CN108165576A - A kind of method for improveing rice seed oxidation resistance - Google Patents
A kind of method for improveing rice seed oxidation resistance Download PDFInfo
- Publication number
- CN108165576A CN108165576A CN201711499313.6A CN201711499313A CN108165576A CN 108165576 A CN108165576 A CN 108165576A CN 201711499313 A CN201711499313 A CN 201711499313A CN 108165576 A CN108165576 A CN 108165576A
- Authority
- CN
- China
- Prior art keywords
- rice
- seed
- oxidation resistance
- plant
- astaxanthin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Abstract
The invention discloses a kind of methods for improveing rice seed oxidation resistance, and the oxidation resistance of rice seed is improved by improving content astaxanthin in rice grain.Further comprise：It by the segment of astaxanthin protein gene, is driven with seed-specific expression promoter, conversion water gene makes specifically expressing in rice seed, to improve content astaxanthin in rice grain in rice.Include the following steps：A. recombinant vector is built；B. transfer-gen plant is obtained；C. individual plant selfing seed is harvested；D. selfing is reserved seed for planting；E. Phenotypic Selection.The method of the present invention improves content astaxanthin in rice grain, improves rice grain quality, creates rice quality improvement new material.
Description
Technical field
The present invention relates to molecular biology field of plant genetic, more particularly to a kind of improvement rice seed is anti-oxidant
The method of ability.
Background technology
Astaxanthin (English：Astaxanthin, also known as astaxanthin or astacin), be carotenoid one kind, be
A kind of stronger natural.As other carotenoid, astaxanthin belongs to a kind of fat-soluble and water-soluble color
Element can be found in the marine organisms such as shrimp, crab, salmon, algae.
The oxidation resistance of astaxanthin is strong, 550 times, 10 times of beta carotene for vitamin e.Different animal species
Different histoorgan astaxanthin existence is different, the skin of salmon, fish scale, roe to be esterified based on the astaxanthin of state,
The astaxanthin of free state is mainly distributed on muscle, blood plasma and internal organs.
In shrimp body based on esterification of astaxanthin.In haematococcus pluvialis (Haematococcus pluvialis) mainly
It is the astaxanthin of esterification, is acknowledged as producing the best biology of natural astaxanthin in nature.Astaxanthin have protection skin and
Eyes resist radiation, cardiovascular Aging, senile dementia and cancer and other effects.
The aging of human body, the oxidation institute mainly caused by free radical is extremely.And the molecule that haematococcus extract is special
Construction, can pass through human body cell outer wall, and the oxygen radical in direct scavenger-cell enhances cytothesis ability, maintains human body
Cell and DNA health are protected in function equilibrium and the accumulation for reducing senile cell from inside to outside.
Rice makes one of most important cereal crops in the world, and even more the most important cereal crops in China, China are the world
The eating rice production state and country of consumption of upper maximum, the compatriots there are about more than 60% are using rice as staple food.
Rice year sown area in China's accounts for the 20% of the world more than 30,000,000 hectares, accounts for national cereal crops sown area
30%.At present, in rice there are no similar report and imagination, therefore realize that this idea will make rice in rice
Nutritive value has the raising of matter, and people is made also to carry out health care while having dinner, and the fitness to improve the people provides
A kind of possibility.
The method of the present invention has not been reported at home and abroad, and our unit also not publicly delivers the article for being related to the content of present invention, this
Invention, which is at home and abroad studied, belongs to blank field.
Invention content
The technical problems to be solved by the invention are, provide a kind of method for improveing rice seed oxidation resistance, will
The segment of astaxanthin protein gene, is driven with seed-specific expression promoter, and conversion water gene makes special table in rice seed in rice
It reaches, to improve content astaxanthin in rice grain, improves rice grain quality, create rice quality improvement new material.
In order to solve the above technical problems, the present invention provides a kind of method for improveing rice seed oxidation resistance, by carrying
The oxidation resistance of content astaxanthin improvement rice seed in high rice grain.
Further comprise：It by the segment of astaxanthin protein gene, is driven with seed-specific expression promoter, the conversion water gene exists
Rice makes specifically expressing in rice seed, to improve content astaxanthin in rice grain.
