phase is acetonitrile/dioxane/contain the methyl alcohol/triethylamine mixed solution of 150mM ammonium acetate, and the volume ratio of 4 kinds of liquid is 82: 10: 8: 0.1, flow velocity is 1ml/min, the application of sample volume is 20 μ l, and the detection wavelength of carotenoid is 450nm, and its content converts by typical curve and obtains.
The charateristic avsorption band of carotenoid is shown in Fig. 2,3 in spectrophotometer scanning rape and the soybean, and main peak is at 445 ± 10nm, and two acromions are respectively at 420 ± 10nm and 470 ± 10nm.Use above-mentioned spectrophotometric rapid assay methods, the carotenoid content of 1000 parts of germ plasm resources of three major types type rape is carried out determination and analysis, screening obtains 38 parts of excellent germplasms, and its carotenoid content is greater than 80 μ ggFW
-1
2) utilize transgenic technology to formulate high carotenoid oil colza matter:
The method of utilizing transgenic technology to formulate high carotenoid oil colza matter is, with the key gene on the carotenoid route of synthesis, comprise phytoene synthase gene PSY, phytoene dehydrogenase gene PDS, lycopene beta cyclase gene LCY, ζ carotene dehydrogenase gene ZDS or GGPP synthase gene GGPS etc., place plant seed special the control of Napin promotor under, carry out overexpression, thereby improve content of carotenoid.
Vector construction: behind HindIII and EcoRI double digestion plasmid vector pCRT-B, electrophoresis reclaims the 1.1kb fragment that comprises phytoene synthetic gene crtB, be connected with pea SSU transhipment polypeptid coding sequence, add the Napin promotor, be inserted into plant expression vector, make up and obtain carrier pCGN3390.
Genetic transformation: selecting cabbage type rape variety Quantum for use is acceptor, 0.1% mercuric chloride sterilization seed was sprouted 4-5 days on the MS substratum, downcut cotyledon petiole and hypocotyl, cultivated altogether 1-2 days with the Agrobacterium bacterium liquid that carries goal gene, be transferred to be added with on the antibiotic substratum and screen.Regeneration plant is implanted in the nutrition pot after root induction, moves into the land for growing field crops when growing to 6 true leaves and produces.Altogether 2 strain regrowths.The strict bagging selfing of transfer-gen plant prevents to go here and there powder.
Genetically modified PCR detects: the total DNA of blade that extracts transfer-gen plant with the SDS method, according to external source crtB gene design and synthetic a pair of primer, the cumulative volume of PCR reaction is 20 μ l, on the PTC-200 thermal cycler, carry out, product is electrophoretic separation on 1% sepharose, through EB dyeing, under ultraviolet lamp, observe the electrophoresis product.
Carotenoid content detects: utilize above-mentioned HPLC method accurately to measure the carotenoid content of transgenosis different generations plant, through screening, for obtaining a strain system (code name is Q-21), its carotenoid content is stabilized in 300-350 μ ggFW at T4
-1, and the relative content of beta carotene be higher than 45%.
3) cultivation of high carotenoid oil vegetable kind
With two No. 4 (carotenoid contents: be receptor parent 25.5 μ ggFW-1) in the rape improved seeds, with swede type rape elite clone CE-24 (carotenoid content: be donor parents 95.1 μ ggFW-1), hybridization obtains F1 generation, again with in backcross for two No. 4, obtained backcrossing 1 generation, code name is BC1F1.
To the whole bagging selfings of 210 strain BC1F1 individual plants, obtain BC1F2 generation.Individual plant results during seed maturity are measured content of carotenoid, identify carotenoid content be higher than 80 μ ggFW-1, other proterties better and with in two No. 4 the most similar 1 BC1F1 individual plant.
The whole bagging selfings of 537 individual plants to BC1F2 colony obtain BC1F3.Individual plant results during seed maturity are measured carotenoid content, adopt the content of erucic acid of methods analyst seed of GB and the sulphur glycosides content of grouts simultaneously.Through identification and analysis, screening obtains that carotenoid content is higher than 80 μ ggFW-1, meets two substandards, other proterties with in two No. 43 suitable BC1F2 individual plants.
In BC1F3 generation, it is preferred to proceed individual plant, and elected individual plant mixes under the isolation condition receives 2 generations of miscegenation, is the high carotenoid new variety (strain) of inheritance stability, and the carotenoid content in its seed is 813 μ ggFW-1.
