CN102321662A - Method for transforming stem tips of plants and special tool thereof - Google Patents
Method for transforming stem tips of plants and special tool thereof Download PDFInfo
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Abstract
The invention discloses a method for transforming stem tips of plants and a special tool thereof. The method for cultivating a transgenic plant comprises the following steps of: 1) puncturing a growing point of a stem tip of a target plant by using a metal brush to obtain a treated target plant; 2) impregnating the treated target plant obtained in step 1) by using a bacterial liquid containing target deoxyribonucleic acid (DNA) to obtain an impregnated seedling; and 3) sequentially coculturing and continuously culturing the impregnated seedling obtained in 2) to obtain the transgenic plant containing the target DNA. Through the experiment, a set of efficient and quick plant transformation system is established by taking meristems of stem tips of seedlings of a monocotyledon, namely wheat and a dicotyledon, namely Salicornia europaea as transformation receptors of Agrobacterium tumefaciens respectively and adopting measures such as retrofit of stem tip damage tools, research on the seedling ages of the seedlings, vacuum-assisted infiltration treatment, application of infiltration promoters and the like, so that the transformation efficiency of the stem tips is improved.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of plant stem apex method for transformation and special tool thereof.
Background technology
Plant genetic transformation method is exactly through specific mode foreign gene to be imported in the recipient cell, makes it to take place directed, permanent heritable variation.At present, people have set up multiple conversion system.As far as most of genetic transformation systems, genetic transforming method is the key link of genetic transformation system, is determining the success or failure and the efficient that transform.The method for transformation that is used for plant has many: agrobacterium-mediated transformation, particle bombardment, electric shocking method, PEG method etc.Agrobacterium-mediated transformation and particle bombardment have become the main method that is used for genetic transformation at present.
Think in the past limited by the Agrobacterium host range, agriculture bacillus mediated genetic transformation only limits to dicotyledons.The successful plant monocotyledons of the employing agrobacterium-mediated transformation of report conversion the earliest is an officinalis, in important crops such as paddy rice, corn, barley, sugarcane, has obtained agriculture bacillus mediated transfer-gen plant in succession afterwards, and competent molecule and hereditary evidence are arranged.Though most of monocotyledonss are not the natural hosts of Agrobacterium,, can realize also that through adding external source semiochemicals such as Syringylethanone (AS) etc. Agrobacterium is to monocotyledonous conversion along with deep understanding to Agrobacterium-mediated Transformation vegetable cell principle.Because Agrobacterium-mediated Transformation utilizes natural integration system to realize the transfer of DNA, this method has that the transgenic copy number is low, inheritance stability, advantage such as with low cost make Agrobacterium become the prefered method of most of genetic transformations.
The selection of plant acceptor is another important factor of genetic transformation system.At present the plant acceptor of Agrobacterium-mediated Transformation mainly comprises explants such as rataria and embryo callus thereof, leaf dish, cotyledon, cotyledonary node, hypocotyl.These acceptors all need pass through the dedifferentiation process and form callus, then through breaking up regeneration plant again.Can tissue culture and genetic transformation depend on to a great extent successfully that the genotype that acceptor gene type, the overwhelming majority have an important economic worth is difficult to utilize, and plant regeneration frequency is very low, and is prone to take place somaclonal variation in the tissue culture procedures.Therefore, select the acceptor of suitable explant, set up the genetic conversion system that not limited by genotype and be still one of emphasis of present research as Agrobacterium-mediated Transformation.
In recent years, the transgenic research work that utilizes the direct regeneration system rapidly of shoot apical meristem that exsomatizes to carry out increases gradually, and makes important progress.1988, the stripped stem apex of usefulness particle gun soybean transformation such as McCabe obtained transfer-gen plant.Gould in 1991 etc. cultivate altogether with stripped stem apex of corn and Agrobacterium and have obtained transformed plant and filial generation.(2002) such as Zapata etc. (1999) and Satyavathi have all obtained transfer-gen plant with the stripped stem apex of agrobacterium-mediated transformation converting cotton respectively.Stripped shoot apical meristem does not need the dedifferentiation process, and stem apex transforms directly regeneration plant of back, and this shortens the transformation period greatly, and has broken the genotype restriction to a certain extent, therefore demonstrates good prospects for application.
It is a difficult problem that agriculture bacillus mediated wheat genetic transforms.According to statistics, before 2002, obtained in the report of wheat transgenic plant, particle bombardment accounts for about 90%, and additive method only accounts for 10%.1997, Cheng etc. utilized the success of agrobacterium-mediated transformation transformed wheat, indicate another milestone that wheat genetic transforms.Owing to Vitro in Wheat receives genotypicly to influence that big, most kind plant regeneration frequencies are low, insensitive to agroinfection, be prone to take place reason such as somaclonal variation in the tissue culture procedures; Make through agriculture bacillus mediated genetic transformation progress slow; Obtain the transgenic wheat plant in batches and still receive the genotype restriction very big, so that the wheat cdna engineering research far lags behind other staple crops.In addition, the genome of wheat has 1.7 * 10
10Bp is 40 times of rice genome, is 6 times of corn gene group, is 100 times of arabidopsis gene group, and huge genome causes the Molecular Identification difficulty of transfer-gen plant big, and particularly the Southern hybridization analysis has influenced offspring's research of transgenic wheat.The factor such as wheat genotypes, explant type, dip-dye and common incubation time, agrobacterium strains and the selective agent etc. that influence the wheat genetic conversion have all obtained broad research in recent years, but transformation efficiency is starkly lower than other staple crops.