Include the following steps：
A. recombinant vector is built；
B. transfer-gen plant is obtained；
C. individual plant selfing seed is harvested；
D. selfing is reserved seed for planting；
E. Phenotypic Selection.
The step further comprises：
A. recombinant vector is built：The one section of astaxanthin protein gene segment detached from haematococcus pluvialis, the segment by
1005 base compositions encode 334 amino acid；Itself and Seeds oil-body-specific promoter digestion are connected to expression vector
On pCAMBIA1300, recombinant vector is built.
The step further comprises：
B. transfer-gen plant is obtained；The transgenic method mediated using agrobacterium tumefaciens lba4404 is imported recombinant vector
In japonica rice, transfer-gen plant is obtained.
The step further comprises：
C. individual plant selfing seed is harvested：Positive detection is carried out for transfer-gen plant to T0 using PCR methods, passes through Northern
The expression of Insert Fragment in hybridization check positive transgenic plant identifies positive transgenic by the method for Real-time PCR
The copy number of plant Insert Fragment, selection harvest single plant containing the Insert Fragment singly copied and normal solid transfer-gen plant
Selfed seed.
The step further comprises：
D. selfing is reserved seed for planting：The seed for the transfer-gen plant selected and remain in above-mentioned steps C is selfed, it is miscellaneous also with Northern
It hands over and Real-time PCR methods identifies the expression of Insert Fragment and copy number in offspring, it is steady with reference to variable rate technology selection phenotype
Transgenosis single plant fixed, astaxanthin expression quantity is high and performance is excellent, selfing are reserved seed for planting.
In order to solve the above technical problems, the present invention also provides a kind of food, water is improved using as described in aforementioned any one
The method of rice seed oxidation resistance directly or indirectly produces.
In order to solve the above technical problems, the anti-oxidant energy of rice seed is improved as described in aforementioned any one the present invention provides a kind of
The method of power is being produced for the application in the edible or rice of food processing.
In order to solve the above technical problems, the anti-oxidant energy of rice seed is improved as described in aforementioned any one the present invention provides a kind of
Application of the method for power in rice strain is established.
Advantageous effect of the present invention includes：
It is easy to implement the method the purpose of the invention is to provide a kind of method for improveing rice grain nutritional quality, operation
Simply, including transgenic method, astaxanthin protein gene segment and Seeds oil-body-specific promoter fusion rice transformation,
Obtain transfer-gen plant；With reference to PCR and molecule hybridizing method, biochemical analysis method etc., after transfer-gen plant
The transgenic line that the expression of selection and breeding astaxanthin is high in generation, single plant yield is constant or is improved creates rice quality improvement new material.
Description of the drawings
Fig. 1 is the nucleotide sequence and amino acid sequence of genetic fragment used in a kind of conversion.
Fig. 2 is a kind of construction of recombinant vector process schematic.
Fig. 3 is T0 for partial transgenic plant positive detection schematic diagram.
Fig. 4 is part T0 for transfer-gen plant Insert Fragment copy number schematic diagram (Southern).
Fig. 5 is part T0 for transfer-gen plant Insert Fragment expression quantity schematic diagram (Northern).
Specific embodiment
The present invention is described in detail with reference to embodiment.To make the objectives, technical solutions, and advantages of the present invention clearer, bright
Really, developing simultaneously referring to the drawings, the present invention is described in more detail for embodiment, but the invention is not limited in these embodiments.
The invention discloses a kind of method for improveing rice grain nutritional quality, what is detached from freshwater shrimp haematococcus pluvialis
One section of astaxanthin protein gene segment constructs recombinant vector with seed-specific expression promoter, utilizes Agrobacterium tumefaciens mediated something lost
Conversion method is passed by recombinant vector Introduced into Rice, transformed plant is obtained, the positive is then carried out to T0 seedlings with PCR
Detection, by the selfing of the seed of the T0 of harvest into family (T1), then to T1 generations positive single-strain seed into T2 for family, therefrom select
Go out the high homozygous single plant of content astaxanthin, selfing is reserved seed for planting, and further performance selection is carried out to the homozygous single plant selected and remain, is cultivated new
Material.Method is simple and practicable, expresses the content that astaxanthin protein gene improves astaxanthin in rice in rice, improves seed
Nutritional quality, can be applied to the improvement of crop quality.
