CN103805631A - Method for efficient gene transformation of photinia serrulata - Google Patents

Abstract

The invention relates to a method for the efficient gene transformation of photinia serrulata, which belongs to the field of plant biotechnologies. The method is implemented by specifically adopting a gene transformation method combining a particle bombardment method with an agrobacterium-mediated method, and comprises the following steps: (1) extracting plasmids DNA; (2) preparing receptor materials; (3) pretreating tungsten powder; (4) preparing microprojectiles; (5) bombarding by using a gene gun; (6) pre-culturing the receptor materials; (7) carrying out agrobacterium infection; (8) co-culturing the obtained product; (9) screening and culturing bacteriostats and kanamycin; and (10) detecting transformation plants PCR and Southern Blotting. According to the invention, due to the adoption of a gene transformation method combining a particle bombardment method with an agrobacterium-mediated method, the cold resistance of obtained genetically modified plant systems is obviously improved, and imported AmGS genes increase the cold resistance of obtained genetically modified plant systems, thereby providing an important basis for cultivating anti-cold varieties.

Description

A kind of method that photinia glabra high efficiency gene transforms

Technical field

The present invention relates to a kind of method that photinia glabra high efficiency gene transforms, belong to plant biotechnology field.

Background technology

Photinia glabra (Photinia xfrasery) is the general designation of Rosaceae Photinia cross-fertilize seed, is to originate from semi-tropical evergreen shrubs seeds.Spring, its young leaves was red gorgeous, turned green summer, and autumn, winter, three seasons of spring present redness, and the heavy look of frost exceedes dense, and low warm colour is better.Do shade tree, its bar Liru torch; Do hedgerow, its shape is sleeping as flue; Scape is made in pruning, and shape can be in different poses and with different expressions, and landscape effect beauty is described as " king of red autumnal leaves hedgerow ".Since last century, the nineties was introduced China, be just listed in and there are the color leaf seeds that higher exploitation is worth.But because the inadaptable north of photinia glabra is as the winter low temperature on the ground such as Shandong, Hebei, Beijing, easily cause freeze injury, limited popularization and development process.Domestic researchist has carried out correlative study to aspects such as photinia glabra winter resistance, Discoloration mechanism, Cold resistances, to screening cold-resistant kind by domestication and cold-resistant adaptability, but does not select yet up to now effectively cold-resistant photinia glabra kind.Along with molecular biological development, utilize genetic engineering technique that anti-freeze gene is forwarded in plant materials, cultivating cold-resistant kind becomes the focus of the cold-resistant breeding research of current xylophyta.

For this reason; by the Differential expression analysis technology of cDNA-AFLP, the evergreen xylophyta Stem and leaf of Mongolian Ammopiptanthus (coming from the national secondary endangered protective plant in the tertiary period in time immemorial) of only depositing from NORTHWEST CHINA Desert Area, filter out 13 low temperature induction up-regulated expressions and transcribe fragment.One of them EST fragment is obtained to its full-length cDNA by RACE, built plant expression vector, arabidopsis thaliana transformation success has also obtained the transgenic arabidopsis plant that frost resistance obviously improves, but transforming xylophyta fails.The present invention selects another anti-freeze gene AmGS to clone, and the genetic transforming method combining with agrobacterium tumefaciens mediated method by particle bombardment imports its in xylophyta photinia glabra, the cold-resistant new variety of seed selection.

Current Genetic Transformation in Higher Plants technology can be divided into two large classes, one class is the direct gene transfer technology take via Particle Bombardment Transformation method as representative, particle bombardment is attached to the micro-bullet of high speed genetic material or other materials and directly injects cell, tissue and organoid, is state-of-the-art gene Transfection Technology in the world at present.The gas percussion ripple that gas gene gun converts to take pressurized gas (helium or nitrogen) is as power, and the gas percussion ripple that makes gas gene gun produce a kind of " cold " enters bombardment chamber, therefore can exempt from the cell injury being caused by " heat " gas percussion ripple.Gas gene gun can obtain in cellular type widely instantaneous, stable and high efficiency transformation.Gas gene gun has a special construction that produces shockwave, in appropriate air pressure range from 3.5MPa-10MPa, have corresponding different can fracturing diaphragm, the particle of coated DNA is passed through, inject in the target cell of bottom, bombardment chamber (maximum target diameter 50mm).Particle gun is applicable to the transgenosis of animals and plants, cell culture, embryo, bacterium and meiofauna.There is quick, easy, safe, efficient feature.Another kind of is the method for transformation of biological mediation, mainly contain agriculture bacillus mediated and virus-mediated two kinds of method for transformation, wherein agriculture bacillus mediated method for transformation is current most widely used gene transformation method, there is feature easy and simple to handle, that cost is low, transformation efficiency is high, be widely used in the genetic transformation of dicotyledons.The present invention organically combines two kinds of genetic transfoumations, for the cold-resistant gene transformation of photinia glabra.