Salicornia europaeal (Salicornia europaea) is the true halophytes of a kind of carnification that belongs to Chenopodiaceae, extensively is distributed near coastal and the inland brine lake, can accumulate high NaCl to dry weight 50%, is considered in the world a kind of higher plant of salt tolerant.Salicornia europaeal has the potentiality that develop into vegetables, oil crops and fodder crop.The salicornia europaeal genetic conversion system of setting up efficient stable is significant for its adversity gene function of Research on Mining and the Improvement in Shape of himself.Shi etc. (2006) are the explant induction callus with the mature embryo and have realized plant regeneration, have set up the external regeneration system of salicornia europaeal.Yet the callus of induce rate and the shoot regeneration frequency of this system are very low, are difficult to genetic transformation in batches.
Summary of the invention
An object of the present invention is to provide a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant provided by the invention comprises the steps:
1) stabs the shoot tip meristem of purpose plant, obtain handling back purpose plant;
2) infect the said processing back purpose plant that step 1) obtains with the reorganization Agrobacterium bacterium liquid that contains target DNA, obtain contaminating the back seedling;
3) culturing step 2) seedling after the dip-dye that obtains, obtain containing the transgenic plant of said target DNA.
In the step 1), said stabbing adopted following metallic brush;
Step 2) in, the said reorganization Agrobacterium bacterium liquid that contains target DNA prepares according to following method:
A) said target DNA is imported agrobacterium tumefaciens through recombinant vectors and obtain the Agrobacterium of recombinating;
B) the said reorganization Agrobacterium that steps A is obtained is resuspended in the substratum that is described below, and the bacterium liquid that obtains is the reorganization Agrobacterium bacterium liquid that contains target DNA;
Said recombinant vectors is the pCAMBIA3300-ubinos that contains the bar gene; Or, obtain expressing the recombinant vectors of target DNA with among the said target DNA insertion expression vector pCAMBIA3300-ubinos.
Said target DNA is SeVP1 or SeVP2;
The nucleotides sequence of said SeVP1 is classified the sequence 2 in the sequence table as;
The nucleotides sequence of said SeVP2 is classified the sequence 3 in the sequence table as.
The nucleotides sequence of said bar is classified the sequence 4 in the sequence table as.
Step 2) in, said infecting at barometric point is to carry out under the 0.05MPa; The said time of infecting is 15-20min, and the said time of infecting is specially 20min.
In the step 1), the plant that obtained in the seed germination 1-7 that said purpose plant is said purpose plant days;
In the step 3), said cultivation is followed successively by common cultivation and continues and cultivates;
Said temperature of cultivating altogether is 20 ℃-22 ℃; The said time of cultivating altogether is 2 days-4 days, and said the cultivation altogether is dark culturing; The said substratum of cultivating altogether is the MS substratum;
Said continuation cultivate for a) or b):
A) temperature of said continuation cultivation is warm 25-30 ℃ of day, and at temperature 18-20 ℃ of night, the relative humidity that said continuation is cultivated is 60-80%, and the illumination condition that said continuation is cultivated is that 16h illumination/8h is dark; The time that said continuation is cultivated is 20-30 days;
B) temperature of said continuation cultivation is warm 22-28 ℃ of day, and at temperature 15-21 ℃ of night, the illumination condition that said continuation is cultivated is natural lighting; The time that said continuation is cultivated is 10-15 days.
In the step 1), the plant that the seed germination that said purpose plant is said purpose plant obtained in 1,2,3 or 7 days;
In the step 3), said temperature of cultivating altogether is 20 ℃ or 22 ℃; The said time of cultivating altogether is 3 days,
Said continuation cultivate for a) or b):
A) temperature of said continuation cultivation is 25 ℃ of day temperature, and night, temperature was 18 ℃, and the relative humidity that said continuation is cultivated is 60%, and the time that said continuation is cultivated is 21 days;
B) temperature of said continuation cultivation is a day warm 22C, and night, temperature was 16 ℃; The time that said continuation is cultivated is 10 days.
The plant that the seed germination of the concrete said purpose plant of said purpose plant obtained in 1,2,3 or 7 days.
Said plant is monocotyledons or dicotyledons, and said monocotyledons is a wheat, and said dicotyledons is a salicornia europaeal.
Second purpose of the present invention provides a kind of metallic brush that is used to stab said purpose plant shoot tip meristem.
Metallic brush provided by the invention comprises steel fiber bundle and the sleeve that is set in the said steel fiber bundle outside, and an end of said steel fiber bundle is located at outside the said sleeve, and the other end of said steel fiber bundle is positioned at sleeve.
The diameter of said steel fiber bundle is (0.3-0.8) centimetre, and the diameter of said steel fiber bundle is specially 0.5 centimetre;
Said steel fiber bundle is that the steel fiber of 20-25 micron is formed by some diameters specifically; Said steel fiber Shu Youqi is that the steel fiber of 20 microns or 25 microns is formed by some diameters specifically;
The said length of being located at the outer steel fiber bundle of said sleeve is (0.2-0.5) centimetre, and the said length of being located at the outer steel fiber bundle of said sleeve is specially 0.5 centimetre.
Said steel fiber bundle is fixedly connected with said sleeve, and said steel fiber bundle is fixedly connected with said sleeve with multi-purpose adhesive through line.
The 3rd purpose of the present invention provides a kind of substratum that is used to prepare said reorganization Agrobacterium bacterium liquid.
Substratum provided by the invention prepares according to following method: Syringylethanone, SilwetL-77 and 1/2MS liquid nutrient medium are mixed, obtain substratum, the concentration of said Syringylethanone in said substratum is 150 μ M-250 μ M; The concentration of said SilwetL-77 in said substratum is 0.005%-0.015% (volumn concentration).
The concentration of said Syringylethanone in said substratum is 200 μ M; The concentration of said SilwetL-77 in said substratum is 0.01% (volumn concentration).
