CN1661020A - Method for cultivating transgenic paddy rice with fragile haulm - Google Patents
Method for cultivating transgenic paddy rice with fragile haulm Download PDFInfo
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Abstract
A method for culturing the stem-crisp rice includes such steps as introducing the gene used to code the amino acid which can not by synthesized in single-stomach animal body to the rice, and directly and selectively culturing or hybridizing to obtain target rice. Its crisp stem is suitable for preparing feed of animal and fowls.
Description
Technical field
The present invention relates to the method for cultivation of the crisp stem paddy rice of the method for cultivation of transgenic paddy rice, particularly transgenosis.
Background technology
China is the big country of Rice Production, year sown area 3176.5 ten thousand hm
2(not comprising Taiwan), account for 21.5% of the total sown area of world paddy rice, and the paddy gross output reaches 2.007 hundred million tons, accounts for world's gross output 36.3%, ranks world's first.Yet in results paddy, also having gathered in the crops straw, is 0.5 rough estimate with harvest index, and the straw of annual harvesting reaches 200,000,000 tons.Before mid-term in 20th century, straw mainly digests on the spot by go back measures such as field after burning bavin, filling out swinery.Since the eighties, along with the raising of life of farmers level, no longer big with volume, that thermo-efficiency is low, potential safety hazard the is high straw in vast rural area is worked as firewood; Meanwhile, the livestock industry in rural area, particularly pig industry are disperseed to raise the scale management of moving towards intensification by single household, also no longer fill out the hurdle with straw, and the content of cellulose height of straw, decomposition rate is slow, is not suitable for direct returning to farmland.The paper quality of straw papermaking is not good enough in addition.Because straw has not had outlet, the peasant has adopted the way that the edge of a field burns.Its result has not only discharged a large amount of greenhouse gases, causes potential harm, and dense smoke curls up, and forces the matters of aggravation of some socked-ins to take place every year.Although governments at all levels three make five early, effective solution for want of eventually, the peasant still burns grass, and the airport can only continue to close, and causes that serious environmental is polluted, great financial loss and bad social influence.
China is herding big country, but because factors such as densely populated and natural condition, the most of geographic livestock feeds in China south rely on the other places to call in.Consider that from economics point no matter feed still is that foreign country is called in from other provinces, has all increased cost, is not good plan, more is not long-range side's meter.Though the maximum byproduct straw comprehensive nutrition of Rice Production, often to some extent with cell walls in materials such as xylogen, Mierocrystalline cellulose, hemicellulose, pectin combine closely, be not easy to be decomposed and utilize, and committed to the flames by animal.Like this, on the one hand crop straw burning is transferred in feed on the one hand, has formed in the agriculture production one and has been difficult to unfolded vicious circle.
Developing extraordinary straw feed is to increase straw added value, protection environment and reduce livestock industry production revolutionary approach.The problem that exists in China's Rice Production can't be looked for answer from developed country because no matter be the U.S. or Japan, plantation all be the single harvest rice, crushing straw does not also influence the production in the coming year in the field during mechanical harvest paddy.China arable land is few, soil multiple crop index height, and mechanization degree is also very low, present stage concerning most of areas, also field solution straw problem pass is unrealistic to rely on crushing straw during results paddy, then because being difficult to abundant corruption macerates slaking, become the rice disease and pest source influences grain-production to the straw direct returning to farmland.Adopt traditional chemistry, physical method to handle straw (for example using the urea ammoniated stalk) and improve its digestion degradation rate, effect is not satisfactory.Some experts once proposed the imagination with microorganism enzymolysis stalk, but enzymolysis time is long, is difficult to practicability.
China Paddy Rice Inst has obtained the low crisp stem rice of rice mutant of crude fiber content in 1996 by radioinduction, and its cauline leaf becomes quite tender and crisp when keeping the lodging resistance shape.Discover that the crude fiber content of this mutant has descended about 30% than original parent, and crude protein, crude fat content improve 60% and 1 times respectively.Now clearly this this proterties is to be controlled by recessive single-gene (fp1-1).Because crude fiber content is low, improved the digestibility of straw feed.United in the nutrient substance agricultural symposial of holding in April, 1998 by rural development research department of Development Research Center of the State Council and Zhejiang Province Agriculture Office, the participant expert teaches and consistently thinks, crisp stem rice seed high yield proterties is constant, cauline leaf becomes quite tender and crisp when keeping the lodging resistance shape, crude fiber content significantly reduces, suitable grain is raised dual-purpose, for improving domestic animal the digestibility of stalk is explored a new approach.