The invention belongs to field of plant genetic.It is more particularly to a kind of side for improveing rice grain nutritional quality
Method specifically contains and one astaxanthin protein gene segment is converted water by the method for Agrobacterium-mediated Transformation using transgenic method
Rice makes the gene be expressed in rice grain, to improve the content of rice grain astaxanthin, creates rice quality improvement new material
Method.
In order to solve the above technical problems, the present invention provides a kind of method for improveing rice seed oxidation resistance, by carrying
The oxidation resistance of content astaxanthin improvement rice seed in high rice grain.
Further comprise：It by the segment of astaxanthin protein gene, is driven with seed-specific expression promoter, the conversion water gene exists
Rice makes specifically expressing in rice seed, to improve content astaxanthin in rice grain.
Include the following steps：
A. recombinant vector is built；
B. transfer-gen plant is obtained；
C. individual plant selfing seed is harvested；
D. selfing is reserved seed for planting；
E. Phenotypic Selection.
The step further comprises：
A. recombinant vector is built：The one section of astaxanthin protein gene segment detached from haematococcus pluvialis, the segment by
1005 base compositions encode 334 amino acid；Itself and Seeds oil-body-specific promoter digestion are connected to expression vector
On pCAMBIA1300, recombinant vector is built.
The step further comprises：
B. transfer-gen plant is obtained；The transgenic method mediated using agrobacterium tumefaciens lba4404 is imported recombinant vector
In japonica rice, transfer-gen plant is obtained.
The step further comprises：
C. individual plant selfing seed is harvested：Positive detection is carried out for transfer-gen plant to T0 using PCR methods, passes through Northern
The expression of Insert Fragment in hybridization check positive transgenic plant identifies positive transgenic by the method for Real-time PCR
The copy number of plant Insert Fragment, selection harvest single plant containing the Insert Fragment singly copied and normal solid transfer-gen plant
Selfed seed.
The step further comprises：
D. selfing is reserved seed for planting：The seed for the transfer-gen plant selected and remain in above-mentioned steps C is selfed, it is miscellaneous also with Northern
It hands over and Real-time PCR methods identifies the expression of Insert Fragment and copy number in offspring, it is steady with reference to variable rate technology selection phenotype
Transgenosis single plant fixed, astaxanthin expression quantity is high and performance is excellent, selfing are reserved seed for planting.
In order to solve the above technical problems, the present invention also provides a kind of food, water is improved using as described in aforementioned any one
The method of rice seed oxidation resistance directly or indirectly produces.
In order to solve the above technical problems, the anti-oxidant energy of rice seed is improved as described in aforementioned any one the present invention provides a kind of
The method of power is being produced for the application in the edible or rice of food processing.
In order to solve the above technical problems, the anti-oxidant energy of rice seed is improved as described in aforementioned any one the present invention provides a kind of
Application of the method for power in rice strain is established.
A kind of method for improveing rice grain nutritional quality, step are：
1. the one section of astaxanthin protein gene segment detached from haematococcus pluvialis, the segment by 1005 base compositions,
Encode 334 amino acid (Fig. 1).It and Seeds oil-body-specific promoter digestion are connected on expression vector pCAMBIA1300
(Cambia Products) build recombinant vector；
2. recombinant vector is imported japonica rice variety force fortune round-grained rice 7 by the transgenic method using agrobacterium tumefaciens lba4404 mediation
In number, transfer-gen plant is obtained；
3. carrying out positive detection for transfer-gen plant to T0 using PCR methods, base is turned by the Northern hybridization check positives
Because of the expression of Insert Fragment in plant, copying for positive transgenic plant Insert Fragment is identified by the method for Real-time PCR
Shellfish number, selection harvest individual plant selfing seed containing the Insert Fragment singly copied and normal solid transfer-gen plant；
4. the seed for the transfer-gen plant selected and remain in above-mentioned 3 is selfed, also with Northern hybridization and Real-time
The expression of Insert Fragment and copy number in PCR method identification offspring, with reference to variable rate technology selection phenotype stabilization, astaxanthin expression quantity
Transgenosis single plant high and that performance is excellent, selfing are reserved seed for planting；
5. pair homozygous single plant selected and remain carries out Phenotypic Selection, new material is cultivated.