Experiment of the present invention proves; Shoot apical meristem with monocotyledons wheat and dicotyledons salicornia europaeal seedling is the Agrobacterium-mediated Transformation acceptor respectively; Through to the transformation of stem apex damage instrument, to the research of seedling seedling age; And measures such as assisted vacuum osmotic treated and penetration-assisting agent use, set up one and overlapped efficiently Plant Transformation system fast, improved the stem apex transformation efficiency; That the advantage of this transformation system is is simple to operate, the cycle short, does not need tissue culture and plant regeneration process, thereby has overcome the problem of ubiquitous genotype dependency and somatic variation.
Description of drawings
Fig. 1 is the structural representation of the brush of steel fiber making
Fig. 2 transform for the wheat stem apex and T0 for resistant plant
Fig. 3 stabs the influence of instrument to weeding for wheat agent resistance rate for different stem apexs
Fig. 4 is the influence of wheat seedling age to the Herbicid resistant rate
Fig. 5 is transgenic wheat and offspring's thereof acquisition
Fig. 6 is for changeing SeVP1 and SeVP2 wheat T1 for the PCR detected result
Fig. 7 is that the acquisition and the vacuum infiltration of salicornia europaeal resistant plant handled the influence to the Herbicid resistant rate
Fig. 8 is the acquisition of salicornia europaeal T1 for resistant plant
Fig. 9 is that the bar gene is at the transcript relative expression quantity of salicornia europaeal T1 for plant
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Following embodiment data statistic analysis carries out (LSD) statistical study of multiple comparisons with the SPSS of statistical software 12.0, and the significance to each processing and strain difference on P＜0.05 and P＜0.01 level compares.
One, the foundation of wheat stem apex transformation system
1, material
A, wheat breed:
Middle wheat 12 wheats (hereinafter to be referred as the wild-type wheat), winter habit; Ability for tillering is stronger, and the percentage of earbearing tiller is higher; The fringe spindle-type, long awns, white shell, white grain, seed cutin; Mu spike number and grain number per spike are more in the output key element, and thousand seed weight is on the low side; Plant is shorter, about 65 centimetres of water saving district examination plant heights, and about 70 centimetres of high fertile district examinations, lodging resistance is good; Winter resistance is better; Anti-, slow stripe rust, slow leaf rust, sense Powdery Mildew and stem rust.
Purchase is middle peasant's gold soil agricultural development research centre seed sales department from Beijing.
B, transfection reorganization bacterium: C58/pCAMBIA3300-ubinos
Reorganization Agrobacterium C58/pCAMBIA3300-ubinos changes the reorganization bacterium that Agrobacterium C58 obtains over to for naming plasmid pCAMBIA3300-ubinos.
Plasmid pCAMBIA3300-ubinos contains ubiquitin promotor and selection markers genes encoding grass fourth phosphinothricin acetyl based transferase (PAT, phosphinothricinacetyltransferase) gene bar.
Agrobacterium C58 bacterial strain disclosed in document " Overexpression of Organellar and Cytosolic AtHSP90 in Arabidopsis thaliana Impairs Plant Tolerance to Oxidative Stress.Hongmiao Song; Pengxiang Fan; Yinxin Li.Plant Mol Biol Rep (2009) 27:342-349. ", and the public can obtain from Chinese Academy of Sciences's plant research.
Plasmid pCAMBIA3300-ubinos inserts the ubiquitin promoter sequence in the Hind of carrier pCAMBIA 3300 III and BamH I; The carrier that obtains; The ubiquitin promoter sequence is seen the sequence 1 in the sequence table; Contain gene bar among the pCAMBIA3300, its nucleotides sequence is classified sequence 4 as.
PCAMBIA3300 is available from the Beijing DingGuo ChangSheng Biology Technology Co., Ltd.
C, steel fiber brush: with about 60,000 diameters is that 20 microns steel fiber bunchy is filled in the aperture of bamboo writing brush pole (the steel fiber beam diameter is 0.5cm), fixes with multi-purpose adhesive and line.In order to keep the rigidity of brush, being exposed at the outer metal fiber length of a pole is 0.5 centimetre.
Steel fiber is available from Xi'an Filter Metal Materials Co., Ltd..Pole is available from the stationer's.Using diameter respectively is that the brush made of 20 and 25 microns steel fiber is as shown in Figure 1.In order to keep the rigidity of brush, the metal fiber length of brush is 0.5 centimetre.
D, substratum
The YEP culture medium preparation:
Yeast extract (yeast extract) 10g/L
Peptone (peptone) 10g/L
NaCl 5g/L
Agar powder (agar) 15g/L (only being used for solid)
NaOH solution with 1M is transferred pH7.0.
The 1/2MS culture medium preparation:
The preparation of substratum mother liquor and preservation
More than in the various mother liquors, the macroelement mother liquor is 20 times of liquid concentrators, micro-mother liquor and mother liquid of iron salt are 200 times of liquid concentrators, the organic composition mother liquor is 100 times of liquid concentrators.When being used to cultivate Arabidopis thaliana, be diluted with water to 1/2MS, add 1% sucrose and 0.488g/L MES, transfer pH=5.8～6.0, add 8% agar powder at last with the NaOH solution of 1M.Autoclaving.
2, the wheat stem apex transforms
A, utilize steel fiber brush to carry out conversion condition to grope
1) preparation of acceptor material
Middle wheat 12 wheat seeds soak 10min with 1% aqueous sodium hypochlorite solution again with 70% alcohol immersion 1min, then with sterilized water washing 5 times.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.The sterilization seed places the petridish that is covered with three metafiltration paper, adds an amount of sterilized water, 25 ℃ of dark culturing 2 days.Peel off coleoptile and spire, stab exposed shoot tip meristem, obtain transformation receptor with the steel fiber brush that obtains in the above-mentioned steps 1.
