CN102144568A - Method for improving quality of rice kernel amino acid - Google Patents
Method for improving quality of rice kernel amino acid Download PDFInfo
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- CN102144568A CN102144568A CN2011101092257A CN201110109225A CN102144568A CN 102144568 A CN102144568 A CN 102144568A CN 2011101092257 A CN2011101092257 A CN 2011101092257A CN 201110109225 A CN201110109225 A CN 201110109225A CN 102144568 A CN102144568 A CN 102144568A
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Abstract
The invention discloses a method for improving the quality of rice kernel amino acid, comprising the following steps of: A, syncretizing a cDNA (complementary deoxyribonucleic acid) segment of dragline silk protein gene separated from a spider with glutelin promoter, and constructing a recombinant carrier; B, introducing the recombined carrier to rice through agrobacterium tumefaciens mediated transgenosis method, and obtaining a converted plant; C, carrying out positive detection on T0 generation transgenic plant by using polymerase chain reaction (PCR), and obtaining positive monoclonal selfedseed; D, growing the selected T0 generation positive monoclonal seed into a family (T1), and carrying out positive detection on T1 generation monoclonal plant; E, continuously planting T1 generation positive monoclonal seed into T2 generation family, selecting pure monoclonal plant with improved amino acid quality, selfing and reserving seed; F, carrying out phenotypic selection on the selected pure monoclonal plant, and culturing new material. The method is simple and easy, and the operation is simple and convenient; the cDNA segment from spider dragline silk protein gene for expressing the rice grins improves the content of amino acid in the rice kernel, and improves nutrition of the kernels; the method improves quality of the rice kernels, and the efficiency is high; and the method is used for improving the crop quality.
Description
Technical field
The invention belongs to the plant gene engineering technology field.More specifically relate to a kind of method that improves rice grain amino acid quality, specifically comprised the applying transgene method cDNA fragment of a spider dragline silk protein gene has been passed through the Agrobacterium-mediated Transformation paddy rice, this fragment is expressed in rice grain, to improve the rice grain amino acid content, create the method for rice quality improvement new material.
Background technology
Paddy rice is one of most important cereal crops in the world, yet particularly lysine and tryptophane are very low for protein and amino acid in its seed, because protein and essential amino acids content are the key factors of its nutritive value of reaction, therefore improve the rice grain amino acid content particularly essential amino acids content become the vital task of rice quality improvement.
At present, do not find the mutant of high-lysine and high tryptophan in paddy rice, therefore, the amino acid content that improves rice grain mainly concentrates on in the engineered method research.The promotor that the full-length cDNA that Lee etc. (2001) react corn lysine to insensitive synthase gene dhps (dihydrodipicolinic acid synthase) connects rice grain selectivity glutelin GluB-1 is built into mosaic gene, rice transformation, free lysine content will be higher than the wild type plant in the immature seed of discovery transgenic line, but free aminoacid content in its mature seed and wild type difference are not obvious.Wu etc. (2003) utilize little bullet method, and the gene of the tRNA-lys that will encode (lysine swing codon) changes paddy rice over to, find that the lysine content of transgenic line seed has improved 6%, but it far are not enough to satisfy the demand of people to its content.Also there are some researches show overexpression tryptophan synthetase gene OASA1D in paddy rice, tryptophan in the rice paddy seed is significantly increased, but defective all appears in performances such as the output of transgenic line, fertility and seed germination, and be difficult to be applied (Wakasa et al, 2006).
Spider (Araneus ventricosue) drags silk-fibroin (Dragline) fiber noticeable with its special dissolution characteristics and mechanical characteristic.Utilize engineered spider dragline silk protein gene can in zooblast, bacterium and plant such as potato and tobacco, produce spider dragline silk albumen (Pan et al, 2006); Someone attempts using spider silk protein gene conversion sea-island cotton and improves cotton fiber quality, but how does not know its effect (Huang et el, 2004).At present, utilizing the spider dragline silk protein gene to improve the crop alimentary Study on Quality does not appear in the newspapers as yet.
The objective of the invention is one section cDNA fragment with spider dragline silk albumen (Dragline) gene, with selectivity glutelin GluB-1 promoters driven, rice transformation, make this section cDNA fragment specifically expressing in rice grain, to improve the rice grain amino acid content, improvement rice grain quality is created rice quality improvement new material.
The present invention does not at home and abroad appear in the newspapers, the yet unexposed article that relates to content of the present invention of delivering of place seminar, the present invention's public's the unknown at home and abroad.