If on the basis of existing crisp stem paddy rice, further improve its stalk and grain protein and indispensable amino acid (can not synthesize in people and the monogastric animal body, need directly to take in from the external world) content by certain approach to keep the amino acid that its normal growth is grown, be highly significant, of great value.
Summary of the invention
The present invention will solve the problem that how to improve crisp stem rice straw, grain protein and essential amino acids content, thereby the method for cultivation of the crisp stem paddy rice of a kind of transgenosis of the present invention is provided, and present method can make existing crisp stem rice straw and/or seed indispensable amino acid protein content significantly improve.
For addressing the above problem, its special character of the technical solution used in the present invention is coding to be rich in the monogastric animal body gene that can not synthesize, need directly to take in from the external world to keep the amino acid protein that its normal growth grows to import crisp stem paddy rice, by seed selection, obtain the crisp stem paddy rice that stalk protein and described aminoacids content are improved.
Amino acid of the present invention can be Methionin, methionine(Met), leucine, Isoleucine, phenylalanine, Threonine, tryptophane and Xie Ansuan.
The method that described importing is adopted can be Agrobacterium-mediated Transformation, particle gun bombardment, protoplastis is cultivated or pollen tube imports.
The gene that the present invention imported can be unit price gene, bivalent gene or multivalent genetic, and can be two or more unit price gene cotransformations.
Seed selection of the present invention can be that transgenic plant are directly carried out seed selection or are the hybridization transformation that the parent carries out with transgenic plant.
The present invention is rich in coding in the monogastric animal body gene that can not synthesize, need directly to take in from the external world to keep the amino acid protein that its normal growth grows and is imported crisp stem paddy rice, and it is feasible cultivating the crisp stem paddy rice that stalk protein and described aminoacids content be improved.The Ti carrier that for example will contain high-lysine protein gene SB401 transforms crisp stem japonica rice and long-grained nonglutinous rice, this gene can stably express in crisp stem rice paddy seed that transforms and blade, the content of crude protein is increased to 8.4% from 7.3% in the japonica rice seed, lysine content is increased to 0.33% from 0.25%, and amplification reaches respectively about 15% and 30%.The content of crude protein is increased to 2.9% from 2.5% in blade, and lysine content is increased to 0.085% from 0.07%, and amplification is respectively 15% and 25% (area, Hangzhou data, other areas can have some variations along with the variation of ecological condition).The present invention is for rice straw becomes high-quality, agreeable to the taste feed is created a new road, its enforcement, can reduce the livestock industry cost greatly, and can increase farmers' income, and can eliminate because of contaminate environment that a large amount of crop straw burnings bring, influence traffic, waste drawbacks such as resource, provide good condition for guaranteeing agricultural sustainable development.Owing in countries in the world grain and the animal husbandry development, generally lack new distinctive nutrition type feed rice varieties, also lack the grain-saving type production technology that adapts with it, so enforcement of the present invention is significant to acquiring an advantage in the international market.
Embodiment
Embodiment 1
By agrobacterium-mediated transformation high-lysine gene SB401 is imported crisp stem japonica rice (tender rice No. 1) and crisp stem long-grained nonglutinous rice nuclear gene group is an example.Present embodiment is not got rid of other test method and measure, comprises test materials etc.
One, the structure of expression vector
1, uses BamHI, goal gene SB401 under the HindIII double digestion.
2, use EcoRI on the basis of initial carrier pCAMBIA1301, two the cutting of KpnI introduced the 35s promotor; Introduce the NOS terminator with PstI and HindIII.
3, SB401 is introduced the binary vector that double digestion is transformed.