2) infect
C58/pCAMBIA3300-ubinos 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum with above-mentioned acquisition make bacterium be in logarithmic phase, and bacterial concentration is OD600=1.0.Centrifugal 5min under 3000rpm abandons supernatant then, and collecting precipitation is thalline, and is again that thalline is resuspended with infecting substratum, obtains infecting bacterium liquid.The wheat seedling of stabbing vegetative point is dipped into infects in the bacterium liquid, vacuumize (0.05MPa) 20min, obtain contaminating the back seedling;
Infect substratum: contain 200 μ M Syringylethanones (acetosyringone, AS) and 0.01%SilwetL-77 (the SilwetL-77 spray adjuvants is based on the alkoxy-modified trisiloxanes that gathers, the tensio-active agent with super spread ability.Spray adjuvants is based on the alkoxy-modified trisiloxanes that gathers, the tensio-active agent with super spread ability.) the 1/2MS liquid nutrient medium;
Syringylethanone is available from Shanghai biotechnology Science and Technology Ltd..
SilwetL-77 (PE) is available from the grand scientific & trading Co., Ltd. of Beijing Kodak.
3) cultivate altogether
To contaminate the back seedling and blot with aseptic filter paper, the outside of belly downwards (like Fig. 2, the A Agrobacterium is infected back seedling (seed)) places on the MS substratum in 20 ℃, cultivates 3 days (Fig. 2, B cultivate seedling after 3 days altogether) under the dark condition altogether, obtains common cultivations seedling afterwards.
4) acquisition of commentaries on classics bar wheat
To cultivate back seedling (aseptic seedling) altogether and be transplanted to (Fig. 2, C are transplanted to the cave and are coiled into seedling alive) in the flowerpot, and let plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 15-21 ℃ of night, the time of continuing to cultivate is 10 days, obtains 188 strain T0 for transformed wheat.
Seedling grows spraying herbicide behind the 2-3 sheet young leaves, and the survival plant is Herbicid resistant plant (Fig. 2, D herbicide screening survive after 15 days plant) after 15 days, obtains 188 strain T0 for changeing the bar wheat.
5) screening of transformed plant and field planting
Be transplanted to T0 in the flowerpot when changeing bar wheat length to tri-leaf period, (solvent is a water, and per-cent is the quality percentage composition evenly to spray the 0.3%basta herbicide solution.), be contrast with wild-type wheat and T0 for changeing bar wheat (dissecting needle), statistics Herbicid resistant rate (with the survival rate note) after 15 days.The experiment triplicate, results averaged, 50 strains of wild-type wheat, 188 strains that T0 obtains more than for commentaries on classics bar wheat being.
Basta weedicide (Sigma): staple is careless fourth phosphorus, available from Xi'an Luo Senbai Science and Technology Ltd..
T0 is for the preparation method that changes bar wheat (dissecting needle): adopt above-mentioned 1 basically)-4) method handle, different is 1) in adopt dissecting needle to stab exposed shoot tip meristem, finally obtain T0 for changeing bar wheat (dissecting needle).
The result is as shown in Figure 3, and Fig. 3 A is with the steel fiber brush with after dissecting pricking wound wheat stem apex, 15 days seedling of herbicide screening; Can find out that after 15 days, the wild-type wheat is dead, T0 is for changeing bar wheat and T0 for changeing all also survivals of bar wheat (dissecting needle).
The statistics survival results is shown in Fig. 3 B; B is the steel fiber brush and dissects the influence of pricking wound wheat stem apex to the Herbicid resistant rate; * represent to compare with the dissecting needle mode of stabbing, the plant resistance rate after the steel fiber brush is stabbed reaches significant difference on P＜0.05 level.Values＝mean±SD(n＝3)。Find out that from figure T0 is 7% for the survival rate of changeing bar wheat (dissecting needle), T0 is 15% for the survival rate of changeing the bar wheat.Statistical analysis shows that both reach the significant difference level.
Can find out, use steel fiber to brush the survival rate height of the transgenic wheat that obtains than dissecting needle.
The influence of B, wheat seedling age antagonism rate
1) preparation of acceptor material
The A of method and step 2 1) basic identical; Different is 25 ℃ of dark culturing 1,2,3,4,5 days, obtains sprouting 1 day wheat acceptor, sprouting 2 days wheat acceptor, 3 days wheat acceptor of sprouting, 4 days wheat acceptor of sprouting, 5 days wheat acceptor of sprouting.
2) infect: the A of method and step 2 2) identical.
3) cultivate altogether: the A of method and step 2 3) identical.
4) change the acquisition of bar wheat: the A of method and step 2 4) identical; The result be the wheat in 1,2,3,4,5 day age as acceptor, obtain T0 respectively for changeing bar wheat (1 day), T0 for changeing bar wheat (2 days), T0 for changeing bar wheat (3 days), T0 for changeing bar wheat (4 days), T0 for changeing bar wheat (5 days).
5) screening of transformed plant and field planting
The A of method and step 2 5) identical, the result is as shown in Figure 4:
After 15 days, T0 is 15% for changeing bar wheat (2 days) survival rate,
T0 is 11% for changeing bar wheat (1 day) and T0 for the survival rate of changeing bar wheat (3 days),
T0 significantly reduces for the survival rate of changeing bar wheat (5 days) for changeing bar wheat (4 days) and T0, all has only 2%.
Values＝mean±SD(n＝3)。The pairing resistance rate of different letter representations reaches significant difference on P＜0.05 level.
Explain that sprouting 1-3 days the seedling in back is more suitable for being used for the stem apex conversion.
Two, utilize wheat stem apex transformation system to obtain transgenic wheat
1, material
A, wheat breed: middle wheat 12.
B, transfection reorganization bacterium:
C58/pCAMBIA3300-ubinos-SeVP1 and C58/pCAMBIA3300-ubinos-SeVP2
Reorganization Agrobacterium C58 pCAMBIA3300-ubinos-SeVP1 changes the reorganization bacterium that Agrobacterium C58 obtains over to for naming plasmid pCAMBIA3300-ubinos-SeVP1.