Summary of the invention
The objective of the invention is to be to provide a kind of method that improves rice grain amino acid quality, easy to implement the method, easy and simple to handle, comprise and pass through transgenic method, the fusion rice transformation of one section cDNA fragment of spider dragline silk albumen (Dragline) gene and rice grain selectivity glutelin GluB-1 promotor, obtain transfer-gen plant; In conjunction with polymerase chain reaction (PCR) (PCR) and molecular hyridization method, the investigation of biochemical analysis method and economical character, seed selection amino acid significantly improves in the transfer-gen plant offspring, single plant yield is constant or the transgenic line of raising is arranged, and creates rice quality improvement new material.
A kind of method that improves rice grain amino acid quality the steps include:
One section cDNA fragment of dragging silk-fibroin (Dragline) gene of 1, separating from spider, this cDNA fragment are by 905 base compositions, and 264 amino acid of encoding are rich in glycine (31%) and alanine (25.8%) (Fig. 1).It and rice grain selectivity glutelin GluB-1 promotor enzyme cut to be connected link expression vector pCAMBIA1300 and go up (Cambia company product), make up recombinant vector;
2, utilize the transgenic method of Agrobacterium tumefaciems (Takara company product) mediation that recombinant vector is imported among the rice varieties Dongjing, obtain transfer-gen plant;
3, utilize polymerase chain reaction (PCR) (PCR) that T0 is carried out positive detection for transfer-gen plant, detect the expression that positive transfer-gen plant changes fragment over to by Northern hybridization, identify the integration copy number of positive transfer-gen plant by Southern hybridization, what selection contained single copy changes fragment and normal solid transfer-gen plant, results individual plant selfing seed over to;
4, the seed kind of the transfer-gen plant that step 3 is selected and remain becomes family (T1 generation), continue to utilize PCR that T1 is carried out positive detection for individual plant, utilize the Northern cross method positive individual plant to be detected the expression that changes fragment over to, select the good transgenosis individual plant of a plurality of performances in conjunction with the field performance, selfing is reserved seed for planting;
5, continuing to plant the target individual plant of selecting and remain in the step 4 becomes T2 for family, utilize PCR that T2 is carried out positive detection for individual plant, positive individual plant is carried out economical character to be investigated and the rice paddy seed amino acid analysis, therefrom select phenotype stable, amino acid quality and economical character have the individual plant that isozygotys of bigger improvement, and selfing is reserved seed for planting;
6, the individual plant of selecting and remain that isozygotys is carried out Phenotypic Selection, cultivate new material.
The present invention compared with prior art has the following advantages and effect:
A, one section cDNA fragment expressing from spider dragline silk albumen (Dragline) gene in rice grain can improve the rice grain amino acid content, essential amino acids content particularly, thereby improved the rice grain nutritional quality, simultaneously, single plant yield also improves;
B, this method adopt transgenic method to improve the rice grain quality, and the improvement cycle is short, the efficient height;
C, this method can directly apply to the improvement practice of other crop qualities;
D, with one section cDNA fragment of spider dragline silk albumen (Dragline) gene, with seed selectivity glutelin GluB-1 promoters driven, rice transformation, make this section cDNA specifically expressing in rice grain, easy to implement the method, easy and simple to handle, improved the rice grain amino acid content, increased single plant yield, for new material has been created in the rice quality improvement.The material that the present invention produced can directly apply to preparation essential amino acid etc. pharmaceutical products and feed product, and promote health and the Sustainable Production of prevent disease and paddy rice etc. is extremely important.
Description of drawings
Fig. 1 is a kind of nucleotide sequence and amino acid sequence that transforms used cDNA fragment.
This cDNA fragment has 905 base compositions, 264 amino acid of encoding.
Fig. 2 is a kind of construction of recombinant vector schematic diagram.
Earlier glutelin GluB-1 promotor (Glb) is connected to expression vector pCAMBIA1300 and goes up the formation intermediate carrier, the cDNA fragment with the spider dragline silk protein gene is connected to intermediate carrier formation recombinant vector again.
Fig. 3 is that a kind of T0 is for transfer-gen plant positive detection schematic diagram.
The 2nd swimming lane is a molecular weight marker, the positive contrast of the 3rd swimming lane (recombinant vector amplification), and the negative contrast of 4-5 swimming lane (wild type Dongjing amplification), the 6-19 swimming lane is that part T0 is for the transfer-gen plant amplification;
Fig. 4 is the expression schematic diagram that a kind of Northern hybridization detects T0 band transfer-gen plant quiding gene fragment.
