CN108239654A - A kind of method for improving rice grain vitamin content - Google Patents
A kind of method for improving rice grain vitamin content Download PDFInfo
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Abstract
This application discloses a kind of methods for improving rice grain vitamin content, include the following steps：A. recombinant vector is built；B. transfer-gen plant is obtained；C. individual plant selfing seed is harvested；D.T1 generation selfings are reserved seed for planting；E.T2 generation selfings are reserved seed for planting；F. Phenotypic Selection.The step further comprises：A. recombinant vector is built：The cDNA segments of gama tocopherol methyl transferases gene are isolated from arabidopsis；It and 1 promoter digestions of rice grain specificity glutelin GluB are connected on expression vector pCAMBIA1300, build recombinant vector.The method of the present invention improves rice grain content of vitamin E, 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 raising rice grain dimension
The method of raw cellulose content.
Background technology
Vitamin E is a kind of fat-soluble antioxidant, be divided into tocopherol (α, β, γ, δ) and tocotrienol (α, β,
γ, δ) two major class, there is important regulating and controlling effect to growth and development of plants.In addition, it also assists in signal transduction in plant cell
(Packer et al., 2001；Munne-Bosch S et al., 2007), adjust (the Landes N et such as expression of gene
Al., 2003；Abate A et al., 2000).
Natural VE mainly synthesizes in plant, and mankind itself can not synthesize, it is necessary to from diet particularly from plant
Middle acquisition, and natural VE is above manually in physiological activity, absorptivity and in vivo in retention time and safety
The vitamin E of synthesis, therefore the content of raising natural plant vitamin E or activity have great importance.
Rice is one of important cereal crops in China, and sown area and total output occupy cereal crops first place, in China
There is very important status, however the content of vitamin E in rice is very low, is insufficient for the mankind to day in grain-production
The demand of right vitamin E.Traditional breeding way has the limitation of variety source for improving content of vitamin E in rice, and
Have many successfully reports in terms of using transgenic technology improvement Nutrition Quality of Rice, it such as will be in beta carotene route of synthesis
3 channel genes rice in render transgenic rice endosperm in synthesize and have accumulated vitamin A precursor --- beta carotene
(Beyer P et al., 2002).
But at present in addition to Sookwong etc. using the method for hybridization improves grinding for tocotrienol content in rice chaff layer
Outside studying carefully, other are yet there are no in relation to improving rice content of vitamin E or changing the report of vitamin E isomers composition research.
Arabidopsis (Arabidopsis thaliana) gama-tocopherol methyl transferase (γ-TMT) is natural VE
Important synzyme in route of synthesis, catalysis δ, gama-tocopherol methyl, generation
Invention content
The technical problems to be solved by the invention are, provide a kind of method for improving rice grain vitamin content,
The method of the present invention is by arabidopsis (Arabidopsis thaliana) gama-tocopherol methyl transferase (γ-TMT) gene
CDNA segments are driven, rice transformation with specificity glutelin GluB-1 promoters, make the cDNA segments special in rice grain
Different expression to improve rice grain content of vitamin E, improves rice grain quality, creates rice quality improvement new material.
In order to solve the above technical problems, the present invention provides a kind of method for improving rice grain vitamin content, including
Following steps：
A. recombinant vector is built；
B. transfer-gen plant is obtained；
C. individual plant selfing seed is harvested；
D.T1 generation selfings are reserved seed for planting；
E.T2 generation selfings are reserved seed for planting；
F. Phenotypic Selection.
The step further comprises：
A. recombinant vector is built：The cDNA segments of gama-tocopherol methyl transferase gene are isolated from arabidopsis；It will
It is connected to rice grain specificity glutelin GluB-1 promoter digestions on expression vector pCAMBIA1300, structure recombination
Carrier.
The step further comprises：
B. transfer-gen plant is obtained：Using Agrobacterium tumefaciens mediated transgenic method by recombinant vector Introduced into Rice,
Obtain transfer-gen plant.
The step further comprises：
C. individual plant selfing seed is harvested：Positive detection is carried out for transfer-gen plant to T0 using PCR, is led to
The expression that Northern hybridization check positive transgenic plant are transferred to segment is crossed, hybridizing the identification positive by Southern turns base
Because of the integration copy number of plant, selection contains the segment that is transferred to singly copied, and normal solid transfer-gen plant, harvests single plant
Selfed seed.