Two, the preparation of Agrobacterium competent cell
The EHA105 that gets-70 ℃ of preservations rules 28 ℃ of cultivations in containing on 50 μ g/ml Rifampins, the kantlex flat board.Picking list colony inoculation in 5ml YM liquid nutrient medium, 28 ℃ of shaking culture 12-16hr of 220rpm.Get 2ml bacterium liquid and transfer in 100ml YM liquid nutrient medium, 28 ℃ of 220rpm shaking culture are to OD
600=0.5.Change aseptic centrifuge tube over to, the centrifugal 5min of 5000rpm removes supernatant liquor.The CaCl that adds the 0.1M of 10ml precooling
2Solution, suspension cell is placed 20min on ice gently.4 ℃ of centrifugal 5min of 5000rpm remove supernatant.The CaCl that adds the 0.1M that contains 15% glycerine of 4ml precooling
2Solution suspends gently.Agrobacterium suspension is sub-packed in the aseptic Eppendorf pipe, and every pipe 200 μ l are frozen in-70 ℃.
Three, binary vector transforms Agrobacterium EHA105
The plasmid DNA of getting about 1 μ g joins in the 200ml EHA105 competent cell, and behind the mixing, ice bath 30min places 10min for-70 ℃.At 37 ℃ of water-bath 5min or 42 ℃ of water-bath 1min, then ice bath 2min adds 28 ℃ of 800mlYM liquid nutrient mediums again, and 175rpm is coated on the YM flat board that contains 50 μ g/ml kantlex after shaking training 3hr.Cultivate for 28 ℃ and form single bacterium colony.Also above-mentioned binary vector plasmid DNA can be imported the EHA105 competent cell with electrization, cultivate for 28 ℃ and form single bacterium colony.The single colony inoculation of the Agrobacterium that picking transforms is in the YM liquid nutrient medium that contains 50 μ g/ml kantlex, and 28 ℃, 220rpm shakes training 16hr, directly carries out PCR with bacterium liquid and identifies.
Four, Agrobacterium tumefaciens mediated rice conversion
About 12-15 days crisp stem paddy rice immature seed placed on the solid inducing culture (NB) through extruding the paddy rice rataria behind the surface sterilization after preparation was bloomed, and secretly cultivated evoked callus.Peel callus after about 5-7 days, change on the freshly prepared subculture medium (NB), succeeding transfer culture is about 5 days under the same conditions; Perhaps prepare the paddy rice mature embryo, evoked callus under the similarity condition, select the yellowish callus in succeeding transfer culture 5-7 days, color and luster and cultivated altogether 15-20 minute, change solid over to altogether on the substratum with an amount of agrobacterium suspension (OD 0.3-0.5), 26 ℃ dark culturing 2-3 days.The callus that is total to after cultivating is placed on the selection substratum that contains the 50mg/l Totomycin, and 26 ℃ of dark cultivations 14 days forward to and continue on the freshly prepared selection substratum to screen 14 days to growing resistant calli.From the resistant calli that after the two-wheeled screening, grows, the resistant calli of selecting the milk yellow densification goes on the division culture medium that contains the 50mg/L Totomycin, dark earlier the cultivation 3 days, and the illumination condition that went to then 12-15 hour/day is cultivated down, through about 15-25 days, grow green point.Further differentiate seedling after 30-40 days.When the seedling of resistant calli differentiation or bud grow to about 2cm, seedling is moved on on the root media, cultivate about two weeks, in the greenhouse, transplant and bury.
Wherein:
YM (liquid) substratum KH
2PO
40.5g/l+ N.F,USP MANNITOL 10g/l+ glutamine 2g/l+NaCl0.2g/l+MgSO
40.2g/l+ yeast extract 0.3g/l, pH 7.0.
Inducing culture MS (long-grained nonglutinous rice)/N6 (japonica rice) is a large amount of+and MS-Fe salt+B5 trace+B5 is organic+and 2,4-D 1.5-4.5mg/l+ proline 3 00-1000mg/l+ glutamine 300-1000mg/l+ caseinhydrolysate 500-1000mg/l+ maltose/sucrose 20-30g/l+Gelrite 2.5-3.0mg/l, pH 5.8.
Subculture medium isogeneous induction substratum, but 2,4-D changes 2.0-3.0mg/l into.