Reorganization Agrobacterium C58 pCAMBIA3300-ubinos-SeVP2 changes the reorganization bacterium that Agrobacterium C58 obtains over to for naming plasmid pCAMBIA3300-ubinos-SeVP2.
The construction process of pCAMBIA3300-ubinos-SeVP1:
Utilize the Sma I and the Sac I of pCAMBIA3300-ubinos carrier MCS, the SeVP1 gene is inserted into pCAMBIA3300-ubinos respectively.The SeVP1 gene order is seen sequence 2.
The construction process of pCAMBIA3300-ubinos-SeVP2:
Utilize the Sma I and the Sac I of pCAMBIA3300-ubinos carrier MCS, the SeVP2 gene is inserted into pCAMBIA3300-ubinos respectively.The SeVP2 gene order is seen sequence 3.
C, steel fiber brush: it is the brush of 25 microns steel fiber making that the method according to is used diameter, and the diameter of steel fiber bundle is specially 0.5 centimetre, and the length of being located at the outer steel fiber bundle of sleeve is 0.5 centimetre.
2, the wheat stem apex transforms
Experimental group:
1) preparation of acceptor material
Middle wheat 12 wheat seeds soak 10min with 1% aqueous sodium hypochlorite solution again with 70% alcohol immersion 1min, then with sterilized water washing 5 times.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.The sterilization seed places the petridish that is covered with three metafiltration paper, adds an amount of sterilized water, 25 ℃ of dark culturing 2 days.Peel off coleoptile and spire, stab exposed shoot tip meristem, obtain transformation receptor with the steel fiber brush that obtains in the above-mentioned steps 1.
2) infect:
With C58/pCAMBIA3300-ubinos-SeVP1 and C58/pCAMBIA3300-ubinos-SeVP2 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum of above-mentioned acquisition, make bacterium be in logarithmic phase respectively, bacterial concentration is OD600=1.0.Centrifugal 5min under 3000rpm abandons supernatant then, and collecting precipitation is thalline, and it is resuspended again thalline to be infected substratum with interpolation, obtains infecting bacterium liquid.The wheat seedling of stabbing vegetative point is dipped into infects in the bacterium liquid, vacuumize (0.05MPa) 20min, obtain contaminating back seedling 1 and contaminate back seedling 2;
Infect substratum: (acetosyringone is AS) with the 1/2MS liquid nutrient medium of 0.01%SilwetL-77 to contain 200 μ M Syringylethanones;
Syringylethanone is available from Shanghai biotechnology Science and Technology Ltd..
SilweetL-77 (PE) is available from the grand scientific & trading Co., Ltd. of Beijing Kodak.
3) cultivate altogether:
To contaminate back seedling 1 respectively and contaminate back seedling 2 usefulness aseptic filter papers and blot, the outside of belly places downwards on the MS substratum in 22 ℃, cultivates seedling 2 after obtaining common cultivations back seedling 1 respectively and cultivating altogether under the dark condition altogether 3 days.
4) change SeVP1 wheat and the acquisition of changeing the SeVP2 wheat:
To cultivate back seedling 1 (aseptic seedling) respectively altogether and cultivate back seedling 2 (aseptic seedling) altogether and be transplanted in the flowerpot; Let plant under natural lighting, grow then; Warm 22 ℃ of day; Night, temperature was 16 ℃, and the time of continuing to cultivate is 10 days, obtained 550 strain T0 respectively for changeing SeVP1 wheat and 730 strain T0 for changeing the SeVP2 wheat.
5) screening of transformed plant
A, the screening of basta Herbicid resistant
Waiting to be transplanted to T0 in the flowerpot for changeing SeVP1 wheat and T0 when changeing SeVP2 wheat length to tri-leaf period, evenly spray 0.3%basta herbicide solution (aqueous solution) respectively, is contrast with the wild-type wheat, the resistant plant that statistics is survived after 15 days.
The result contrasts and is the wild-type wheat shown in Fig. 5 A (herbicide screening of transformed plant), 1,2 be T0 for changeing the SeVP1 wheat, 3 be that T0 is for commentaries on classics SeVP2 wheat.Obtain the positive T0 of 60 strain resistance screenings for changeing SeVP1 wheat and 90 strain resistance screening T0 for changeing the SeVP2 wheat.The wild-type wheat is all dead.
B, T1 detect for the PCR of plant
Respectively gather in the crops seed with resistance screening T0 for changeing on the SeVP2 wheat for changeing the SeVP1 wheat from the positive T0 of resistance screening, sowing, obtain T1 for change SeVP1 wheat and T1 for commentaries on classics SeVP2 wheat (as the solid T1 of obtaining of Fig. 5 B resistant plant for).
T1 is seeded into the land for growing field crops for plant, survives the winter naturally, normally tiller (Fig. 5 C T1 is seeded into the land for growing field crops for plant, survives the winter naturally, normally tillers).
Adopt CTAB method trace to extract T1 respectively for changeing SeVP1 wheat and T1 for the DNA that changes the SeVP2 wheat leaf blade.
PCR detects used upstream primer according to the design of ubiquitin promoter sequence, and downstream primer designs according to target gene sequences.
Detect the primer of SeVP1:
ubi-1：5’-GCTTTCCCTTCCTCGCCC-3’
vp1-2?5’-GTCATAGGTGCACTCCTGGGTA-3’
Detect the primer of SeVP2:
ubi：5’-TTTAGCCCTGCCTTCATACGC-3’
vp2-1：5’-CCAACATCAGCAGCCTTTGTGTA-3’
T1 detects for changeing SeVP1 wheat PCR: is template with T1 for the DNA of the blade that changes the SeVP1 wheat, as primer, carries out pcr amplification with ubi-1 and vp1-2, the result shown in the last figure of Fig. 6, Marker, DL2000 plus; Plasmid, pCAMBIA3300-ubinos-SeVP1; WT is the wild-type wheat, and the positive T1 that obtains 1527bp obtains from the positive T1 of 50 strains of 25 strain systems for changeing the SeVP1 wheat for changeing the SeVP1 wheat altogether.The wild-type wheat does not have the purpose fragment.