The step further comprises：
D.T1 generation selfings are reserved seed for planting：By the seed kind of the step C transfer-gen plants selected and remain into family T1 generations, PCR is continued with
Positive detection is carried out for single plant to T1, is transferred to the expression of segment, knot to being detected to positive single plant using Northern hybridizing methods
Variable rate technology is closed, the transgenosis single plant for selecting multiple performances excellent, selfing is reserved seed for planting.
The step further comprises：
E.T2 generation selfings are reserved seed for planting：Continue the target single plant selected and remain of 4 kinds of plantation step into T2 for family, using PCR to T2 generations
Single plant carries out positive detection, and agronomic shape investigation is carried out to positive single plant and rice paddy seed vitamin content is analyzed, is therefrom selected
Phenotype stabilization, content of vitamin E and agronomic shape have the homozygous single plant of larger improvement, and selfing is reserved seed for planting.
The step further comprises：
F. Phenotypic Selection：Phenotypic Selection is carried out to the homozygous single plant selected and remain, cultivates new material.
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 vitamin content directly or indirectly produces.
In order to solve the above technical problems, rice grain dimension is improved as described in aforementioned any one invention further provides a kind of
The method of raw cellulose content is being produced for the application in the edible or rice of food processing.
It is tieed up in order to solve the above technical problems, the present invention separately provides a kind of rice grain that improved as described in aforementioned any one
Application of the method for raw cellulose content in rice strain is established.
Advantageous effect of the present invention includes：
A. cDNA piece of the expression from arabidopsis gama-tocopherol methyl transferase (γ-TMT) gene in rice grain
Section can improve rice grain content of vitamin E, so as to improve rice grain nutritional quality, meanwhile, single plant yield also has one
It is fixed to improve；
B. this method improves rice grain quality using transgenic method, and the improvement period is short, efficient；
C. this method may be directly applied to the improvement practice of other crop qualities；
D. by the cDNA segments rice grain specificity of arabidopsis gama-tocopherol methyl transferase (γ-TMT) gene
Glutelin GluB-1 promoters drive, and rice transformation makes its cDNA specifically expressings in rice grain, easy to implement the method, operation letter
Just, rice grain content of vitamin E is improved, increases single plant yield, new material is created for rice quality improvement.This hair
Material caused by bright can directly apply to and prepares the pharmaceutical products such as vitamin E, to promoting health and rice is held
Continuous production etc. is extremely important.
Description of the drawings
Fig. 1 is a kind of nucleotide sequence of cDNA segments used in conversion, which has 1047 base compositions.
Fig. 2 is a kind of T0 for transfer-gen plant positive detection schematic diagram：
1st swimming lane is molecular weight marker, and the 2nd swimming lane is positive control (recombinant vector amplification), and the 3rd swimming lane is the moon
Property control (wild type force educates No. 7 amplifications of round-grained rice), 4-5 swimming lanes be blank control, 6-18 swimming lanes for part T0 generation turn base
Because of the amplification of plant.
Fig. 3 is a kind of expression quantity schematic diagrames of Northern hybridization checks T0 for transfer-gen plant quiding gene segment：
Upper row is rRNA, and lower row is each single plant seed RNA and probe results of hybridization.
Fig. 4 left sides are a kind of copy number schematic diagram of Southern hybridization checks gene integration：There are 3 to be converted for single copy
Plant.
Fig. 4 right sides are a kind of copy number schematic diagram of Southern hybridization checks gene integration：There are 3 to be converted for single copy
Plant.
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 are implemented
Example.
The invention belongs to field of plant genetic.It is more particularly to a kind of raising rice grain vitamin content
Method, specifically contain and lead to the cDNA segments of gama-tocopherol methyl transferase (γ-TMT) gene using transgenic method
Agrobacterium-mediated Transformation rice is crossed, the segment is made to be expressed in rice grain, to improve rice grain vitamin content, creates rice
Quality-improving new material.
In order to solve the above technical problems, the present invention provides a kind of method for improving rice grain vitamin content, including
Following steps：
A. recombinant vector is built；
B. transfer-gen plant is obtained；
C. individual plant selfing seed is harvested；
D.T1 generation selfings are reserved seed for planting；
E.T2 generation selfings are reserved seed for planting；
F. Phenotypic Selection.
The step further comprises：
A. recombinant vector is built：The cDNA segments of gama-tocopherol methyl transferase gene are isolated from arabidopsis；It will
It is connected to rice grain specificity glutelin GluB-1 promoter digestions on expression vector pCAMBIA1300, structure recombination
Carrier.