Claims (3)
1. the method for cultivation that has the oil crops of high carotenoid is characterized in that its concrete steps are as follows:
1) acquisition of basic germplasm, undertaken by following approach:
The oil crops germ plasm resource that China preserves is screened in a large number, and screening method adopts spectrophotometry, uses the spectrophotometric determination optical density value, the substitution formula
X-carotenoid content in the formula
The maximum absorbance value that A-measures at 445 ± 10nm place
The amount of the extracting solution that y-is used
The weight of g-analytic sample
Ask and calculate content of carotenoid X in the seed, select X and be higher than 60 μ ggFW
-1Resource as the basic germplasm of high carotenoid breeding;
Spectrophotometry is, every part of resource takes by weighing the 1g seed respectively, fully dry, in mortar, grind, change triangular flask over to, add the 30ml extracting solution, extracting solution is the mixed solution of sherwood oil and acetone, the volume ratio of sherwood oil and acetone is 1: 1, vibration was extracted 6 hours under the lucifuge condition, and spectrophotometer is confirmed the existence of carotenoid in the interscan of 300-800nm wavelength region when observing three characteristic peaks, measure the optical density value at main peak 445 ± 10nm place, substitution formula compute classes carotene carotene content;
2) breed of variety, carry out in two kinds of situation:
1. 60 μ ggFW have been higher than for carotenoid content
-1, and other proterties meet the germ plasm resource of production requirement, direct utilization and extention;
2. 60 μ ggFW have been higher than for carotenoid content
-1, but other proterties does not meet the germ plasm resource of production requirement, then carries out genetic improvement, method is with improved seeds; Or the sterile line of cross-fertilize seed, maintenance line and recovery system; As receptor parent, high carotenoid basic germplasm is a donor parents, hybridizes transformation, high carotenoid content is carried out many for directive breeding, and the high carotenoid gene of enrichment obtains the kind or the hybrid parents of high carotenoid;
3) preparation of commodity kind:
1. for the kind of high carotenoid, expanding propagation under the isolation condition is prepared the oil crop seeds by using with high carotenoid;
2. for cross-fertilize seed:
Two-series hybrid: according to a conventional method, with sterile dual-purpose with recover system capable by 3: 1 than planting under the isolation condition, from dual-purpose system, pull out flowering period and can educate strain, account for 50%, gather in the crops seed from sterile strain during seed maturity, be the oil crop seeds by using with high carotenoid of two-series hybrid;
Three line hybrid seed according to a conventional method, is sowed in the isolation condition and to be planted sterile line and to recover system, row is than being 2: 1, and flowering period, supple-mentary pollination cut off the male parent row after the sterile line pollination, gather in the crops seed from sterile strain during seed maturity, be the oil crop seeds by using with high carotenoid of three line hybrid seed;
4) technical indicator: the technical indicator of the seed carotenoid content of seed selection kind is at 60-300 μ ggFW
-1Between, utilize the HPLC method accurately to measure or the spectrophotometry rapid determination, each crop seed carotenoid content of selection is in above-mentioned scope.
2. the method for cultivation with oil crops of high carotenoid according to claim 1, it is characterized in that the HPLC method is, every part of seed selection kind takes by weighing the 100mg seed respectively, add the 3ml extracting solution, extracting solution is that volume ratio is a normal hexane: acetone: the mixed solution of ethanol=50: 25: 25, residue repeatedly extracting of 2ml extracting solution, till leach liquor is colourless, merge leach liquor, centrifugation 3min, getting supernatant liquor transfers in the Glass tubing, nitrogen dries up, with 3ml normal hexane and 1ml dissolve with methanol settling, add the saturated NaCl solution of 1ml again, abundant mixing, centrifugal 3min, supernatant liquor is transferred in the new Glass tubing, the liquid of bottom with the extracting of 2ml normal hexane repeatedly, till supernatant liquor is colourless, merge supernatant liquor, nitrogen dries up, the settling acetonitrile, the mixed solution 2ml dissolving of methylene dichloride and methyl alcohol, mixeding liquid volume is than being acetonitrile: methylene dichloride: methyl alcohol=50: 40: 10, centrifugal 3min, get supernatant liquor by 0.45 μ membrane filtration, the filtrate sealing is preserved, and is separator column with the anti-phase C-18 post of SpherisorbODS2, and two probes carry out determination and analysis, moving phase is acetonitrile/dioxane/contain methyl alcohol/triethylamine mixed solution of 150mM ammonium acetate, the volume ratio of 4 kinds of liquid is 82: 10: 8: 0.1, and flow velocity is 1ml/min, the application of sample volume is 20 μ l, the detection wavelength of carotenoid is 450nm, and its content converts by typical curve and obtains.
3. the method for cultivation with oil crops of high carotenoid according to claim 1 is characterized in that oil crops have rape, soybean, peanut, sesame, Sunflower Receptacle, safflower, flax and corn.
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| EP2538770A4 (en) * | 2010-02-22 | 2013-08-07 | Israel State | Melon plants comprising tetra-cis-lycopene |
| US9879236B2 (en) * | 2010-10-08 | 2018-01-30 | Korea Research Institute Of Bioscience And Biotechnology | GGPS gene for promoting higher growth or biomass of plant and use thereof |
| CN102778436B (en) * | 2011-12-20 | 2015-04-01 | 金华市农业科学研究院 | Method for breeding freshly eaten corn with eye health-care effect |
| CN104297407B (en) * | 2014-10-23 | 2016-01-20 | 中国农业科学院作物科学研究所 | The Ultra Performance Liquid Chromatography assay method of carotenoid content in wheat |
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| Title |
|---|
| 杜咏梅，等.光度法测定烟草中总类胡萝卜素的方法研究.《中国烟草科学》.2003,(第3期),28-29. * |
| 胡建中,等.高效液相色谱法测定柑橘汁中类胡萝卜素.《食品科学》.2006,第27卷(第12期),634-636. * |
| 高桂珍,等.油菜种子类胡萝卜素总量测定方法的研究.《植物遗传资源学报》.2005,第6卷(第4期),414-417. * |
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| US8748826B2 (en) | 2004-11-17 | 2014-06-10 | Biosensor International Group, Ltd. | Radioimaging methods using teboroxime and thallium |
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