Last row is rRNA, and following row is each individual plant seed RNA and probe hybridization result.
Fig. 5 A is the copy number schematic diagram that a kind of Southern hybridization detects gene integration.
There are 3 to be single copy transformed plant.
Fig. 5 B is the copy number schematic diagram that a kind of Southern hybridization detects gene integration.
3 single copy plant are arranged.
Fig. 6 A is that a kind of Northern hybridization detects the target gene expression schematic diagram of the T1 of single copy individual plant for family.
There is the fragment that changes over to of 9 transformed plants to obtain expressing.
Fig. 6 B is that a kind of Northern hybridization detects the target gene expression schematic diagram of the T1 of single copy individual plant for family.
There is the fragment that changes over to of 6 transformed plants to obtain expressing.
Fig. 6 C is that a kind of Northern hybridization detects the target gene expression schematic diagram of the T1 of single copy individual plant for family.
There is the fragment that changes over to of 6 transformed plants to obtain expressing.
Embodiment
The present invention provides by following detailed embodiment.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.
Embodiment 1: the foundation of the structure of recombinant vector and conversion Agrobacterium:
(1), technology path according to Fig. 2, will (carrying out pcr amplification according to EMBL sequence X 5433314 be cloned on the pUC18 carrier (Takara company product) with the plasmid pUC18-Glb of glutelin GluB-1 promotor (Glb), method is with reference to Lee etal, 2001, Constitutive and seed-specific expression of a maize lysinefeedback-insensitivedihydrodipioolinate synthase gene leads to increased free lysine levels in rice seedsMolecular Breeding 8:75-84) with EcoR I and Hind III double digestion, separate and reclaim the purpose fragment, with be connected to form intermediate carrier with the pCAMBIA1300 behind EcoR I and the Hind III double digestion (Cambia company product) with the T4 ligase, again will be from spider the cDNA fragment (sequence is seen Fig. 1) of one section spider dragline silk albumen (Dragline) gene of separating clone with Kpn I and Sac I double digestion, separate and reclaim target fragment, with cross intermediate carrier with Kpn I and Sac I double digestion and be connected to form recombinant vector with the T4 ligase, above restriction enzyme (EcoR I Hind IIIKpn I and Sac I) and T4 ligase are all purchased the company in Takara;
(2), with recombinant vector transformed into escherichia coli competence DH5 α (Takara company product), on the resistance medium that contains X-Gal, IPTG and kanamycin (Shanghai give birth to worker company product), select white single bacterium colony;
(3), the white single bacterium colony enrichment extracting plasmid that will select, detect with EcoR I and Hind III double digestion rear electrophoresis, select and connect correct plasmid and transform among the Agrobacterium EHA105 (Takara company product) the bacterial strain called after S1 after the conversion;
Above-mentioned molecular cloning method and agent prescription are with reference to J. Sa nurse Brooker etc., molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, 2002.
Embodiment 2: agriculture bacillus mediated genetic transformation:
(1), induce:
Rice varieties (the disclosed japonica rice variety of the Dongjign Korea S) seed of maturation is shelled, use the Ethanol Treatment 1 minute of 70% volume ratio then successively, the mercury chloride (HgCl of 0.15% concentration
2) the surface of the seed sterilization 15 minutes; Wash seed 4-5 time with sterilization; Seed is placed on the japonica rice inducing culture; Place dark place to cultivate 4 weeks, 25 ± 1 ℃ of temperature postvaccinal medium.
(2), subculture:
Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark 2-3 week, 25 ± 1 ℃ of the temperature of cultivating down on the japonica rice subculture medium.
(3), the pre-cultivation:
Select the embryo callus subculture of consolidation and relatively dry, be put on the japonica rice pre-culture medium and trained 4-5 days 25 ± 1 ℃ of temperature under the dark.
(4), Agrobacterium is cultivated:
(the LA culture medium preparation is with reference to J. Sa nurse Brooker etc., molecular cloning experiment guide, the third edition having the LA medium that kalamycin resistance (Shanghai give birth to worker company product) selects, Jin Dongyan etc. (translating), Science Press, 2002) went up pre-cultivation Agrobacterium strain S1 two days, 28 ℃ of temperature; Scrape and get Agrobacterium suspension culture to the suspension culture base, 28 ℃ of temperature.
(5), infect:
Pre-incubated callus is transferred in the bottle of the bacterium of having gone out; The suspension of regulating Agrobacterium S1 is to OD
6000.8-1.0; Callus was soaked in agrobacterium suspension 30 minutes; Shifting callus blots to the good filter paper of sterilization; Be placed on japonica rice then and cultivated temperature 19-20 ℃ 3 days on the medium altogether.