The step further comprises：
B. transfer-gen plant is obtained：Using Agrobacterium tumefaciens mediated transgenic method by recombinant vector Introduced into Rice,
Obtain transfer-gen plant.
The step further comprises：
C. individual plant selfing seed is harvested：Positive detection is carried out for transfer-gen plant to T0 using PCR, is led to
The expression that Northern hybridization check positive transgenic plant are transferred to segment is crossed, hybridizing the identification positive by Southern turns base
Because of the integration copy number of plant, selection contains the segment that is transferred to singly copied, and normal solid transfer-gen plant, harvests single plant
Selfed seed.
The step further comprises：
D.T1 generation selfings are reserved seed for planting：By the seed kind of the step C transfer-gen plants selected and remain into family T1 generations, PCR is continued with
Positive detection is carried out for single plant to T1, is transferred to the expression of segment, knot to being detected to positive single plant using Northern hybridizing methods
Variable rate technology is closed, the transgenosis single plant for selecting multiple performances excellent, selfing is reserved seed for planting.
The step further comprises：
E.T2 generation selfings are reserved seed for planting：Continue the target single plant selected and remain of 4 kinds of plantation step into T2 for family, using PCR to T2 generations
Single plant carries out positive detection, and agronomic shape investigation is carried out to positive single plant and rice paddy seed vitamin content is analyzed, is therefrom selected
Phenotype stabilization, content of vitamin E and agronomic shape have the homozygous single plant of larger improvement, and selfing is reserved seed for planting.
The step further comprises：
F. Phenotypic Selection：Phenotypic Selection is carried out to the homozygous single plant selected and remain, cultivates new material.
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 vitamin content directly or indirectly produces.
In order to solve the above technical problems, rice grain dimension is improved as described in aforementioned any one invention further provides a kind of
The method of raw cellulose content is being produced for the application in the edible or rice of food processing.
It is tieed up in order to solve the above technical problems, the present invention separately provides a kind of rice grain that improved as described in aforementioned any one
Application of the method for raw cellulose content in rice strain is established.
It is easy to implement the method the purpose of the invention is to provide a kind of method for improving rice grain vitamin content, behaviour
Make it is easy, including by transgenic method, arabidopsis (Arabidopsis thaliana) gama-tocopherol methyl transferase
The cDNA segments of (γ-TMT) gene and the fusion rice transformation of specificity glutelin GluB-1 promoters, acquisition turn base
Because of plant；With reference to PCR (PCR) and molecular hybridization, the investigation of biochemical analysis method and agronomic shape,
The transgenic line that selection and breeding content of vitamin E significantly improves in transfer-gen plant offspring, single plant yield is constant or is improved, wound
Crop corn quality-improving new material.
A kind of method for improving rice grain vitamin content, laboratory operating procedures include：
1. the cDNA segments of gama-tocopherol methyl transferase (γ-TMT) gene, the cDNA pieces are isolated from arabidopsis
Section is by 1047 base compositions (Fig. 1).It and rice grain specificity glutelin GluB-1 promoter digestions are connected to expression
On carrier pCAMBIA1300 (Cambia Products), recombinant vector is built；
2. the transgenic method that Agrobacterium tumefaciems (Takara Products) mediates is utilized by recombinant vector Introduced into Rice product
Kind force is educated in round-grained rice 7, obtains transfer-gen plant；
3. positive detection is carried out for transfer-gen plant to T0 using PCR (PCR), it is miscellaneous by Northern
Detection positive transgenic plant is handed over to be transferred to the expression of segment, the integration of identification positive transgenic plant is hybridized by Southern
Copy number, selection contain the segment that is transferred to singly copied, and normal solid transfer-gen plant, harvest individual plant selfing seed.