Cultivate altogether (solid) substratum MS (long-grained nonglutinous rice)/N6 (japonica rice) a large amount of+MS-Fe salt+B5 trace+B5 is organic+2,4-D 2.0-3.0mg/l+ caseinhydrolysate 500-1000mg/l+ inositol 500-2000mg/l+ Syringylethanone 100 μ M+ maltose/sucrose 20-30g/l+Gelrite 2.5-3.0mg/l, pH 5.5.
(liquid culture medium does not altogether have Gelrite).
Select substratum MS (long-grained nonglutinous rice)/N6 (japonica rice) a large amount of+MS-Fe salt+B5 trace+B5 is organic+2,4-D 2.0-3.0mg/l+ proline 3 00-500mg/l+ glutamine 300-1000mg/l+ caseinhydrolysate 500-1000mg/l+ maltose/sucrose 20-30g/l+ cephamycin 250mg/l+ Totomycin 50mg/l+Gelrite 2.5-3.0mg/l, pH 5.8.
Division culture medium MS (long-grained nonglutinous rice)/N6 (japonica rice) is a large amount of+and MS-Fe salt+B5 trace+B5 is organic+NAA 0.1-0.5mg/l+KT 4-8mg/l+ proline 3 00-500mg/l+ glutamine 300-1000mg/l+ caseinhydrolysate 500-1000mg/l+ maltose/sucrose 20-30g/l+ cephamycin 250mg/l+ Totomycin 50mg/l+Gelrite 2.5-3.0mg/l, pH 5.8.
Root media 1/2 MS/N6 is a large amount of+MS-Fe salt+B5 trace+sucrose 10-20g/l+ agar 0.8%, and pH 5.8.
Five, the detection of transfer-gen plant and seed selection
After transfer-gen plant (T0) survived, sampling was carried out PCR (polymerase chainreaction, polymerase chain reaction) and is detected, and kept PCR male plant, Routine Management.It is the basic detection technique that any Molecular Biology Lab can both carry out that PCR detects.The purification of transgenic line and seed selection are carried out simultaneously.
Scheme 1, the tassel of getting transgenic positive plant (T0) carry out anther culture, and anther cultural technology is used widely, and common laboratory and technician can operate, and concrete grammar is seen: rice in China science, 1993,7:227-231; 1996,10:37-42 or Plant Breeding, 1996,115:295-300.The anther culture regeneration plant is by the individual plant seed of gathering.Every strain system gets 50-100 grain seed and ties up in the Totomycin aqueous solution that contains 50mg/l by strain and germinate the examination percentage of germination.Select the chitting piece sowing of the similar strain system of percentage of germination and contrast.Insert for single or many, Routine Management is contrast with the original parent.Select the good strain system of crisp stem, economical character, every strain ties up to gets 10 strains before the heading and falls the blade (2 of every strains) of 2 leaves, and biased sample is measured indispensable amino acid and Protein content with automatic analyzer for amino acids.With the crisp stem paddy rice of primary is contrast.Target amino acid (as Methionin) and total protein content comparison are the seed selection program that enters new variety according to the strain that increases more than 10%.
Scheme 2, transgenic positive plant (T0) are by the individual plant seed of gathering.Every strain is to get 50-100 grain seed (T1) to germinate in containing the Totomycin aqueous solution of 50mg/l, and the examination percentage of germination is contrast with the original parent.Select percentage of germination to be about the chitting piece sowing of the strain system of contrast 3/4.Insert Routine Management for single or many.Select the good strain system of crisp stem, economical character, every strain ties up to fringe phase beginning and gets 10 strains and fall the blade (2 of every strains) of 2 leaves, and biased sample is measured indispensable amino acid and Protein content with automatic analyzer for amino acids.With the crisp stem paddy rice of primary is contrast.The comparison of target amino acid (as Methionin) and total protein content is by the individual plant seed (T2) of gathering according to the roguing of going into that increases by 10% or more.Seed germinates in containing the Totomycin aqueous solution of 50mg/l, and the normal strain of percentage of germination system enters the seed selection program of new variety.