(6), screening:
Wash callus 8 times with aqua sterilisa; Be immersed in the aqua sterilisa that contains 400 milligrams/L carbenicillin (CN) (Shanghai give birth to worker company product) 30 minutes; Shifting callus blots to the good filter paper of sterilization; Shifting callus selects to cultivate each 2 weeks 2-3 time to containing on 250mg/L carbenicillin (CN), 50mg/L hygromycin (Hn) (Roche company product) the japonica rice selection medium.
(7), differentiation:
Kanamycin-resistant callus tissue is transferred on the japonica rice differential medium, and illumination is cultivated down, 26 ℃ of temperature.
(8), take root:
Cut the root that the regrowth differentiation phase produces; Then it is transferred to and cultivates 2-3 week, 26 ℃ of temperature in the root media under the illumination.
(9), transplant:
Wash the residual medium on the regeneration plant root off, potted plant in the immigration alms bowl, the while divides moistening at initial several Tian Bao water holding, treats immigration land for growing field crops, the healthy and strong back of plant survival.
Embodiment 3:
Get rotaring gene plant blade extracting DNA, carry out polymerase chain reaction (PCR) (PCR), the PCR program: 94 ℃ of pre-sex change 5 minutes; 33 circulations (94 ℃ of sex change 1 minute; Annealed 1 minute for 55 ℃; 72 ℃ were extended 2 minutes), 72 ℃ were extended 7 minutes; ) detecting positive transformed plant, the individual plant that can amplify the 1.2kb size strip is a positive conversion individual plant (Fig. 3).The RNA of the positive individual plant seed of extracting carries out Northern hybridization detection and changes the expression of fragment in seed over to, and the fragment that changes over to of positive individual plant is all expressed (Fig. 4).The positive individual plant DNA of extracting full-page proof uses Sac I and Xba I (Takara company product) enzyme to cut respectively, carries out the positive fragment that transforms individual plant of Southern hybridization evaluation and integrates copy number, obtains 3 single independent transformed plants (Fig. 5) that copy.Correlation techniques such as DNA extracting, RNA extracting, PCR reaction system, Southern and Northern hybridization are with reference to J. Sa nurse Brooker etc., molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, 2002.
Embodiment 4:
Become T1 for family 3 single seed kinds that copy the transgenosis individual plants of selecting and remain, because T1 separates for meeting, therefore further utilize polymerase chain reaction (PCR) (PCR) (method is with embodiment 3) to detect positive individual plant in each family, positive individual plant extracting seed RNA is carried out Northern hybridization to be detected the expression that changes fragment over to (method is with embodiment 3, Fig. 6), show each family in conjunction with the field and select an individual plant that contains target fragment and target fragment normal expression, acceptance is planted certainly;
Embodiment 5:
Become T2 for family the seed kind of 3 individual plants selecting and remain among the embodiment 4, continue to utilize PCR (method is with embodiment 3) to detect positive individual plant in each family, wherein family 3 and family 5 isozygotied stable (table 1).Investigate the Other Main Agronomic Characters of family 3 and family 5 and analyze the seed amino acid content, therefrom select 3 amino acid contents to improve and the good individual plant that isozygotys (SPD1, SPD2 and SPD3) of field performance.
Table 1 T2 is for positive plant rate (positive plant number/detection plant number)
It is as follows that Other Main Agronomic Characters is investigated standard:
Plant height: results are preceding in the height (cm) of the highest fringe grain husk of field test individual plant point apart from ground;
The real grain of every fringe number: whenever select good strains in the field for seed and get 3 big fringes, count all real grain numbers of every fringe;
Ripening rate: the real grain of every fringe number spends number to multiply by 100 (%) divided by every fringe grain husk;
Single plant yield: all real grain of individual plant heavy (g)
Seed amino acid content assay method passes through hydrochloric acid hydrolysis (with reference to Wang Lingqiang, 2007, the genetic analysis of molecular labeling genetic map construction and rice quality proterties, doctorate paper: 166-167), utilize amino-acid analyzer (the full-automatic amino-acid analyzer of the L-8800 of Hitachi) to measure; The amino-acid analyzer operational procedure is seen amino acid fully-automatic analyzer specification.
Ground rice preparation: get about 25 gram paddy and going out on the rough machine to shell, go out essence with the husk rice of homemade JMJ-100 type rice polisher then, use cyclone type cracker (Udy corporation again, Colorado, USA) polished rice is broken into powder, cross the plastic bag sealing of packing into behind 100 mesh sieves, it is standby to place-20 ℃ of refrigerators to preserve.