4. the seed kind for the transfer-gen plant that step 3 is selected and remain continues with PCR to T1 for single plant into family (T1 generations)
Positive detection is carried out, the expression of segment is transferred to being detected to positive single plant using Northern hybridizing methods, with reference to field table
Transgenosis single plant that is existing, selecting multiple performances excellent, selfing are reserved seed for planting；
5. the target single plant that 4 kinds of plantation step of continuation is selected and remain, for family, carries out T2 for single plant using PCR positive into T2
Detection, carries out positive single plant agronomic shape investigation and rice paddy seed vitamin content is analyzed, and therefrom phenotype is selected to stablize, dimension
Raw element E
Content and agronomic shape have the homozygous single plant of larger improvement, and selfing is reserved seed for planting；
6. 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 pUC18-Glb with glutelin GluB-1 promoters (Glb) (according to
EMBL sequence Xs 5433314 carry out PCR amplification and are cloned on pUC18 carriers (Takara products), method reference Lee et al.,
2001).With EcoR I and HindIII double digestions, target fragment is separated and recovered, and with after EcoR I and Hind III double digestions
PCAMBIA1300 (Cambia Products) connect to form intermediate carrier by T4 ligases, then from arabidopsis will divide
CDNA segments (sequence is shown in Fig. 1) Bam H I and the Sac I of gama-tocopherol methyl transferase (γ-TMT) gene from clone
Double digestion, separates and recovers target fragment, and the intermediate carrier with being crossed with Bam H I and Sac I double digestions is formed by T4 ligases
Recombinant vector, more than restriction enzyme (EcoR I, HindIII, Bam H I and Sac I) and T4 ligases are purchased from
Takara companies；
2. by recombinant vector conversion E. coli competent DH5 α (Takara Products), containing X-Ga1, IPTG and
White single bacterium colony is selected on the resistance culture base of kanamycins (Shanghai Sangon Biotech Company's product)；
3. the white single bacterium colony selected is enriched with extracting plasmid, detected with EcoR I and HindIII double digestions rear electrophoresis,
It selects in the correct plasmid conversion Agrobacterium EHA105 (Takara Products) of connection, the strain was named S1 after conversion；
Embodiment 2：The genetic transformation of Agrobacterium：
1. induction：
By ripe rice varieties (force educates round-grained rice 7) seed decladding, then successively at the absolute ethyl alcohol with 70% volume ratio
1min, sodium hypochlorite (NaClO) seed concussion disinfection 20min of 60.15% concentration are managed, then with aseptic water washing 4-5 times,
Then seed is placed on japonica rice inducing culture (N6D2), is placed in cultivating 4 weeks at dark, 25 ± 1 DEG C of temperature.
2. subculture：
The embryo callus subculture of glassy yellow, consolidation and relatively dry is selected, is put on japonica rice subculture medium, is trained under dark
It supports 2-3 weeks, 25 ± 1 DEG C of temperature.
3. preculture：
The embryo callus subculture of consolidation and relatively dry is selected, is put on japonica rice pre-culture medium dark lower culture 4-5 days, temperature
25±1℃。
4. Agrobacterium is cultivated：
In the LA culture mediums selected with kalamycin resistance (Shanghai life work), (preparation of LA culture mediums is with reference to J. Pehanorms
(translating) such as Brooker etc., Molecular Cloning:A Laboratory guide, the third edition, golden winter wild geese, Science Press, 2002) preculture Agrobacterium on
Strain S1 two days, 28 DEG C of temperature scrape the culture that suspends in Agrobacterium to suspension medium, 28 DEG C of temperature.
5. it infects：
The callus of preculture is transferred in the bottle for bacterium of having gone out；The suspension of Agrobacterium S1 is adjusted to OD6000.8-
1.0；Callus is impregnated into 30min in agrobacterium suspension；It is blotted in transfer callus to the filter paper to have sterilized；It is then placed within
Japonica rice co-cultures
It is cultivated 3 days on base, 19-20 DEG C of temperature.
6. screening：
With sterile water washing callus 8 times；It is immersed in going out for carbenicillin containing 400mg/L (CN) (Shanghai life work)
30min in bacterium water；It is blotted in transfer callus to the filter paper to have sterilized；Transfer callus to the filter paper to have sterilized blots；Shift callus
To on containing 250mg/L carbenicillins (CN), 50mg/L hygromycin (Hn) (Roche Products) japonica rice Selective agar medium
Selection culture 2-3 times, every time 2 weeks.
7. differentiation：
Kanamycin-resistant callus tissue is transferred on japonica rice differential medium, is cultivated under illumination, 26 DEG C of temperature.
8. it takes root：
Cut the root generated during regrowth differentiation；It is then transferred in root media under illumination and cultivates 2-3 weeks,
26 DEG C of temperature.
9. transplanting
The remaining medium on regeneration plant root is washed off, moves into potting in alms bowl, while kept moisture wet at initial several days
Profit moves into crop field after plant to be planted survival is healthy and strong.