T1 detects for changeing SeVP2 wheat PCR: is template with T1 for the DNA of the blade that changes the SeVP2 wheat, as primer, carries out pcr amplification with ubi and vp2-1, the result shown in Fig. 6 figure below, Marker, DL2000 plus; Plasmid, pCAMBIA3300-ubinos-SeVP2; WT is the wild-type wheat, and the positive T1 that obtains 847bp obtains from the positive T1 of 90 strains of 29 strain systems for changeing the SeVP2 wheat for changeing the SeVP2 wheat altogether.The wild-type wheat does not have the purpose fragment.
The above results proves that above-mentioned transformation system can obtain transgenic wheat.
One, experimental group
1, material
A, salicornia europaeal (S.europeae L.) seed (below be called the wild-type salicornia europaeal) are available from the brilliant grand marine industries Development Co., Ltd in Dafeng City, Jiangsu Province.
B, transfection reorganization bacterium: C58/pCAMBIA3300-ubinos
Reorganization Agrobacterium C58/pCAMBIA3300-ubinos changes the reorganization bacterium that Agrobacterium C58 obtains over to for naming plasmid pCAMBIA3300-ubinos.
Plasmid pCAMBIA3300-ubinos contains ubiquitin promotor and selection markers genes encoding grass fourth phosphinothricin acetyl based transferase (PAT, phosphinothricinacetyltransferase) gene bar.
Agrobacterium C58 bacterial strain disclosed in document " Overexpression of Organellar and Cytosolic AtHSP90 in Arabidopsis thaliana Impairs Plant Tolerance to Oxidative Stress.Hongmiao Song; Pengxiang Fan; Yinxin Li.Plant Mol Biol Rep (2009) 27:342-349. ", and the public can obtain from Chinese Academy of Sciences's plant research.
The construction process of plasmid pCAMBIA3300-ubinos is: utilize the HindIII and the BamHI of the MCS of carrier pCAMBIA 3300, insert the ubiquitin promoter sequence.Sequence 1 in the sequence table of ubiquitin promoter sequence.
PCAMBIA3300 is available from the Beijing DingGuo ChangSheng Biology Technology Co., Ltd.
C, steel fiber brush: using about 40,000 diameters is the brush of 25 microns steel fiber composition steel fiber bundle (diameter is 0.5 centimetre) making, and the telescopic metal fiber length of exposing of brush is 0.5 centimetre.
D, substratum: with embodiment 1.
2, salicornia europaeal transforms
1) preparation of acceptor material
The salicornia europaeal seed is evenly sowed in vermiculite, treated seed germination, water with the l/2Hoagland nutritive medium.Seedling is cultivated in plant institute of Chinese Academy of Sciences greenhouse, and day temperature maintains 25-30 ℃, and nocturnal temperature is at 18-20 ℃, and relative humidity maintains 60-80%, and illumination condition is that 16h illumination/8h is dark.Sprout back 7 days seedling, when two cotyledons have just launched, stab two cotyledon intermediary shoot tip meristems with the steel fiber brush that above-mentioned C obtains.
2) infect
With C58/pCAMBIA3300-ubinos 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum, make bacterium be in logarithmic phase.Centrifugal 5min under 3000rpm abandons supernatant then.Again that thalline is resuspended with infecting substratum, obtain infecting bacterium liquid.
Being divided into following two groups handles:
A, the aseptic cotton balls that will dip in resuspended bacterium liquid place the stem apex position of damage, vacuumize (0.05MPa) 15min, obtain contaminating the back seedling;
B, the aseptic cotton balls that will dip in resuspended bacterium liquid place the stem apex position of damage, and 15min obtains contaminating back seedling 1;
Infect substratum: contain 200 μ M Syringylethanones (acetosyringone, AS) and 0.01%SilweetL-77 (the SilwetL-77 spray adjuvants is based on the alkoxy-modified trisiloxanes that gathers, the tensio-active agent with super spread ability.Spray adjuvants is based on the alkoxy-modified trisiloxanes that gathers, the tensio-active agent with super spread ability.) the 1/2MS liquid nutrient medium;
Syringylethanone is available from Shanghai biotechnology Science and Technology Ltd..
SilweetL-77 (PE) is available from the grand scientific & trading Co., Ltd. of Beijing Kodak.
3) cultivate altogether
Take off cotton balls, will contaminate back seedling 1 respectively and contaminate back seedling 2 in 20 ℃, dark condition was cultivated (still in vermiculite) 3 days down altogether, obtained common cultivation back seedling 1 and cultivated back seedling 2 altogether.
4) acquisition of commentaries on classics bar salicornia europaeal
To cultivate back seedling 1 respectively altogether and cultivate back seedling 2 altogether and transfer to that (day temperature maintains 25 ℃ in the greenhouse; Nocturnal temperature is at 18 ℃; Relative humidity maintains 60%; Illumination condition is that 16h illumination/8h is dark), continue to cultivate 21 days, obtain 441 strain T0 for changeing bar salicornia europaeal 1 (vacuumizing) and 337 strain T0 for changeing bar salicornia europaeal 2 (not vacuumizing).
Syringylethanone is available from Shanghai biotechnology Science and Technology Ltd..
SilweetL-77 (PE) is available from the grand scientific & trading Co., Ltd. of Beijing Kodak.