Hydrochloric acid hydrolysis method determination step is as follows:
(1), notes the little spoon sampling of long handle when sample weighting amount is approximately about 100mg (sensibility reciprocal of balance is 1/10000, generally controls 80-110mg) weighing sample, then sample is sent into hydrolysis pipe bottom, and carried out record.To wear gloves during operation.
(2), the hydrochloric acid that in the hydrolysis pipe that sample is housed, adds the 6mol/L of 6-8mL with the adjustable quantitative charger.Note adding about 0.5mL earlier soaking.
(3), vacuumizing the decompression back seals with the calcination of butane blowtorch.
(4), hydrolysis 22 hours in 110 ℃ of baking ovens.
(5), shake up liquid in the hydrolysis pipe, (11 * 11cm) are filtered in the 50mL volumetric flask and with distilled water and are settled to 50mL with filter paper.Attention must be more than the distilled water rinse hydrolysis Guan Sanci, and guarantees to add water filtration next time again after water has been filtered to volumetric flask on the filter paper, to guarantee that no amino acid is residual on hydrolysis pipe and the filter paper.
(6), get 1mL after shaking up in the 2.5mL plastic centrifuge tube, 4-6 hour extremely nearly drying regime of freeze dryer vacuum concentration.(also available Other Instruments replaces freeze dryer, as long as can carry out vacuum drying)
(7), adding 1mL 0.02mol/L hydrochloric acid shakes in centrifuge tube evenly.
(8), the centrifugal 15min of 14000rpm sucts clear liquid 0.8mL in the amino acid determining instrument sample bottle, and last machine is measured.Each sample determination 2 times.
According to the content of every seed amino acid in the standard specimen conversion sample, unit is mg/g.Tryptophan is destroyed in hydrochloric acid hydrolysis, this method undetermined; Glutamic acid (Glu) and asparatate (Asp) content have comprised the part that comes from glutaminase (Gln) and aspargine (Asn) degraded.Therefore, essential amino acids content (Eaa) is these 7 kinds of essential amino acids content sums of threonine, valine, methionine, isoleucine, leucine, phenyl alanine and lysine.All total amino acid contents (Total) are 17 seed amino acid content sums.
Embodiment 6:
The previous generation select isozygoty individual plant SPD1 with contrast wild type Dongjing respectively in June, 2008 and December planting into family with Hainan in Wuhan, analyze the seed amino acid content (method is with embodiment 5) of transfer-gen plant and contrast, compared with the control, family SPD1 seed essential amino acids content when Wuhan and Hainan plantation has all improved more than 17%, total amino acid content has all improved more than 20%, proline wherein, alanine, lysine, glutamic acid, glycine, 7 seed amino acid content such as arginine and leucine all have raising largely compared with the control, just little (table 2) of planting than Wuhan at the increase rate of Hainan plantation.Investigate the transgenosis family and the Other Main Agronomic Characters (method is with embodiment 5) of contrast of Hainan plantation simultaneously, compared with the control, plant height no significant difference, and ripening rate, the real number of every fringe and single plant yield all be significantly increased (table 3).
Table 2 transgenosis family SPD1 with to impinge upon Wuhan and Hainan the plantation analysis of amino acids (mg/g)
Annotate: amplification %=(transgenosis family-contrast)/contrast * 100
Table 3 transgenosis family SPD1 and the performance of contrast Hainan plantation Other Main Agronomic Characters
*, * *, the significance of 0.05 and 0.01 level
Embodiment 7:
The previous generation select isozygoty individual plant SPD2 with contrast wild type Dongjing respectively in June, 2008 and December planting into family with Hainan in Wuhan, analyze the seed amino acid content (the same embodiment 5 of method) of transfer-gen plant and contrast, compared with the control, family SPD2 has all improved about 17% in Wuhan and Hainan plantation seed essential amino acids content, total amino acid content has all improved more than 20%, proline wherein, alanine, glutamic acid, lysine, 6 seed amino acid content such as glycine and valine all have raising largely compared with the control, just little (table 4) of planting than Wuhan at the increase rate of Hainan plantation.Investigate the transgenosis family and the Other Main Agronomic Characters (method is with embodiment 5) of contrast of Hainan plantation simultaneously, compared with the control, real number no significant difference of plant height and every fringe, and ripening rate and single plant yield all be significantly increased (table 5).