Embodiment 3：
Rotaring gene plant blade is taken to extract DNA, carries out PCR (PCR), PCR programs：94 DEG C of pre-degenerations
5min；35 cycle (94 DEG C of denaturation 1min；55 DEG C of annealing 1min；72 DEG C of extension 2min), 72 DEG C of extension 7min.Detection is positive
Transformed plant, the single plant that can amplify 1064bp size strips are positive transformants single plant (Fig. 3).The positive single plant seed of extracting
RNA carry out Northern hybridization checks and be transferred to expression of the segment in seed, the segment that is transferred to of positive single plant is all expressed
(Fig. 4).Positive single plant DNA full-page proofs are extracted respectively with Sac I and Xba I (Takara Products) digestion, and it is miscellaneous to carry out Southern
The segment of identification positive transformants single plant is handed over to integrate copy number, obtains 6 independent transformation plant singly copied.DNA extractings, RNA
The relevant technologies such as extracting, PCR reaction systems, Southern and Northern hybridization are with reference to J. Pehanorm Brookers etc., molecular cloning
(translating) such as experiment guide, the third edition, golden winter wild geese, Science Press, 2002.
Embodiment 4：
By select and remain 6 single seed kinds for copying transgenosis single plant into T1 for family, since in T1 generations, can detach, into
One step detects the positive single plant in each family using PCR (PCR) (method is with embodiment 3), to positive single plant
It takes out
It carries seed RNA and carries out the expression (method is with embodiment 3) that Northern hybridization checks are transferred to segment, with reference to field
It shows each family and selects a single plant containing target fragment and target fragment normal expression, selfing is reserved seed for planting.
Embodiment 5：
4 kinds of seed kinds of 6 single plants selected and remain of embodiment are continued with into PCR (the same embodiments of method into T2 for family
3) the positive single plant in each family is detected, wherein family 3, family 6 and family 7 are homozygous stablizes (table 1).Investigation family 3,
The Other Main Agronomic Characters of family 6 and family 7 simultaneously analyze seed vatimin E content, therefrom select 3 content of vitamin E improve and
The good homozygous single plant (SPD1, SPD2 and SPD3) of variable rate technology.
1 T2 of table is for positive plant rate (positive plant number/detection plant number)
It is as follows that Other Main Agronomic Characters investigate standard：
Plant height：In height (cm) of the field test single plant highest fringe grain husk point apart from ground before harvest；
Defined daily doses：It often selects good strains in the field for seed and takes 3 big fringes, count per all bear fruit grains of fringe；
Setting percentage：Defined daily doses divided by grains per panicle are multiplied by 100 (%)；
Single plant yield：All real grain is heavy (g) for single plant
The measure of content of vitamin E uses high performance liquid chromatography (HPLC), and (Ex=is detected using fluorescence detector
295nm, Em=330nm), using α-, β-, γ-, Delta-Tocopherol as standard items, addition tocol be internal standard, calculate soybean in tie up
The content and total content of raw element E Isomers.
It is prepared by rice flour：About 25g paddy is taken to shell on rough machine is gone out, is then gone out with domestic JMJ-100 types rice polisher husk rice
Essence, then polished rice is broken into powder with whirlwind type crushing machine (Udy corporation, Colorado, USA), it is filled after sieving with 100 mesh sieve
Enter plastic bag sealing, be placed in -20 DEG C of refrigerators and save backup.
HPLC is analyzed：Using 1200 type high performance liquid chromatograph of Agilent companies of the U.S., chromatographic column Agilent
Eclipse XDB-C18 column(4.6×150mm length；5 μm), mobile phase methanol: water 95: 5 (V/V), flow velocity
1.5mL/min, excitation wavelength and launch wavelength are respectively 290nm and 330nm, 10 μ L of sample size.
Embodiment 6：
The homozygous single plant SPD1 of previous generation's selection is educated round-grained rice 7 with compareing wild type force respectively in April, 2016 and November to exist
Qingdao and Hainan kind analyze the vitamin E content of transfer-gen plant and control into family (method is with embodiment 5).With to photograph
Than in the chaff layer of family SPDl, the ratio that the highest alpha-tocopherol of Vitamin E activity accounts for total tocopherol is increased to by 90%
The content of 95%, α-tocotrienol is 2.23 times of empty vector control, and the ratio for accounting for total tocotrienol is improved by 48%
To 86%.In endosperm, alpha-tocopherol accounts for the content that the ratio of total tocopherol is increased to 98%, α-tocotrienol by 93%
It is 3.44 times of control, the ratio for accounting for total tocotrienol is increased to 89%, and 67% is improved compared with compareing (22%)
(table 2).The transgenic lines of Hainan plantation and the Other Main Agronomic Characters (method is with embodiment 5) compareed are investigated simultaneously, with compareing
It compares, plant height and Defined daily doses no significant difference, and setting percentage and mass of 1000 kernel are significantly increased (table 3).