L/2 Hoagland nutrient solution prescription is following:
5) screening of transformed plant and field planting
A, the screening of basta Herbicid resistant
T0 evenly sprays the 0.1%basta herbicide solution for changeing bar salicornia europaeal 1 (vacuumizing) and T0 for changeing bar salicornia europaeal 2 (not vacuumizing) continue 3 weeks of cultivation in the greenhouse after.With the wild-type salicornia europaeal is contrast.Basta weedicide (Sigma): staple is careless fourth phosphorus, available from Xi'an Luo Senbai Science and Technology Ltd..Vacuum treated 441 strains, not vacuum treated 337 strains, wild-type contrasts 127 strains.
Statistics Herbicid resistant rate after 20 days.
The result can find out that shown in Fig. 7 A the survival digital display work of transformed plant is higher than the wild-type contrast, and wherein the survival number through vacuum treated transformed plant is higher than the survival number that does not pass through vacuum treated transformed plant.
Statistics Herbicid resistant rate, result can find out shown in Fig. 7 B,
T0 is 49% for changeing bar salicornia europaeal 1 (vacuumizing) Herbicid resistant rate, obtains the positive T0 of 218 strain Herbicid resistants for changeing bar salicornia europaeal 1 (vacuumizing);
T0 is 40% for changeing bar salicornia europaeal 2 (not vacuumizing) Herbicid resistant rate, obtains the positive T0 of 137 strain Herbicid resistants for changeing bar salicornia europaeal 2 (not vacuumizing);
Wild-type salicornia europaeal Herbicid resistant rate is 13%.
Values＝mean±SD(n＝3)。The pairing resistance rate of different letter representations reaches significant difference on P＜0.05 level.
Show that the vacuum infiltration processing can significantly improve the Herbicid resistant rate.
Herbicid resistant male T0 is transplanted to Jiangsu Da Feng for changeing bar salicornia europaeal 1 (vacuumizing), and under the natural condition, selfing, solid obtains changeing bar salicornia europaeal T1 for seed.
To change bar salicornia europaeal T1 and evenly be seeded in the vermiculite, treat seed germination, water with the l/2Hoagland nutritive medium for seed.Seedling is cultivated in plant institute of Chinese Academy of Sciences greenhouse, and day temperature maintains 25-30 ℃, and nocturnal temperature is at 18-20 ℃, and relative humidity maintains 60-80%, and illumination condition is that 16h illumination/8h is dark.After sprouting for 4 weeks, screen with the 0.2%basta herbicide solution.With the wild-type salicornia europaeal is contrast.The experiment triplicate, results averaged, each strain is 100 strains.
The result is as shown in Figure 8, can find out the The selection result of part transformed plant.Obtain the positive T1 of 200 strain resistance screenings altogether for changeing the bar salicornia europaeal.The wild-type wheat is all dead.
B, T1 detect for the fluorescence quantitative RT-RCR of plant
Use Trizol reagent to extract positive T1 for changeing the total RNA of bar salicornia europaeal over-ground part; The total RNA that extracts digests through the DNase I; After TransScript First-Strand cDNA Synthesis SuperMix reverse transcription; (Toyobo Japan) carries out fluorescence quantitative RT-RCR and detects, and is contrast with the wild-type salicornia europaeal to utilize SYBR Green Realtime PCR Master Mix.
With salicornia europaeal α-tubulin gene as confidential reference items, through 2
-Δ Δ CtMethod relative quantification is carried out in bar genetic expression.
The primer sequence is following in the fluorescence quantitative RT-RCR:
α-tubulin gene test primer:
Tubup：5’-CAGTGCCTTTGAGCCATCTTC-3’
Tubdn：5’-CTGAATGGTTCGCTTGGTCTT-3’
Bar gene test primer:
BarRT1：5’-CACCATCGTCAACCACTACATCG-3’
BarRT2：5’-GCGACGAGCCAGGGATAGC-3’
Trizol reagent is available from TIANGEN Biotech (Beijing) Co., Ltd..DNase I (Fermentas) is available from Beijing Hua Lvyuan Bioisystech Co., Ltd.
TransScript First-Strand cDNA Synthesis SuperMix is available from Beijing Quan Shi gold Bioisystech Co., Ltd.
(Toyobo is Japan) available from Beijing Hua Lvyuan Bioisystech Co., Ltd for SYBR Green Realtime PCR Master Mix.
Detected 10 positive T1 altogether for changeing bar gene transcription horizontal expression situation in the contrast of bar salicornia europaeal strain system and wild-type, tested triplicate, results averaged, wild-type are got 10 strains systems.
The result is as shown in Figure 9; L1-L10 is respectively 10 positive T1 for changeing bar salicornia europaeal strain system, and WT is the wild-type salicornia europaeal, from figure, finds out; Except L1 and L5; The bar gene transcripts of other 8 transgenic lines is 6-157 times of wild-type, explains that the bar gene has forwarded in these 8 transfer-gen plants and expression, explains that above-mentioned transformation system is good.
Claims (10)
1. a method of cultivating transgenic plant comprises the steps:
1) stabs the shoot tip meristem of purpose plant, obtain handling back purpose plant;
2) infect the said processing back purpose plant that step 1) obtains with the reorganization Agrobacterium bacterium liquid that contains target DNA, obtain contaminating the back seedling;
3) culturing step 2) seedling after the dip-dye that obtains, obtain containing the transgenic plant of said target DNA.
2. method according to claim 1 is characterized in that:
In the step 1), said stabbing adopted arbitrary described metallic brush among the following claim 6-8;
Step 2) in, the said reorganization Agrobacterium bacterium liquid that contains target DNA prepares according to following method:
A) said target DNA is imported agrobacterium tumefaciens through recombinant vectors and obtain the Agrobacterium of recombinating;
B) the said reorganization Agrobacterium that steps A is obtained is resuspended in following claim 9 or 10 said substratum, and the bacterium liquid that obtains is the reorganization Agrobacterium bacterium liquid that contains target DNA.