Embodiment 1：The structure of recombinant vector and the foundation for converting Agrobacterium：
(1) technology path according to fig. 2, by the plasmid pUC19-LTP2 with astaxanthin gene-seed-specific expression promoter into
Row PCR amplification is cloned into pUC18 carriers, and method is with reference to molecular cloning, with EcoRI and HindIII double digestions, electrophoresis recycling purpose
Segment connect to form intermediate carrier with the pCAMBIA1300 after EcoRI and HindIII double digestions with T4 ligases, then will be from
The segment KpnI and SacI double digestions of the astaxanthin protein gene of clone, electrophoresis recycling target patch are detached in haematococcus pluvialis
Section forms recombinant vector, more than restriction enzyme and T4 with crossing intermediate carrier T4 ligases with KpnI and SacI double digestions
Ligase is purchased from Promega companies.
(2) recombinant vector is converted into E. coli competent DH5 α, in the resistance training containing X-Gal, IPTG and kanamycins
It supports and selects white single bacterium colony on base；
(3) the white single bacterium colony selected is enriched with extracting plasmid, is detected, chosen with EcoRI and HindIII double digestions rear electrophoresis
Choosing is connected in correct plasmid conversion Agrobacterium LBA4404, the strain was named YC1 after conversion；
Embodiment 2：Agrobacterium-mediated genetic transformation
(1) callus induces
Impregnate 2-3 minutes after rice paddy seed decladding that will be ripe inside 95% ethyl alcohol, then 40% secondary chlorine
Concussion rinsing 15 minutes in acid sodium solution replace a rinsing liquid and continue concussion rinsing 15 minutes；Finally floated with sterile distilled water
Wash seed 4 times.In 28 DEG C of illumination box evoked callus 14 days.
(2) Agrobacterium-mediated Transformation
Picking single bacterium colony is seeded to the freshly prepared Agrobacterium culture mediums of 25mL (addition antibiotic), 28 DEG C of 250-
300rpm shakes training overnight, and the Agrobacterium that spreads cultivation is to 0.3 ＜ OD550 ＜ 1.0；
Bacterium solution is centrifuged into 5 minutes (4 DEG C) with 6000g, soft resuspension Agrobacterium to OD550=0.1 (100 μM of AS of addition,
The mother liquid concentration that AS is dissolved in DMSO is 100mmol/L)；
It infects the callus of preculture 5-10 minutes, gently overturns or shake at regular intervals culture bottle；
Agrobacterium bacterium solution is removed, dry in the shade callus on filter paper；
Callus is placed on the co-cultivation base tablet for being covered with filter paper, per ware placement amount had the gap between callus for
It is accurate；
In 21-25 DEG C of incubator dark culturing 72h；
After conventional method rinsing callus, dry in the shade and be transferred to screening and culturing medium, in 28 DEG C of dark culturings.
(3) resistance screening
Picking co-cultures the callus of 2d in culture dish, if callus surface can be rushed with the presence of thalline with sterile water
It washes 3-5 times, stands 1h with containing 500mg/LCef sterile waters for the last time；
Standing is placed on drying 30 minutes on aseptic filter paper, until callus surface exists without visible Agrobacterium；
Callus is transferred to the screening of kanamycins concentration 200mg/L and 400mg/L Timentin after the completion of processing
Culture medium carries out screening kanamycin-resistant callus tissue；
28 DEG C of light cultures, 15d screenings are primary, screen altogether twice.
(4) break up
By the callus of screening twice in long 55 ＞ 0.2mm on screening and culturing medium, it is transferred to pre- differential medium, dark item
Lower 28 DEG C of part is cultivated 7-10 days；
Callus is transferred to differential medium, visible young shoot after 2-3 weeks；
When seedling grows to 7-8cm, root media is transferred to, hardening, transplanting after 7 days.
Embodiment 3：
Rotaring gene plant blade is taken to extract DNA, carries out PCR (PCR), PCR amplification program is 94 DEG C
10min；94 DEG C of 1min, 62 DEG C of 1min, 72 DEG C of 30s；35 cycles；72℃10min.Positive transformants plant is detected, can be amplified
The single plant of 750bp size strips is positive transformants single plant.The RNA of the positive single plant seed of extracting carries out Northern hybridization checks
Expression of the Insert Fragment in seed, the Insert Fragment of positive single plant are all expressed.The DNA of a large amount of positive single plants of extracting, then
With SacI and XbaI enzyme cutting, the copy number that Southern hybridization identification positive transformants single plant Insert Fragments are integrated is carried out, obtains two
A independent transformation strain singly copied.The relevant technologies ginsengs such as DNA extractions, RNA extractions, Northern hybridization and Southern hybridization
Examine Molecular Cloning:A Laboratory guide.