2 transgenic lines SPD1 of table and the content of vitamin E (μ g/g) to impinging upon Qingdao and Hainan plantation
3 transgenic lines SPD1 of table is with compareing Hainan plantation Other Main Agronomic Characters performance
*, * *：0.05 and 0.01 horizontal conspicuousness.
Embodiment 7：
The homozygous single plant SPD2 of previous generation's selection is educated round-grained rice 7 with compareing wild type force respectively in April, 2016 and November to exist
Qingdao and Hainan kind analyze the vitamin E content of transfer-gen plant and control into family (method is with embodiment 5).With to photograph
Than in the chaff layer of family SPD2, the ratio that the highest alpha-tocopherol of Vitamin E activity accounts for total tocopherol is increased to by 90%
The content of 94%, α-tocotrienol is 1.99 times of empty vector control, and the ratio for accounting for total tocotrienol is improved by 48%
To 84%.In endosperm, alpha-tocopherol accounts for the content that the ratio of total tocopherol is increased to 99%, α-tocotrienol by 93%
It is 3.03 times of control, the ratio for accounting for total tocotrienol is increased to 89%, and 67% is improved compared with compareing (22%)
(table 4).The transgenic lines of Hainan plantation and the Other Main Agronomic Characters (method is with embodiment 5) compareed are investigated simultaneously, with compareing
It compares, plant height and Defined daily doses no significant difference, and setting percentage and mass of 1000 kernel are significantly increased (table 5).
4 transgenic lines SPD2 of table and the content of vitamin E (μ g/g) to impinging upon Qingdao and Hainan plantation
5 transgenic lines SPD2 of table is with compareing Hainan plantation Other Main Agronomic Characters performance
*, * *：0.05 and 0.01 horizontal conspicuousness.
Embodiment 8：
The homozygous single plant SPD3 of previous generation's selection is educated round-grained rice 7 with compareing wild type force respectively in April, 2016 and November to exist
Qingdao and Hainan kind analyze the vitamin E content of transfer-gen plant and control into family (method is with embodiment 5).With to photograph
Than in the chaff layer of family SPD3, the ratio that the highest alpha-tocopherol of Vitamin E activity accounts for total tocopherol is increased to by 90%
The content of 93%, α-tocotrienol is 1.70 times of empty vector control, and the ratio for accounting for total tocotrienol is improved by 48%
To 82%.In endosperm, alpha-tocopherol accounts for the content that the ratio of total tocopherol is increased to 96%, α-tocotrienol by 93%
It is 3.52 times of control, the ratio for accounting for total tocotrienol is increased to 90%, and 68% is improved compared with compareing (22%)
(table 6).The transgenic lines of Hainan plantation and the Other Main Agronomic Characters (method is with embodiment 5) compareed are investigated simultaneously, with compareing
It compares, plant height and setting percentage no significant difference, and Defined daily doses and mass of 1000 kernel are significantly increased (table 7).
6 transgenic lines SPD3 of table and the content of vitamin E (μ g/g) to impinging upon Qingdao and Hainan plantation
7 transgenic lines SPD3 of table is with compareing Hainan plantation Other Main Agronomic Characters performance
*, * *：0.05 and 0.01 horizontal conspicuousness.
The culture medium and its preparation method of genetic transformation used in the present invention are as described below：
1. reagent and solution abbreviation
The abbreviations table of plant hormone in the present invention used in culture medium represents as follows：
6-BA (6-Benzylaminopurine, 6-benzyladenine)；
CN (Carbenicillin, carbenicillin)；
KT (Kinetin, kinetin)；
NAA (Naphthalene acetic acid, methyl α-naphthyl acetate)；
IAA (Indole-3-acetic acid, heteroauxin)；
2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4- dichlorphenoxyacetic acids)；
AS (Acetosringone, acetosyringone)；
CH (Casein Enzymatic Hydrolysate, hydrolysis network albumen)；
HN (Hygromycin B, hygromycin)；
N6max (N6 a great number of elements ingredient solution)；
N6min (N6 Trace Elements solution)；
MSmax (MS a great number of elements ingredient solution)；
MSmin (MS Trace Elements solution).
2. main solution formula
(1) N6 culture mediums a great number of elements mother liquor (being prepared according to 10 times of 10 × concentrates)：
Mentioned reagent is dissolved one by one, then under room temperature condition (20-25 DEG C, same as below), is settled to distilled water
1000mL。
(2) N6 culture mediums trace element mother liquor (being prepared according to 100 times of 100 × concentrates)：
Mentioned reagent is dissolved at room temperature and is settled to 1000mL with distilled water.