3. method according to claim 1 and 2 is characterized in that:
Step 2) in, said infecting at barometric point is to carry out under the 0.05MPa; The said time of infecting is 15-20min, and the said time of infecting is specially 15min.
4. according to arbitrary described method among the claim 1-3, it is characterized in that:
In the step 1), the plant that obtained in the seed germination 1-7 that said purpose plant is said purpose plant days.
5. according to arbitrary described method among the claim 1-4, it is characterized in that:
In the step 1), the plant that the seed germination that said purpose plant is said purpose plant obtained in 1,2,3 or 7 days.
6. metallic brush that is used to stab said purpose plant shoot tip meristem; Comprise steel fiber bundle and the sleeve that is set in the said steel fiber bundle outside; One end of said steel fiber bundle is located at outside the said sleeve, and the other end of said steel fiber bundle is positioned at sleeve.
7. metallic brush according to claim 6 is characterized in that:
The diameter of said steel fiber bundle is 0.3-0.8 centimetre, and the diameter of said steel fiber bundle is specially 0.5 centimetre;
Said steel fiber bundle is that the steel fiber of 20-25 micron is formed by some diameters specifically; Said steel fiber Shu Youqi is that the steel fiber of 20 microns or 25 microns is formed by some diameters specifically;
The said length of being located at the outer steel fiber bundle of said sleeve is 0.2-0.5 centimetre, and the said length of being located at the outer steel fiber bundle of said sleeve is specially 0.5 centimetre.
8. according to claim 6 or 7 described metallic brushs, it is characterized in that:
Said steel fiber bundle is fixedly connected with said sleeve.
9. substratum that is used to prepare said reorganization Agrobacterium bacterium liquid; Prepare according to following method: Syringylethanone, SilweetL-77 and 1/2MS liquid nutrient medium are mixed; Obtain substratum, the concentration of said Syringylethanone in said substratum is 150 μ M-250 μ M; The concentration of said SilweetL-77 in said substratum is 0.005%-0.015% (volume percent).
10. substratum according to claim 9 is characterized in that: the concentration of said Syringylethanone in said substratum is 200 μ M; The concentration of said SilweetL-77 in said substratum is 0.01% (volume percent).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014029045A1 (en) * | 2012-08-22 | 2014-02-27 | 河北省农林科学院遗传生理研究所 | Dicotyledon transgenic method for invading growing points of seed sprouts or seedling stems minimally and fully |
| WO2014029044A1 (en) * | 2012-08-22 | 2014-02-27 | 河北省农林科学院遗传生理研究所 | Monocotyledon transgenic method for invading growing points of seed buds minimally and fully |
| CN103849644A (en) * | 2012-12-07 | 2014-06-11 | 安徽科技学院 | Sorghum germ inoculation transgenic technology |
| CN114946653A (en) * | 2022-05-09 | 2022-08-30 | 鲁东大学 | Pineapple seedling regeneration method and axillary bud growth point transformation method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596617A (en) * | 2004-08-06 | 2005-03-23 | 山东大学 | Method of establishing early-maturing ripe hereditary transform system and application |
| CN101016544A (en) * | 2007-01-19 | 2007-08-15 | 山东大学 | Method of establishing winter jujube genetic conversion system for receptor by using stem tip |
| CN101643745A (en) * | 2009-09-09 | 2010-02-10 | 山东大学 | Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof |
| CN101698854A (en) * | 2009-05-07 | 2010-04-28 | 山东大学 | Application of transcription thellungiella halophila CBF1 gene in improving drought resistance and salt tolerance of corn and wheat |
| CN201459148U (en) * | 2009-06-12 | 2010-05-12 | 河北省农林科学院遗传生理研究所 | Minimally invasive transgenic brush of plant bud growing points |
-
2011
- 2011-07-14 CN CN201110197273A patent/CN102321662A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596617A (en) * | 2004-08-06 | 2005-03-23 | 山东大学 | Method of establishing early-maturing ripe hereditary transform system and application |
| CN101016544A (en) * | 2007-01-19 | 2007-08-15 | 山东大学 | Method of establishing winter jujube genetic conversion system for receptor by using stem tip |
| CN101698854A (en) * | 2009-05-07 | 2010-04-28 | 山东大学 | Application of transcription thellungiella halophila CBF1 gene in improving drought resistance and salt tolerance of corn and wheat |
| CN201459148U (en) * | 2009-06-12 | 2010-05-12 | 河北省农林科学院遗传生理研究所 | Minimally invasive transgenic brush of plant bud growing points |
| CN101643745A (en) * | 2009-09-09 | 2010-02-10 | 山东大学 | Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof |
Non-Patent Citations (1)
| Title |
|---|
| 董福双: "小麦茎尖生长点转化技术研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014029045A1 (en) * | 2012-08-22 | 2014-02-27 | 河北省农林科学院遗传生理研究所 | Dicotyledon transgenic method for invading growing points of seed sprouts or seedling stems minimally and fully |
| WO2014029044A1 (en) * | 2012-08-22 | 2014-02-27 | 河北省农林科学院遗传生理研究所 | Monocotyledon transgenic method for invading growing points of seed buds minimally and fully |
| US20150344897A1 (en) * | 2012-08-22 | 2015-12-03 | Hebei Academy Of Agriculture And Forestry Sciences, Institute Of Genetics And Physiology | Monocotyledon transgenic method for invading growing points of seed buds minimally and fully |
| US20160135379A1 (en) * | 2012-08-22 | 2016-05-19 | Haibo Wang | Dicotyledon transgenic method for invading growing points of seed sprouts or seedling stems minimally and fully |
| CN103849644A (en) * | 2012-12-07 | 2014-06-11 | 安徽科技学院 | Sorghum germ inoculation transgenic technology |
| CN114946653A (en) * | 2022-05-09 | 2022-08-30 | 鲁东大学 | Pineapple seedling regeneration method and axillary bud growth point transformation method |
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