Embodiment 4：
The seed of select and remain 2 single copy genes is sowed into plantation, forms two different familys, because in T1 generations, can detach,
So the method using PCR carries out the identification of each family positive single plant, select one containing target gene with reference to variable rate technology and
Normal single plant is expressed, is reserved seed for planting after selfing.
Embodiment 5：
2 single-strain seeds selected and remain in embodiment 4 plantation is continued with into PCR detection family middle-jiao yang, function of the spleen and stomach into T2 for family
Property single plant, wherein family 1 and family 6 tied homozygous stabilization, analyzed the content of astaxanthin in seed, therefrom selects 1 astaxanthin
Content is higher and the good homozygous single plant (YCLine1) of variable rate technology
1 T2 of table is for positive plant rate (positive plant number/detection plant number)
Seed content astaxanthin measures
The assay method of content astaxanthin is yellow with reference to angle in SN/T2327-2009 Imported and exported animals derived foods in seed
The detection method of element, astaxanthin.
It is prepared by rice flour：The paddy after 500g shellings is taken, paddy is broken into powder with whirlwind type crushing machine, is entered after sieving with 100 mesh sieve
Plastic bag sealing is placed in -20 DEG C of refrigerators and saves backup.
Extraction and purification：Sample about 5g is weighed in 50mL centrifuge tubes, adds in 30mL acetonitriles extractant and 10g anhydrous slufuric acids
After sodium, vortex mixed 1min, water bath sonicator extracts 10min below 35 DEG C, then centrifuges 5min with 3000r/min.By supernatant
Immigration is preset in the 125mL separatory funnels of 20mL n-hexanes, after concussion shakes up 0.5min, stratification；Collect lower floor's acetonitrile
Phase, n-hexane mutually stay in separatory funnel, treat that the follow-up degreasing of sample is reused.
Extraction is repeated twice with 20mL acetonitrile extractants every time to the residue in centrifuge tube by above-mentioned steps, gained carries
Take liquid above-mentioned n-hexane mutually degreasing successively.Merge acetonitrile phase collected after degreasing three times, 5mL normal propyl alcohols are added in, in 40 DEG C
Lower rotation is concentrated by evaporation near and does, and is dissolved with acetonitrile and is settled to 5.0mL, 0.20 μm of membrane filtration, and filtrate is to be measured.
Strong light should be kept away, be carried out continuously by measuring operation.
It measures：According to measured object content situation in sample, choose the suitable standard working solution of response and carry out chromatography.
The response of astaxanthin should be in instrument linear response range in standard working solution and the sample solution to be tested.Standard working solution is with treating test sample
The isometric sample introduction of liquid.Under above-mentioned chromatographic condition, astaxanthin is trans-, cis-isomer retention time respectively may be about 8.4min,
10.6min, according to peak area quantified by external standard method (mg/g).
Genetic transformation culture medium and its preparation method used in the present invention is as follows：
Main solution formula
N6 culture medium a great number of elements mother liquor (is prepared) according to 10 times of concentrates
Reagent name dosage (g)
Potassium nitrate 28.3
Sodium dihydrogen phosphate 4
Ammonium sulfate 4.63
Magnesium sulfate 1.85
Calcium chloride 1.66
Mentioned reagent is dissolved one by one, is then settled to 1000mL with distilled water under room temperature (20-25 DEG C, similarly hereinafter).
2) N6 culture mediums trace element mother liquor (being prepared according to 100 times of concentrates)
Reagent name dosage (g)
Potassium iodide 0.08
Boric acid 0.16
Manganese sulfate 0.44
Zinc sulfate 0.15
Mentioned reagent is dissolved at room temperature and is settled to 1000mL with distilled water.
3) molysite storage liquid (being prepared by 100 times of concentrates)
3.73g disodium ethylene diamine tetraacetates and 2.78g green vitriols are dissolved respectively, mix and determined with distilled water
Hold to 1000mL, until 70 DEG C of warm bath 2 hours, 4 DEG C save backup.