(3) molysite (Fe2EDTA) storage liquid (being prepared according to 100 times of 100 × concentrates)
3.73g disodium ethylene diamine tetraacetates (Na2EDTA2H2O) and 2.78g Fe SO47H2O are dissolved respectively,
It mixes and is settled to 1000mL with distilled water, until 70 DEG C of warm bath 2h, 4 DEG C save backup.
(4) vitamins stock liquid (being prepared according to 100 times of 100 × concentrates)
Distilled water is added to be settled to 1000mL, 4 DEG C save backup.
(5) MS culture mediums a great number of elements mother liquor (MSmax) (being prepared according to 10 times of concentrates)
Mentioned reagent is dissolved, and be settled to 1000mL with distilled water at room temperature.
(6) MS culture mediums trace element mother liquor (MSmin) (being prepared according to 100 times of concentrates)
Mentioned reagent is dissolved, and be settled to 1000mL with distilled water at room temperature.
The preparation of (7) 2,4-D storage liquid (1mg/mL)：
2,4-D 100mg are weighed, dissolve 5min with 1mL1N potassium hydroxide, after then addition 10mL distillations water dissolution is complete
100mL is settled to, is preserved at room temperature.
(8) 6-BA stores the preparation of liquid (1mg/mL)：
6-BA 100mg are weighed, dissolve 5min with 1mL1N potassium hydroxide, after then addition 10mL distillations water dissolution is complete
100mL is settled to, is preserved at room temperature.
(9) preparation of methyl α-naphthyl acetate (NAA) storage liquid (1mg/mL)：
NAA 100mg are weighed, dissolve 5min with 1mL1N potassium hydroxide, it is completely fixed afterwards then to add in 10mL distillations water dissolution
Hold to 100mL, 4 DEG C save backup.
(10) preparation of heteroauxin (IAA) storage liquid (1mg/mL)：
IAA 100mg are weighed, dissolve 5min with 1mL1N potassium hydroxide, it is completely fixed afterwards then to add in 10mL distillations water dissolution
Hold to 100mL, 4 DEG C save backup.
(11) preparation of glucose storage liquid (0.5mg/mL)：
Glucose 125g is weighed, 250mL is settled to distilled water, is saved backup for 4 DEG C after sterilizing.
(12) AS stores the preparation of liquid：
AS 0.392g are weighed, DSMO10mL dissolvings is added in, dispenses to 1.5mL centrifuge tubes, 4 DEG C save backup.
(13) 1N potassium hydroxide storage liquid
Potassium hydroxide 5.6g is weighed, is settled to 100mL with distillation water dissolution, room temperature preservation is spare.
3. for the culture medium prescription of rice transformation
(1) inducing culture
Adding distilled water, 1N sodium hydroxides adjust pH value to 5.9, boil and are settled to 1000mL, are dispensed into 900mL
In 50mL conical flasks (25mL/ bottles), conventionally sterilize after sealing (such as sterilize 25 minutes at 121 DEG C, following trainings
It is identical with the sterilizing methods of basal culture medium to support base sterilizing methods).
(2) subculture medium
Adding distilled water, 1N sodium hydroxides adjust pH value to 5.9, boil and are settled to 1000mL, are dispensed into 900mL
In 50mL conical flasks (25mL/ bottles), sealing sterilizes as stated above.
(3) pre-culture medium
Add distilled water to 250mL, 1N sodium hydroxides adjust pH value to 5.6, and sealing sterilizes as stated above.
Using preceding heated solution culture medium and 5mL glucose storages liquid and 250 μ L AS storage liquid are added in, training is poured into packing
It supports in ware (25mL/ wares).
(4) base is co-cultured
Add distilled water to 250mL, 1N sodium hydroxides adjust pH value to 5.6, and sealing sterilizes as stated above.
Using preceding heated solution culture medium and 5mL glucose storages liquid and 250 μ L AS storage liquid are added in, training is poured into packing
It supports in ware (25mL/ wares).
(5) suspension medium
Add distilled water to 100mL, 1N sodium hydroxides adjust pH value to 5.4, are dispensed into the conical flask of two 100mL, seal
Mouthful, it sterilizes as stated above.
Use preceding addition 1mL glucose storages liquid and 100 μ L AS storage liquid.