The japonica rice plant of scheme 3, transgenic positive and good Java type recovery system (kind or strain system) hybridization, it is that the male individual plant carries out anther culture that the F1 plant detects through PCR, anther cultural technology is used widely, common laboratory and technician can operate, concrete grammar is seen: the rice in China science, 1993,7:227-231; 1996,10:37-42 or Plant Breeding, 1996,115:295-300.The anther culture regeneration plant is by the individual plant seed of gathering.Every strain system gets 50-100 grain seed and ties up in the Totomycin aqueous solution that contains 50mg/l by strain and germinate the examination percentage of germination.Select the chitting piece sowing of the similar strain system of percentage of germination and contrast.Single this is inserted, and Routine Management is contrast with the original parent.Select crisp stem, economical character is good and the strain of inclined to one side Java type system, every strain ties up to gets 10 strains before the heading and falls the blade (2 of every strains) of 2 leaves, biased sample is measured indispensable amino acid and Protein content with automatic analyzer for amino acids.With Java type parental rice is contrast.Target amino acid (as Methionin) and total protein content comparison are measured restorability according to the roguing system of going into that increases more than 10%, and surveying extensive is the basic skills that all breeding of hybridized rice men can both carry out.Select the similar crisp stem of restorability and Java type parental rice, high-lysine strain system as recovering the seed selection program that system enters the new combination of crisp stem hybrid rice.
The long-grained nonglutinous rice plant of transgenic positive hybridizes with good indica cms line and corresponding maintenance line respectively.Detecting through PCR with the F1 plant of sterile line hybridization is that the long-grained nonglutinous rice plant of the sterile individual plant of male and transgenic positive carries out successive and backcrosses and detect selection economical character and similar, the crisp stem of original sterile line, the sterile individual plant of PCR male.
In the F1 plant of maintenance line hybridization, selecting PCR to detect is that the male plant carries out anther culture, and anther cultural technology is used widely, and common laboratory and technician can operate, and concrete grammar is seen: rice in China science, 1993,7:227-231; 1996,10:37-42 or Plant Breeding, 1996,115:295-300.The anther culture regeneration plant is by the individual plant seed of gathering.Every strain system gets 50-100 grain seed and ties up in the Totomycin aqueous solution that contains 50mg/l by strain and germinate the examination percentage of germination.Select the chitting piece sowing of the similar strain system of percentage of germination and contrast.Single this is inserted, and Routine Management is contrast with the original parent.Select the similar strain system of crisp stem, economical character and original maintenance line, every strain is got the blade (2 of every strains) of 2 leaves of 10 strains before tying up to heading, and biased sample is measured indispensable amino acid and Protein content with automatic analyzer for amino acids.With original maintenance is contrast.It is to measure hold facility that the roguing of going into that increases more than 10% is shone in target amino acid (as Methionin) and total protein content comparison, and surveying the guarantor is the basic skills that all breeding of hybridized rice men can both carry out.Similar crisp stem, the high-lysine paddy rice of hold facility and original maintenance line parent enters the new seed selection program that makes up of crisp stem hybrid rice as crisp stem maintenance line.
Crisp stem maintenance line is hybridized with crisp stem, the sterile individual plant of PCR male respectively, selects economical character and consistent, the crisp stem of crisp stem maintenance line, PCR male high-lysine sterile line.
The Java type of crisp stem, high-lysine indica cms line and crisp stem, high-lysine recovers system's hybridization acquisition Xian pawl and hands over crisp stem, high-lysine hybrid rice.
Embodiment 2
By the particle gun blast technique high-lysine protein gene SB401 and homocysteine protein gene HcpB are imported No. 1 cell nucleus gene group of tender rice simultaneously is example.Present embodiment is not got rid of other test method and measure, comprises test materials etc.
One, the structure of expression vector
What be different from example 1 is to make up goal gene HcpB and SB401 respectively, and all the other are identical with example 1.