4) MS culture mediums trace element mother liquor (being prepared according to 100 times of concentrations)
Reagent name dosage (g)
Manganese sulfate 2.23
Zinc sulfate 0.86
Boric acid 0.62
Potassium iodide 0.083
Sodium molybdate 0.025
Copper sulphate 0.0025
Cobalt chloride 0.0025
Mentioned reagent is dissolved, and be settled to 1000mL with distilled water at room temperature
(2) for the culture medium prescription of rice transformation
1) inducing culture
Reagent name dosage (g/mL)
The a large amount of mother liquor 100mL of N6
The micro mother liquor 10mL of N6
Molysite storage liquid 10mL
Vitamins stock liquid 10mL
2,4-D storage liquid 2.5mL
Proline 0.3g
CH 0.6g
Sucrose 30g
Phytagel 3g
Adding distilled water, 1N potassium hydroxide adjusts pH value to 5.9, boils and is settled to 1000mL, is dispensed into 50mL to 900mL
Triangular flask, sterilize according to a conventional method after sealing (such as sterilize 25 minutes at 121 DEG C, following medium sterilization methods and this culture
The sterilizing methods of base are identical).
2) subculture medium
Reagent name dosage (g/mL)
The a large amount of mother liquor 100mL of N6
The micro mother liquor 10mL of N6
Molysite storage liquid 10mL
Vitamins stock liquid 10mL
2,4-D storage liquid 2.0mL
Proline 0.5g
CH 0.6g
Sucrose 30g
Phytagel 3g
Adding distilled water, 1N potassium hydroxide adjusts pH value to 5.9, boils and is settled to 1000mL, is dispensed into 50mL to 900mL
Triangular flask sterilizes according to a conventional method after sealing.
And culture medium 3)
Reagent name dosage (g/mL)
The a large amount of mother liquor 12.5mL of N6
The micro mother liquor 1.25mL of N6
Molysite storage liquid 2.5mL
Vitamins stock liquid 2.5mL
2,4-D storage liquid 2.0mL
CH 0.15g
Sucrose 5g
Agar powder 1.75g
Adding distilled water, 1N potassium hydroxide adjusts pH value to 5.6, sterilizes according to a conventional method after sealing to 250mL.
Using preceding heating for dissolving culture medium and 5mL glucose storages liquid and 250mLAS storage liquid are added in, culture is poured into packing
In ware.
4) base is co-cultured
Reagent name dosage (g/mL)
The a large amount of mother liquor 12.5mL of N6
The micro mother liquor 1.25mL of N6
Molysite storage liquid 2.5mL
Vitamins stock liquid 2.5mL
2,4-D storage liquid 0.75mL
CH 0.2g
Sucrose 5g
Agar powder 5g
Adding distilled water, 1N potassium hydroxide adjusts pH value to 5.6, sterilizes according to a conventional method after sealing to 250mL.
Using preceding heating for dissolving culture medium and 5mL glucose storages liquid and 250mLAS storage liquid are added in, culture is poured into packing
In ware.
5) Selective agar medium
Reagent name dosage (g/mL)
The a large amount of mother liquor 25mL of N6
The micro mother liquor 2.5mL of N6
Molysite storage liquid 2.5mL
Vitamins stock liquid 2.5mL
2,4-D storage liquid 0.625mL
CH 0.15g
Sucrose 7.5g
Agar powder 1.75g
Distilled water is added to adjust pH value to 6.0, sealing sterilizes as stated above to 250mL.
Using preceding dissolving culture medium, add in 250mLHN and 400mL packing and pour into culture dish.
6) pre- differential medium
Reagent name dosage (g/mL)
The a large amount of mother liquor 25mL of N6
The micro mother liquor 2.5mL of N6
Molysite storage liquid 2.5mL
Vitamins stock liquid 2.5mL
6-BA storage liquid 0.5mL
KT storage liquid 0.5g
50 μ L of NAA storage liquid
50 μ L of IAA storage liquid
CH 0.15g
Sucrose 7.5g
Agar powder 1.75g
Adding distilled water, 1N potassium hydroxide adjusts pH value to 5.9, sterilizes according to a conventional method after sealing to 250mL.
Using preceding heating for dissolving culture medium, 250mLHN and 250mLCN storage liquid is added in, packing is poured into culture dish.