(6) Selective agar medium
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 250 μ LHN (50mg/mL) and add in 400 μ LCN (250mg/mL), packing is fallen
Enter in culture dish (25mL/ wares).(note：First time a concentration of 400mg/L of Selective agar medium carbenicillin, second and after
A concentration of 250mg/L of Selective agar medium carbenicillin).
(7) pre- differential medium
Distilled water is added to adjust pH value to 5.9, sealing sterilizes as stated above to 250mL.
Using preceding dissolving culture medium, add in 250 μ LHN (50mg/mL) and add in 250 μ LCN (250mg/mL), packing is fallen
Enter in culture dish (25mL/ wares).
(8) differential medium
Distilled water is added to adjust pH value to 6.0 to 900mL.
It boils and is settled to 1000mL with distilled water, be dispensed into 50mL conical flasks (25mL/ bottles), seal, as stated above
Sterilizing.
(9) root media
Adding distilled water, 1N sodium hydroxides adjust pH value to 5.8 to 900mL.
It boils and is settled to 1000mL with distilled water, be dispensed into pipe of taking root (25mL/ pipes), seal, go out as stated above
Bacterium.
The above is only several embodiments of the present invention, any type of limitation is not done to the present invention, although this
Invention is disclosed as above with preferred embodiment, however not to limit the present invention, any person skilled in the art,
In the range of not departing from technical solution of the present invention, make a little variation using the technology contents of the disclosure above or modification is impartial
Equivalence enforcement case is same as, is belonged in technical solution of the present invention protection domain.
Claims (10)
- A kind of 1. method for improving rice grain vitamin content, 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.T1 generation selfings are reserved seed for planting；E.T2 generation selfings are reserved seed for planting；F. Phenotypic Selection.
- 2. the method for rice grain vitamin content is improved according to claim 1, which is characterized in that the step is further Including：A. recombinant vector is built：The cDNA segments of gama-tocopherol methyl transferase gene are isolated from arabidopsis；By it and water Rice seed specificity glutelin GluB-1 promoter digestions are connected on expression vector pCAMBIA1300, build recombinant vector.
- 3. the method for rice grain vitamin content is improved according to claim 1, which is characterized in that the step is further Including：B. transfer-gen plant is obtained：Using Agrobacterium tumefaciens mediated transgenic method by recombinant vector Introduced into Rice, obtain Transfer-gen plant.
- 4. the method for rice grain vitamin content is improved according to claim 1, which is characterized in that the step is further Including：C. individual plant selfing seed is harvested：Positive detection is carried out for transfer-gen plant to T0 using PCR, is passed through Northern hybridization check positive transgenic plant are transferred to the expression of segment, and hybridizing identification positive transgenic by Southern plants The integration copy number of strain, selection contain the segment that is transferred to singly copied, and normal solid transfer-gen plant, harvest individual plant selfing kind Son.
- 5. the method for rice grain vitamin content is improved according to claim 1, which is characterized in that the step is further Including：D.T1 generation selfings are reserved seed for planting：By the seed kind of the step C transfer-gen plants selected and remain into family T1 generations, PCR is continued with to T1 Positive detection is carried out for single plant, the expression of segment is transferred to being detected to positive single plant using Northern hybridizing methods, with reference to field Between show, select the excellent transgenosis single plant of multiple performances, selfing is reserved seed for planting.
- 6. the method for rice grain vitamin content is improved according to claim 1, which is characterized in that the step is further Including：E.T2 generation selfings are reserved seed for planting：Continue the target single plant selected and remain of 4 kinds of plantation step into T2 for family, using PCR to T2 for single plant Positive detection is carried out, agronomic shape investigation is carried out to positive single plant and rice paddy seed vitamin content is analyzed, therefrom selects phenotype Stabilization, content of vitamin E and agronomic shape have the homozygous single plant of larger improvement, and selfing is reserved seed for planting.
- 7. the method for rice grain vitamin content is improved according to claim 1, which is characterized in that the step is further Including：F. Phenotypic Selection：Phenotypic Selection is carried out to the homozygous single plant selected and remain, cultivates new material.
- 8. a kind of food, which is characterized in that contained using rice grain vitamin is improved as described in any one of claim 1~7 The method of amount directly or indirectly produces.
- 9. a kind of method that rice grain vitamin content is improved as described in any one of claim 1~7 is in production for eating With or the rice of food processing in application.
- 10. a kind of method that rice grain vitamin content is improved as described in any one of claim 1~7 is establishing rice product Application in system.
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| CN118460783A (en) * | 2024-07-10 | 2024-08-09 | 海南大学三亚南繁研究院 | A SNP site associated with rice α-tocopherol content and its application |
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