Table 4 transgenosis family SPD2 with to impinging upon the analysis of amino acids (mg/g) of Hainan plantation
Annotate: amplification %=(transgenosis family-contrast)/contrast * 100
Table 5 transgenosis family SPD2 and the performance of contrast Hainan plantation Other Main Agronomic Characters
*, * *, the significance of 0.05 and 0.01 level
Embodiment 8:
The previous generation select isozygoty individual plant SPD3 with contrast wild type Dongjing respectively in June, 2008 and December planting into family with Hainan in Wuhan, analyze the seed amino acid content (method is with embodiment 5) of transfer-gen plant and contrast, compared with the control, family SPD3 has improved 18% and 10% respectively in Wuhan and Hainan plantation seed essential amino acids content, total amino acid content has improved 25% and 17% respectively, proline wherein, alanine, glutamic acid, lysine, 6 seed amino acid content such as glycine and valine are all compared according to having largely and are improved, just little (table 6) of planting than Wuhan at the increase rate of Hainan plantation.Investigate the transgenosis family and the Other Main Agronomic Characters (method is with embodiment 5) of contrast of Hainan plantation simultaneously, compared with the control, plant height and ripening rate no significant difference, and real number of every fringe and single plant yield all be significantly increased (table 7).
Table 6 transgenosis family SPD3 with to impinging upon the analysis of amino acids (mg/g) of Hainan plantation
Annotate: amplification %=(transgenosis family-contrast)/contrast * 100
Table 7 transgenosis family SPD3 and the performance of contrast Hainan plantation Other Main Agronomic Characters
*, * *, the significance of 0.05 and 0.01 level
The medium of the used genetic transformation of the present invention and the method for preparation thereof are as described below:
(1) reagent and solution abbreviation
The abbreviation of the used plant hormone of medium is expressed as follows among the present invention:
6-BA (6-BenzylaminoPurine, 6-benzyladenine);
CN (Carbenicillin, carbenicillin);
KT (Kinetin, kinetin);
NAA (Napthalene acetic acid, methyl);
IAA (Indole-3-acetic acid, heteroauxin);
2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid);
AS (Acetosringone, acetosyringone);
CH (Casein Enzymatic Hydrolysate, caseinhydrolysate);
HN (Hygromycin B, hygromycin);
DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO));
N6max (N6 macroelement composition solution);
N6mix (N6 trace element composition solution);
MSmax (MS macroelement composition solution);
MSmix (MS trace element composition solution)
(2) main solution formula
1) N6 medium macroelement mother liquor (according to 10 times of concentrates (10X) preparation):
Mentioned reagent is dissolved one by one, be settled to 1000 milliliters with distilled water under the room temperature (20-25 ℃, below identical) then.
2) N6 medium trace element mother liquor (is prepared according to 100 times of concentrates (100X)
Mentioned reagent is at room temperature dissolved and be settled to 1000 milliliters with distilled water.
3) molysite (Fe
2EDTA) stock solution (according to the preparation of 100X concentrate)
With 3.73 gram b diammonium disodium edta (Na
2EDTA2H
2O) and 2.78 the gram FeSO
47H
2O dissolves respectively, mixes and is settled to 1000 milliliters with distilled water, bathes 2 hours to 70 ℃ of temperature, and 4 ℃ of preservations are standby.
4) vitamin stock solution (according to the preparation of 100X concentrate)
Adding distil water is settled to 1000 milliliters, and 4 ℃ of preservations are standby.
5) MS medium macroelement mother liquor (MSmax mother liquor) (according to the preparation of 10X concentrate)
Mentioned reagent is at room temperature dissolved, and be settled to 1000 milliliters with distilled water.
6) MS medium trace element mother liquor (MSmin mother liquor) (according to the preparation of 100X concentrate)
Mentioned reagent is at room temperature dissolved, and be settled to 1000 milliliters with distilled water.
7) 2, the preparation of 4-D stock solution (1 mg/ml):
Weigh 2,100 milligrams of 4-D dissolved 5 minutes with 1 milliliter of 1N potassium hydroxide, added then to be settled to 100 milliliters after 10 ml distilled waters dissolve fully, preserved under room temperature.
8) preparation of 6-BA stock solution (1 mg/ml):
Weigh 100 milligrams of 6-BA,, be settled to 100 milliliters, room temperature preservation after adding the dissolving fully of 10 ml distilled waters then with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes.
9) preparation of methyl (NAA) stock solution (1 mg/ml):
Weigh 100 milligrams of NAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 4 ℃ of preservations are standby.