7) differential medium
Reagent name dosage (g/mL)
The a large amount of mother liquor 100mL of N6
The micro mother liquor 10mL of N6
Molysite storage liquid 10mL
Vitamins stock liquid 10mL
6-BA storage liquid 2mL
KT storage liquid 2g
NAA storage liquid 0.2mL
IAA storage liquid 0.2mL
CH 1g
Sucrose 30g
Phytagel 3g
Adding distilled water, 1N potassium hydroxide adjusts pH value to 6.0 to 900mL.
It boils and is settled to 1000mL with distilled water, be dispensed into 50mL triangular flasks, seal, sterilize as stated above.
8) root media
Reagent name dosage (g/mL)
The a large amount of mother liquor 50mL of MS
The micro mother liquor 5mL of MS
Molysite storage liquid 5mL
Vitamins stock liquid 5mL
Sucrose 20g
Phytagel 3g
Adding distilled water, 1N potassium hydroxide adjusts pH value to 5.8 to 900mL.
It boils and is settled to 1000mL with distilled water, be dispensed into 50mL triangular flasks, seal, sterilize as stated above.
The above is only several embodiments of the present invention, any type of limitation is not done to the present invention, although this hair
It is bright to be disclosed as above with preferred embodiment, however not to limit the present invention, any person skilled in the art is not taking off
In the range of technical solution of the present invention, make a little variation using the technology contents of the disclosure above or modification is equal to
Case study on implementation is imitated, is belonged in technical solution of the present invention protection domain.
Claims (10)
- A kind of 1. method for improveing rice seed oxidation resistance, which is characterized in that by improving content astaxanthin in rice grain Improve the oxidation resistance of rice seed.
- 2. the method for rice seed oxidation resistance is improved according to claim 1, which is characterized in that further comprise：By shrimp The segment of green fibroin gene, is driven with seed-specific expression promoter, and conversion water gene makes special table in rice seed in rice It reaches, to improve content astaxanthin in rice grain.
- 3. the method for rice seed oxidation resistance is improved according to claim 1, which is characterized in that include the following steps：A. recombinant vector is built；B. transfer-gen plant is obtained；C. individual plant selfing seed is harvested；D. selfing is reserved seed for planting；E. Phenotypic Selection.
- 4. the method for rice seed oxidation resistance is improved according to claim 3, which is characterized in that the step is further wrapped It includes：A. recombinant vector is built：The one section of astaxanthin protein gene segment detached from haematococcus pluvialis, the segment is by 1005 Base composition encodes 334 amino acid；Itself and Seeds oil-body-specific promoter digestion are connected to expression vector On pCAMBIA1300, recombinant vector is built.
- 5. the method for rice seed oxidation resistance is improved according to claim 3, which is characterized in that the step is further wrapped It includes：B. transfer-gen plant is obtained；Recombinant vector is imported japonica rice by the transgenic method mediated using agrobacterium tumefaciens lba4404 In, obtain transfer-gen plant.
- 6. the method for rice seed oxidation resistance is improved according to claim 3, which is characterized in that the step is further wrapped It includes：C. individual plant selfing seed is harvested：Positive detection is carried out for transfer-gen plant to T0 using PCR methods, is hybridized by Northern The expression of Insert Fragment in positive transgenic plant is detected, positive transgenic plant is identified by the method for Real-time PCR The copy number of Insert Fragment, selection harvest individual plant selfing containing the Insert Fragment singly copied and normal solid transfer-gen plant Seed.
- 7. the method for rice seed oxidation resistance is improved according to claim 3, which is characterized in that the step is further wrapped It includes：D. selfing is reserved seed for planting：The seed of the transfer-gen plant selected and remain in above-mentioned steps C is selfed, also with Northern hybridization and The expression of Insert Fragment and copy number in Real-time PCR methods identification offspring, with reference to variable rate technology selection phenotype stabilization, shrimp Green element expression quantity height is reserved seed for planting with excellent transgenosis single plant, selfing is showed.
- 8. a kind of food, which is characterized in that using the improvement rice seed oxidation resistance as described in any one of claim 1~7 Method directly or indirectly produce.
- 9. a kind of method that rice seed oxidation resistance is improved as described in any one of claim 1~7 is in production for eating Or the application in the rice of food processing.