Two, the extraction of expression vector transformed into escherichia coli and plasmid
1, the preparation of competent cell
Electric shock transforms the preparation with competent cell: from LB substratum (Tryptone 10g/l, Yeast extract 5g/l, NaCl 5g/l, agar 20g/l, pH 7.0) the single bacterium colony of the new activatory intestinal bacteria of picking on the flat board, be inoculated in the triangular flask of the 250ml that 50ml LB liquid nutrient medium is housed, 37 ℃ of shaking culture 12-16hr are until the logarithmic growth later stage.From this bacteria suspension, take out 2.0ml, change in the bacteria culture bottle of the 500ml that 200ml LB liquid nutrient medium is housed and (add) by 1: 100 volume ratio, 37 ℃ the more about 3hr to OD600 of shaking culture reach about 0.5.Change nutrient solution over to centrifuge tube (the flat centrifuge tube of 250ml), place 10min on ice, 4 ℃ then, 4, the centrifugal 15min of 000g (swing bucket rotor is best) removes clean supernatant liquor as far as possible.10% the glycerine that adds 200ml (with the original bacteria liquid equal-volume) sterilization precooling, put upside down mixing gently after, 4 ℃, 4, the centrifugal 15min of 000g.Outwell supernatant, will precipitate with 10% glycerine of 100ml (original bacteria liquid volume 1/2) sterilization precooling and wash one time.4 ℃, 4, the centrifugal 15min of 000g, to precipitate with 10% glycerine of 50ml (original bacteria liquid volume 1/4) sterilization precooling and wash again one time, outwell supernatant, add 10% glycerine of 10ml left and right sides precooling, divide rapidly behind the mixing and install in the EP pipe, every pipe 40 μ l put into the liquid nitrogen quick-frozen rapidly, be put at last-80 ℃ standby.
Also can prepare and be used for the competent cell that thermal shock transforms: activation of bacterium and bacterium liquid are cultivated the same, and the cultured bacterium liquid of 200ml is placed 10min on ice, then in 4 ℃, and 4, the centrifugal 5min of 000g adds ice-cold CaCl2 solution (the 0.1M CaCl of 40ml
2, 0.01M Tris, pH 7.0,10mM DTT) precipitation that suspends gently, ice bath 20min behind the mixing.4 ℃, 4, the centrifugal 5min of 000g adds the ice-cold 0.1M CaCl of 4ml
2The solution precipitation that suspends gently, ice bath 2-5hr behind the mixing.Add 15% (v/v) of sterile glycerol, ice bath 12-18hr behind the mixing to cumulative volume.Took out in second day to divide rapidly behind the mixing and install in the EP pipe, every pipe 100 μ l put into the liquid nitrogen quick-frozen, be put at last-80 ℃ standby.
2, transform
Connect product and be used for will passing through ethanol sedimentation before electric shock transforms, 70% ethanol cleans, and is dissolved in the aseptic ddH of 4 μ l after the vacuum-drying
2O.It is as follows that electric shock transforms concrete steps: open the electric shock conversion instrument, parameter is set to voltage 2.5KV, electric capacity 25 μ F, resistance 200 Ω.From-80 ℃ of refrigerators, take out competent cell, place on ice, treat that it melts after, add 2 μ l and connect product, inhale gently fast and tell mixing, ice bath 1min.The electric shock that changes last mixture over to-20 ℃ of precoolings transforms in the cup (gently get rid of after adding, bacterium liquid is paved with glass at the bottom of), cleans electric shock with the water that thieving paper is outer with wall of cup.Take out and transform cup, add 1ml SOC substratum (LB+10mMMgCl immediately
2+ 10mM MgSO
4The glucose of+20mM filtration sterilization) and mixing, said mixture is changed in the centrifuge tube of a sterilization, 37 ℃, 200rpm shaking culture 1hr gets 200 μ l coated plates.
Connect product and can be directly used in thermal shock and transform, take out competent cells from-80 ℃, place on ice, treat that it melts after, will connect product and all add wherein, mixing, ice bath 30min are told in suction gently fast.In 42 ℃ of water-baths, place 1min, take out about ice bath 2min.Add 500 μ l SOC substratum, 37 ℃, 200rpm shaking culture 1hr.Get 200 μ l coated plates.