Summary of the invention

A kind of method that the object of the present invention is to provide photinia glabra high efficiency gene to transform, to transform for the cold-resistant gene of photinia glabra.

To achieve these goals, technical scheme of the present invention is as follows.

The method that photinia glabra high efficiency gene transforms, what specifically adopt is the gene transformation method of particle bombardment and agrobacterium-mediated transformation combination, its step comprises: the extraction of (1) plasmid DNA; (2) preparation of acceptor material; (3) tungsten powder pre-treatment; (4) micro-bullet preparation; (5) particle gun bombardment; (6) preculture of acceptor material; (7) Agrobacterium is infected; (8) cultivate altogether; (9) antibacterial and kantlex screening and culturing; (10) transformed plant PCR and Southern Blotting detect.

Each step is specific as follows:

Step (1): the extraction of plasmid DNA: the agrobacterium strains LBA4404 that contains goal gene used is the binary vector pCAMBIA2300-35S-OCS (Genetics and Developmental Biology Institute of the Chinese Academy of Sciences is so kind as to give) that contains 35S promoter, goal gene, terminator NOS and marker gene NPTII.The cultivation of the thalline inoculation YEP solid medium that picking is preserved on Bechtop, bacterium plate is placed in constant incubator, cultivates 2～3d for 28 ℃, to single bacterium colony generation; The cultivation of picking list colony inoculation YEP liquid nutrient medium, on constant-temperature table, 28 ℃, 200～210r/min incubated overnight (24h left and right is to the logarithmic growth later stage), be to can be used for the extraction of plasmid DNA at 0.5～0.8 o'clock until OD600 value.From connect Agrobacterium cell the liquid YEP substratum containing 20mg/l Kanamycin and 15mg/l Gentamycin5-8ml, then cultivate 24h at 28 ℃, 200-220rpm.Then carry out plasmid extraction.Fresh preparation Sol II solution (880 μ l H2O, 100 μ lSDS10%, 20 μ l10N NaOH), and 20 0 μ lSol I (P1) solution that comprise 4mg/ml N,O-Diacetylmuramidase Lysozyme.Under 3600rpm speed, carry out centrifugal 12min to cultivating circadian Falcon test tube, abandon supernatant.With the resuspended agrobatcerium cell of 200 μ l5M NaCl, utilize vortex that agrobatcerium cell is mixed.Transfer in Eppendorf pipe.Add 20 μ l10%N-Lauroylsarcosine sodium solution, turned upside down mixes (being inverted from different directions) for 6-8 times.The lower centrifugal 2min of top speed (13000rpm), loses supernatant liquor.The resuspended precipitation of SolI (P1) (SolI:50mM glucose, 25mM Tris-Cl (8.0), 10M EDTA (8.0)) with 200 μ l containing 4mg/mlLysozyme, vortex is until mix.Be placed on ice.Add 400 μ l Sol II (P2) (Sol II:880 μ l H2O, 100 μ lSDS10%, 20 μ l10N NaOH), turned upside down mixes (being inverted from different directions) for 6-8 times.Place 5min on ice.Add 300 μ lSol III (P3), vibrate with hand.Place 10min on ice.SolIII=Buffer P3, the lower centrifugal 12min of Neutrilization solution. top speed (12000rpm).By supernatant liquor, (700 μ l) transfer in new Eppendorf pipe.With 1 × phenol/chloroform (Phenol/Chloroforum:350 μ lPhenol+350 μ l Chlororum, " Choloforum ": 24 times of volume chloroforum and 1 times of volume isoamylalcohol) extracting.In stink cupboard, in Eppendorf pipe, add phenol and chloroform (chloroform adds fashionable will being swift in motion, otherwise can suck in micropipet).Vortex 15s, the lower centrifugal 2min of top speed (12000rpm).By upper strata phase, (700 μ l) carefully move in new Eppendorf pipe.Tilt to draw, prevent from sucking the phenol/chloroform of lower floor.Notice that all used suction nozzles all put into the poisonous dustbin of stink cupboard.Phenol/chloroform debris is poured into below stink cupboard in the phenol/chloroform waste liquid bottle in cupboard.By 700 μ lisopropanel precipitations, under room temperature, place 5min.The lower centrifugal 15min of top speed (12000rpmg), carefully removes supernatant.Can in two steps, first remove most of supernatant with large transfer pipet, residue 50 μ l left and right centrifugal 5min again.Noting precipitating bead might not firmly inhale on tube wall, does not carefully siphon away precipitation.With 70% alcohol washing.Add 1ml70% alcohol, the lower centrifugal 5min of top speed (12000rpm), carefully removes supernatant.Under room temperature, dry.The resuspended precipitation of 20 μ l TE+10mg/ml RNase A.Cold house place diel, after store in-20 ℃ of refrigerators.By 100 times of DNA extraction liquid dilutions, measure its absorption value under wavelength 260nm and 280nm wavelength with UV-2450 ultraviolet-visible pectrophotometer, OD260/OD280=1.8～1.9 are high purity DNA, and being greater than 1.9 has RNA to pollute, and being less than 1.8 explanations has protein contamination.DNA concentration is calculated as follows: DNA concentration (ng/ μ L)=OD260 × 50 × extension rate.Be 200 μ g/mL, 500 μ g/mL, tri-kinds of concentration of 800 μ g/mL by plasmid DNA with 0.i%TE solution dilution, point install be housed in-20 ℃ of refrigerators for subsequent use.