- 10. a kind of method that rice seed oxidation resistance is improved as described in any one of claim 1~7 is establishing rice strain In application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711499313.6A CN108165576A (en) | 2017-12-31 | 2017-12-31 | A kind of method for improveing rice seed oxidation resistance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711499313.6A CN108165576A (en) | 2017-12-31 | 2017-12-31 | A kind of method for improveing rice seed oxidation resistance |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108165576A true CN108165576A (en) | 2018-06-15 |
Family
ID=62516985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711499313.6A Pending CN108165576A (en) | 2017-12-31 | 2017-12-31 | A kind of method for improveing rice seed oxidation resistance |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108165576A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206042A (en) * | 2020-02-25 | 2020-05-29 | 中国科学院昆明植物研究所 | Fusion gene for expressing ketocarotenoid, recombinant vector and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101229885B1 (en) * | 2011-09-26 | 2013-02-08 | 대한민국 | Polynucleotide for biosynthesis of astaxanthin and transformed plant using the same |
| CN102144568B (en) * | 2011-04-22 | 2013-03-06 | 华中农业大学 | Method for improving quality of rice kernel amino acid |
| CN105907780A (en) * | 2016-04-29 | 2016-08-31 | 华南农业大学 | Transgenic breeding method producing astaxanthin in crop seed endosperm |
-
2017
- 2017-12-31 CN CN201711499313.6A patent/CN108165576A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102144568B (en) * | 2011-04-22 | 2013-03-06 | 华中农业大学 | Method for improving quality of rice kernel amino acid |
| KR101229885B1 (en) * | 2011-09-26 | 2013-02-08 | 대한민국 | Polynucleotide for biosynthesis of astaxanthin and transformed plant using the same |
| CN105907780A (en) * | 2016-04-29 | 2016-08-31 | 华南农业大学 | Transgenic breeding method producing astaxanthin in crop seed endosperm |
Non-Patent Citations (1)
| Title |
|---|
| 高俊全等: "《虾青素 健康新世纪的奥秘》", 31 July 2013, 北京：中国医药科技出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206042A (en) * | 2020-02-25 | 2020-05-29 | 中国科学院昆明植物研究所 | Fusion gene for expressing ketocarotenoid, recombinant vector and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Negbi | Saffron cultivation: past, present and future prospects | |
| Hallmann | Algae biotechnology–green cell-factories on the rise | |
| KR101896243B1 (en) | Chlamydomonas mutants produced usingrgen rnp and method for preparing pigment using the same | |
| CN108164588A (en) | Application of the cotton transport protein GhBASS5 genes in plant salt tolerance | |
| CN101653103B (en) | Breeding method of ultra-large nuclear-free pearl with good quality | |
| CN102144568B (en) | Method for improving quality of rice kernel amino acid | |
| CN108660140A (en) | Application of the SlSL4 genes in regulating and controlling Fruit Ripening of Tomato | |
| CN105837671B (en) | Obtained from the flavonols modulin MsMYB22 and its encoding gene of functional form apple and application | |
| CN111926036B (en) | Combined expression vector and application thereof in expression of astaxanthin by corn kernels | |
| CN108165576A (en) | A kind of method for improveing rice seed oxidation resistance | |
| CN107384907A (en) | A kind of preparation method of selenium-rich astaxanthin chlorella powder | |
| CN105949289B (en) | Obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application | |
| CN106883292B (en) | Obtained from the MsLBD13 albumen and its encoding gene of functional form apple and application | |
| CN105837669B (en) | Obtained from the anthocyanin modulin MsARF3 and its encoding gene of functional form apple and application | |
| CN108192911A (en) | A kind of method of starch quality in improvement rice | |
| CN107033228A (en) | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 of functional form apple and its encoding gene and application | |
| JP6165876B2 (en) | Resveratrol biosynthesis rice and its use | |
| CN107022014B (en) | Obtained from the MsTMT1L albumen and its encoding gene of functional form apple and application | |
| CN109007442A (en) | A kind of fish feed and preparation method thereof improving immunity | |
| CN104830871B (en) | A kind of paddy gene OsAP2 6 and preparation method and application | |
| CN107969575A (en) | A kind of red cream crab feed containing natural pigment raw material | |
| Rout et al. | Plant Disease Management by Modern Applications RNA Interference | |
| Kalia et al. | Genome Designing for Nutritional Quality in Vegetable Brassicas | |
| Goh | ORAL AS1 | |
| CN108239654A (en) | A kind of method for improving rice grain vitamin content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180615 |
|
| RJ01 | Rejection of invention patent application after publication |