3, the screening of recombinant clone and evaluation
In containing corresponding antibiotic LB liquid nutrient medium, 37 ℃ of shaking culture 16hr are used for plasmid and extract with single colony inoculation of growing after the aseptic toothpick picking conversion.By digested plasmid, electrophoresis detection, the reorganization transformant is identified then.
4, the extraction of plasmid
Change the bacterium of incubated overnight over to 1.5ml EP pipe, 13, the centrifugal 30s of 000rpm collects thalline; Outwell most of supernatant, stay about 50-100 μ l (comprising sedimentary thalline); Add 300 μ lTENS (the TE damping fluid that contains 0.1N NaOH and 0.5%SDS), vortex is mixed to be clamminess until mixture; Add 150 μ l 3M sodium-acetates (pH 5.2), mixing; 13, the centrifugal 10min of 000rpm gets supernatant, adds 900 μ l dehydrated alcohols, mixing; Place-20 ℃ of 30min; 13, the centrifugal 5min of 000rpm washes twice with 70% ethanol with precipitation, is dissolved in an amount of TE damping fluid after the vacuum-drying.
Three, the particle gun bombardment imports foreign gene
Take away the crisp stem paddy rice immature seed of spending back about 12-15 days, place on the solid inducing culture (NB), secretly cultivate evoked callus through extruding the paddy rice rataria after the surface sterilization.Peel callus after about 5-7 days, change on the freshly prepared subculture medium (NB), succeeding transfer culture is about 5 days under the same conditions.Callus is transferred to height ooze on the substratum, callus closely comes the central authorities of culture dish, overnight incubation.
The bronze that takes by weighing 60mg diameter l μ m adds the 1ml raw spirit in the centrifuge tube of 1.5ml, vibration 1min, and centrifugal 10 seconds of 10000rpm abandons supernatant, repeats once, bronze is suspended in the 1ml sterilized water-20 ℃ of preservations.Draw 50 μ l bronze suspension, add 20 μ l 0.1M spermidines, 50 μ l 2.5M CaCl
2, 2.5 μ g HcpB plasmid DNA and 2.5 μ g SB401 plasmids.Vibration 3min, centrifugal 20 seconds of 10000rpm abandons supernatant, and raw spirit rinsing 2 times adds 60 μ l raw spirit constant volumes.Get 20 μ l bronze suspensions and place on the split film of 1100psi type, dry back BiolisticTM PDS-1000 particle gun, vacuum tightness 28, pressure 1100psi bombards callus.Every ware callus bombardment 2 times.
Material after the bombardment changes freshly prepared subculture medium (NB) over to and goes up the dark cultivation that recovers.Change over to after 2-7 days on the selection substratum that contains the 50mg/l Totomycin and cultivate kanamycin-resistant callus tissue, 26 ℃ of dark cultivations 14 days forward to and continue on the freshly prepared selection substratum to screen 14 days to growing resistant calli.From the resistant calli that after the two-wheeled screening, grows, the resistant calli of selecting the milk yellow densification goes on the division culture medium that contains the 50mg/L Totomycin, dark earlier the cultivation 3 days, and the illumination condition that went to then 12-15 hour/day is cultivated down, through about 15-25 days, grow green point.Further differentiate seedling after 30-40 days.When the seedling of resistant calli differentiation or bud grow to about 2cm, seedling is moved on on the root media, cultivate about two weeks, in the greenhouse, transplant and bury.
Wherein:
Inducing culture, subculture medium, selection substratum, division culture medium and root culture agent are identical with example 1, and it is inducing culture+2 that height oozes substratum, 4-D 1.5-4.5mg/l+0.5mol/l N.F,USP MANNITOL, and pH 5.8.
The detection of transfer-gen plant and seed selection can be with example 1 identical, selecting the individual plant or the strain that detect HcpB and SB401 simultaneously after the Molecular Detection is the seed selection of offerings kind.
Embodiment 3
Importing No. 1 cell nucleus gene group of tender rice with the bivalent gene that will contain high-lysine protein gene SB401 and high tryptophan protein gene WRB by the pollen tube introductory technique is example.Present embodiment is not got rid of other test method and measure, comprises test materials etc.