10) preparation of heteroauxin (IAA) stock solution (1 mg/ml):
Weigh 100 milligrams of IAA, with 1 milliliter of 1N potassium hydroxide dissolving 5 minutes, be settled to 100 milliliters after adding the dissolving fully of 10 ml distilled waters then, 4 ℃ of preservations are standby.
11) preparation of glucose stock solution (0.5 grams per milliliter):
Weigh glucose 125 grams, be settled to 250 milliliters with dissolved in distilled water then, the back 4 ℃ of preservations of sterilizing are standby.
12) preparation of AS stock solution:
Weigh AS 0.392 gram, add 10 milliliters of dissolvings of DMSO, divide to be filled in 1.5 milliliters of centrifuge tubes, 4 ℃ of preservations are standby.
13) 1N potassium hydroxide stock solution
Weigh potassium hydroxide 5.6 grams, be settled to 100 milliliters with dissolved in distilled water, room temperature preservation is standby.
(3) be used for the culture medium prescription that rice genetic transforms
1) inducing culture
Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boil and be settled to 1000 milliliters, divide and install to 50 milliliters of triangular flasks (25 milliliters/bottle), sterilization according to a conventional method after sealing (for example sterilized 25 minutes down for 121 ℃, following medium sterilization method is identical with the sterilizing methods of basal culture medium).
2) subculture medium
Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000 milliliters, divides to install to 50 milliliters of triangular flasks (25 milliliters/bottle), seals, as stated above sterilization.
3) pre-culture medium
Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, seals, as stated above sterilization.
Use preceding heating for dissolving medium and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/ware) in the culture dish are poured in packing into.
4) be total to medium
Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.6, seals, as stated above sterilization.
Use preceding heating for dissolving medium and add 5 milliliters of glucose stock solutions and 250 microlitre AS stock solutions, (25 milliliters/every ware) in the culture dish are poured in packing into.
5) suspension culture base
Adding distil water to 100 milliliter is regulated pH value to 5.4, divides in the triangular flask that installs to two 100 milliliters, seals, as stated above sterilization.
Add 1 milliliter of aseptic glucose stock solution and 100 microlitre AS stock solutions before using.
6) select medium
Adding distil water to 250 milliliter is regulated pH value to 6.0, seals, as stated above sterilization.
Dissolving medium before using adds 250 microlitre HN (50 mg/ml) and (25 milliliters/ware) in the culture dish are poured in 400 microlitre CN (250 mg/ml) packing into.(annotate: selecting medium carbenicillin concentration for the first time is 400 mg/litre, and selecting medium carbenicillin concentration for the second time and later on is 250 mg/litre).
7) break up medium in advance
Adding distil water to 250 milliliter, 1N potassium hydroxide is regulated pH value to 5.9, seals, as stated above sterilization.
Dissolving medium before using, 250 microlitre HN (50 mg/ml), 250 microlitre CN (250 mg/ml), (25 milliliters/ware) in the culture dish are poured in packing into.
8) differential medium
Adding distil water to 900 milliliter, 1N potassium hydroxide is regulated pH value to 6.0.
Boil and be settled to 1000 milliliters, divide to install to 50 milliliters of triangular flasks (50 milliliters/bottle), seal, as stated above sterilization with distilled water.
9) root media
Adding distil water to 900 milliliter is regulated pH value to 5.8 with 1N potassium hydroxide.
Boil and be settled to 1000 milliliters, divide to install to (25 milliliters/pipe) in the pipe of taking root, seal, as stated above sterilization with distilled water.