Step (2), the preparation of acceptor material: photinia glabra stem segment length should be 1-1.5cm, do the use of particle gun bombardment through 0.4mol/L osmotic pressure substratum (MS substratum adds 0.2mol/L N.F,USP MANNITOL, 0.2mol/L sorbyl alcohol) alcohol processing 4-6h.

Step (3) tungsten powder pre-treatment: get the tungsten powder that 60mg diameter is 1 μ m, add 1ml dehydrated alcohol, 95 ℃ of incubation 1h, supersound process 3 times under peak power, each 5min (or ultrasonic hot to feel with ultrasonic grinder), the centrifugal supernatant of removing; Add 1ml dehydrated alcohol, vortex concussion 3-5min, the centrifugal supernatant of removing; Add 1ml sterilized water, vortex concussion, centrifugal, abandon supernatant, repeat 3 times; With the sterilized water suspension containing 50% glycerine, making its concentration is 60mg/ml.

The micro-bullet of step (4) preparation: get step (3) suspension 50 μ l, add 6 μ l plasmid DNA (1 μ g/ μ l), 20 μ l spermidines (0.1M), 50 μ lCaCl2 (2.5M).Concussion 1min, leaves standstill 1min, centrifugal, abandons supernatant; Add 70% ethanol to 600 μ l, supersound process 4-6 times under 200W power, each 1 second, centrifugal, abandon supernatant; Add 150 μ l dehydrated alcohols, do not destroy precipitation, leave standstill 1min, abandon supernatant; Add 60 μ l dehydrated alcohols, concussion, each bombardment amount 5-10 μ l.

Step (5) particle gun bombardment: particle gun is Bio-Rad PDS1000/He type, gets bronze suspension (60mg, the bronze of diameter 1.5-3.0 μ m, sterilize with dehydrated alcohol, be suspended in 1ml sterilized water) 25 μ l, add 5 μ gAmGS plasmid DNA, 50 μ l2.5mol/L Cacl2,20 μ l0.1mol/L spermidines, fully mix, centrifugal, dehydrated alcohol precipitation, again be suspended from 60 μ l dehydrated alcohols, for subsequent use.Can split film and select 1550psi.Can split film, carrier film and use in advance for 70% alcohol immersion half an hour, dry at Bechtop.Each 10 μ lDNA-bronze complex bodys of drawing are coated carrier film, fully dry.Choose vigorous, the of the same size callus of growth conditions and bombard, target material distance can be split film 5-10cm, every ware acceptor material bombardment 1 time.Callus after bombardment forwards in time on MS solid medium and to recover growth 4-7 days, screens, and the good stem section of choosing growth conditions is carried out Agrobacterium and infected conversion.