One, the structure of bivalent gene expression vector
1, on the basis of initial carrier pCAMBIA1301,, stays original promotor of Gus gene and terminator with NcoI and BstEII double digestion excision Gus gene;
2, design primer according to high tryptophan protein gene WRB gene order, and ' introduce the NcoI restriction enzyme site in the end primer, ' introduce the BstEII restriction enzyme sites in the end primer 35;
3, with PCR method amplification high tryptophan protein gene WRB, introduce the binary vector that step 1 double digestion is transformed;
4, use EcoRI, KpnI is two to be cut the carrier that step 3 finishes and introduces the 35s promotor, cuts the carrier introducing NOS terminator that step 3 is finished with PstI and HindIII are two;
5, use BamHI, HindIII downcuts the high acid albumin gene SB401 of relying from the original plasmid double digestion that contains the high acid albumin gene SB401 of relying;
6, use BamHI, the two carrier that step 4 is finished, the high acid albumin gene SB401 that rely of introducing of cutting of HindIII.
Two, the extraction of expression vector transformed into escherichia coli and plasmid
1, the preparation of competent cell
Identical with example 2.
2, transform
Identical with example 2.
3, the screening of recombinant clone and evaluation
Identical with example 2.
4, the extraction of plasmid
Identical with example 2.
Three, pollen tube imports foreign gene
Originally operate in paddy rice (tender rice No. 1) heading and carry out when blooming, need to observe tender rice No. 1 in advance and take time and flowering peak times in the beginning of locality.Selecting the same day before paddy rice begins to spend in the morning will have the tassel of about 20 little tassel blossoms, cut off all small ears of having bloomed.In the peak period of paddy rice flowering, cut off the small ear of all not blooming.After 1 hour, each small ear cuts off about 2/3 inner glume, and column cap and about 1/3 style keep coetonium.With liquid-transfering gun 10 μ l plasmid DNA are dropped on the otch of style, the concentration of plasmid DNA is 50-300 μ g/ml.Plasmid DNA with same volume and concentration after 1 hour is dripped once again, then bagging and listing.The tassel threshing of gathering after 25-30 days, preserve the dry back of seed (T0).The rice plant that is grown by the T0 seed is called T0 for plant.
The detection of transfer-gen plant and seed selection can be with example 1 identical.Selecting the individual plant or the strain that detect WRB and SB401 simultaneously after the Molecular Detection is the seed selection of offerings kind.
Claims (5)
1, the method for cultivation of the crisp stem paddy rice of a kind of transgenosis, it is characterized in that coding is rich in the monogastric animal body gene that can not synthesize, need directly to take in and import crisp stem paddy rice to keep the amino acid protein that its normal growth grows from the external world, by seed selection, obtain the crisp stem paddy rice that stalk protein and described aminoacids content are improved.
2, the method for claim 1 is characterized in that described amino acid is Methionin, methionine(Met), leucine, Isoleucine, phenylalanine, Threonine, tryptophane and Xie Ansuan.
3, method as claimed in claim 1 or 2 is characterized in that Agrobacterium-mediated Transformation, particle gun bombardment are adopted in described importing, protoplastis is cultivated or the method for pollen tube importing.
4, method as claimed in claim 3 is characterized in that the gene that is imported is unit price gene, bivalent gene or multivalent genetic.
5, method as claimed in claim 4 is characterized in that described seed selection is that transgenic plant are directly carried out seed selection or are the hybridization transformation that the parent carries out with transgenic plant.
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| CN2004100075574A CN1661020A (en) | 2004-02-26 | 2004-02-26 | Method for cultivating transgenic paddy rice with fragile haulm |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103229713A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Selective breeding method for high-nutrition rice varieties |
| CN106106157A (en) * | 2016-06-29 | 2016-11-16 | 无锡南理工科技发展有限公司 | A kind of breeding method of crisp stem Fructus Hordei Vulgaris |
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2004
- 2004-02-26 CN CN2004100075574A patent/CN1661020A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103229713A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Selective breeding method for high-nutrition rice varieties |
| CN106106157A (en) * | 2016-06-29 | 2016-11-16 | 无锡南理工科技发展有限公司 | A kind of breeding method of crisp stem Fructus Hordei Vulgaris |
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