Claims (1)
1. a method that improves rice grain amino acid quality the steps include:
A, one section cDNA fragment of dragging the silk-fibroin gene of from spider, separating, this cDNA fragment is by 905 base compositions, 264 amino acid of encoding, be rich in glycine and alanine, it is cut to be connected with rice grain selectivity glutelin GluB-1 promotor enzyme link on the expression vector pCAMBIA1300, make up recombinant vector;
B, utilize Agrobacterium tumefaciens mediated transgenic method that recombinant vector is imported in the paddy rice, obtain transfer-gen plant;
C, utilize polymerase chain reaction (PCR) that T0 is carried out positive detection for transfer-gen plant, detect the expression that positive transfer-gen plant changes fragment over to by Northern hybridization, identify the integration copy number of positive transfer-gen plant by Southern hybridization, what selection contained single copy changes fragment and normal solid transfer-gen plant, results individual plant selfing seed over to;
The seed kind of D, transfer-gen plant that step (C) is selected and remain becomes family T1 generation, continue to utilize polymerase chain reaction PCR that T1 is carried out positive detection for individual plant, utilize Northern hybridization to detect the expression that positive individual plant changes fragment over to, change the transfer-gen plant of fragment expression in conjunction with the field Phenotypic Selection, selfing is reserved seed for planting;
E, the target individual plant that continues to select and remain in the plantation step (D) become T2 for family, utilize polymerase chain reaction PCR that T2 is carried out positive detection for individual plant, positive individual plant is carried out economical character to be investigated and the seed amino acid analysis, therefrom select phenotype stable, amino acid quality and economical character have the individual plant that isozygotys of bigger improvement, and selfing is reserved seed for planting;
F, the individual plant of selecting and remain that isozygotys is further carried out Phenotypic Selection, cultivate new material.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192911A (en) * | 2017-12-31 | 2018-06-22 | 青岛袁策生物科技有限公司 | A kind of method of starch quality in improvement rice |
| CN108239654A (en) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | A kind of method for improving rice grain vitamin content |
| CN108239655A (en) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | The method for promoting rice grain starch quality |
| CN110951702A (en) * | 2019-12-21 | 2020-04-03 | 华中农业大学 | Rice DMNT and TMTT synthesis related protein OsCYP92C21, and coding gene and application thereof |
| CN111662926A (en) * | 2020-07-17 | 2020-09-15 | 南通大学 | Application of rice histone demethylase JMJ708 in rice breeding |
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| CN108165576A (en) * | 2017-12-31 | 2018-06-15 | 青岛袁米农业科技有限公司 | A kind of method for improveing rice seed oxidation resistance |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362520A (en) * | 2001-01-02 | 2002-08-07 | 成都天友发展有限公司 | Constitution process of blank expression system for transferring spider's dragline protein gene into Bombyx mori |
| CN1380418A (en) * | 2002-01-24 | 2002-11-20 | 中国科学院遗传与发育生物学研究所 | Cotton fibrocyte expression vector plasmid of spider silk gene |
| CN1450169A (en) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | Separation purification method for preparing sex-gene recombination spider dragline silk protein |
| CN1842601A (en) * | 2003-08-27 | 2006-10-04 | 奥夫莱夫塔埃克尼公司 | Enhancing accumulation of heterologous polypeptides in plant seeds through targeted suppression of endogenous storage proteins |
-
2011
- 2011-04-22 CN CN 201110109225 patent/CN102144568B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1362520A (en) * | 2001-01-02 | 2002-08-07 | 成都天友发展有限公司 | Constitution process of blank expression system for transferring spider's dragline protein gene into Bombyx mori |
| CN1380418A (en) * | 2002-01-24 | 2002-11-20 | 中国科学院遗传与发育生物学研究所 | Cotton fibrocyte expression vector plasmid of spider silk gene |
| CN1450169A (en) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | Separation purification method for preparing sex-gene recombination spider dragline silk protein |
| CN1842601A (en) * | 2003-08-27 | 2006-10-04 | 奥夫莱夫塔埃克尼公司 | Enhancing accumulation of heterologous polypeptides in plant seeds through targeted suppression of endogenous storage proteins |
Non-Patent Citations (2)
| Title |
|---|
| 《Plant Biotechnology Journal》 20041231 Rima Menassa等 "Spider dragline silk proteins in transgenic tobacco leaves:accumulation and field production" 第431-438 1 , 第2期 * |
| RIMA MENASSA等: ""Spider dragline silk proteins in transgenic tobacco leaves:accumulation and field production"", 《PLANT BIOTECHNOLOGY JOURNAL》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108192911A (en) * | 2017-12-31 | 2018-06-22 | 青岛袁策生物科技有限公司 | A kind of method of starch quality in improvement rice |
| CN108239654A (en) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | A kind of method for improving rice grain vitamin content |
| CN108239655A (en) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | The method for promoting rice grain starch quality |
| CN110951702A (en) * | 2019-12-21 | 2020-04-03 | 华中农业大学 | Rice DMNT and TMTT synthesis related protein OsCYP92C21, and coding gene and application thereof |
| CN110951702B (en) * | 2019-12-21 | 2021-07-16 | 华中农业大学 | Rice DMNT and TMTT synthesis related protein OsCYP92C21, and coding gene and application thereof |
| CN111662926A (en) * | 2020-07-17 | 2020-09-15 | 南通大学 | Application of rice histone demethylase JMJ708 in rice breeding |
| CN111662926B (en) * | 2020-07-17 | 2022-02-15 | 南通大学 | Application of rice histone demethylase JMJ708 in rice breeding |
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