The preculture of step (6) acceptor material: recover the stem section after screening in selecting step (3), length should be 1-1.5cm, at least retains a bud, by the shallow stem section differentiation culture primary surface that is embedded in.Substratum thickness 1-2cm, every bottle of approximately 15-20ml of culture volume, every bottle of substratum can be put 6-8 stem sections.The stem section that embeds substratum is placed in to the dark 2-6d of cultivation under dark surrounds, 25 ℃ of left and right of culture temperature.

Step (7), Agrobacterium is infected: first the Agrobacterium LBA4404 bacterium liquid preparing and MS mother liquor are mixed by 1/3-1/2 volume ratio, mixed solution is poured into empty sterile culture flask and regulated pH6.2-6.7; Pre-incubated acceptor material stem section is taken out again, be placed in aseptic empty culture dish, soak acceptor stem section immediately with the liquid that infects preparing, carry out afterwards suitably carving wound under aseptic condition, control material is carved injury reason simultaneously equally.Carve after injury reason, acceptor material be transferred to fill in the culturing bottle that infects liquid and infect 15-20min, infect liquid long-pending the material of not infecting to be advisable, during infecting, shake intermittence and to infect bottle with hand so that material with infect liquid and fully contact.

Step (8) is cultivated altogether: by the acceptor stem section after infecting, blot surface liquid with aseptic filter paper, turn in surface and add on the Double-Medium of the wetting aseptic filter paper of lid layer MS mother liquor, still place by 6-8 stem sections of every bottle of substratum.The culturing bottle of sealing mouthful is placed in and under dark condition, cultivates altogether 2-4d.

Antibacterial and the kantlex screening and culturing of step (9): acceptor material is transferred and continued to cultivate in the screening culture medium that contains Kanamycin (kantlex) and ablastins Cefradine (cephamycin), and point 3 stages are carried out (pH6.2-7.0).First stage Kan10-15mg/L, Cef350-400mg/L, cultivates 5-8d; Subordinate phase Kan40-45mg/L, Cef300-350mg/L, cultivates 14-21d; Phase III card Kan45-50mg/L, Cef150-200mg/L, cultivates 20-30d.

Step (10), transformed plant PCR and Southern Blotting detect: through antibacterial and screening and culturing, acceptor stem segment table reveals obvious separation growth conditions, exceed 1/4 stem section occur by chrysanthemum to withered or variation, approximately 3/4 stem section sprouts green resistance sprouting, and CTAB method is extracted the genomic dna performing PCR augmentation detection of going forward side by side.Goal gene primer sequence is as follows: AmGS-F:TCA TGG CAC CTG ATA TCA CCA CCG CT; AmGS-R:TAT TAG GCA GCG GAT GGG GCG GGA A;

Reaction system: ddH2O (38.5 μ L); Buffer (6 μ L); DNTP (2 μ L); Primerl (1 μ L); Primer2 (1 μ L); Template DNA (1 μ L); TaqE (0.5 μ L); Total (50 μ L).

Amplification program: response procedures: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30sec; Renaturation, 55 ℃, 40sec; Amplification, 72 ℃, 1Min, 30cycle; Extend, 72 ℃, 10Min.

Transfer-gen plant to the PCR detected result positive and not genetically modified adjoining tree are delivered to Beijing biotechnology service department of Mei Laibo medical science and technology company limited simultaneously and adopt PCR labelling method to carry out Southern detection.Concrete grammar: with NotI and SacI double digestion pATCAmGS plasmid DNA, DIG-dUTP mark (digoxin hybridization check test kit), the fragment that obtains 800bp is label probe; Prehybridization: get the efficient hybridization solution of 10.0mlHyb (Hyb-100), add in hybrid pipe, prehybridization 2h in 60 ℃ of hybrid heaters.Probe sex change: probe 100 ℃ of sex change 10 minutes in PCR instrument, put immediately the cooling 5min of ice-water bath; Hybridization: drain prehybridization solution, add the good probe of the new sex change of 4.0 μ l at the efficient hybridization solution of 10.0mlHyb (Hyb-100), mix, hybridize and spend the night in 60 ℃ of hybridization instruments; Wash film, signal detection: use Mei Laibo DIGD-110 " digoxin hybridization check test kit I I (chemoluminescence method) ".

This beneficial effect of the invention is: the present invention adopts the gene transformation method of particle bombardment and agrobacterium-mediated transformation combination, the transgenic line winter resistance obtaining is significantly improved, the AmGS gene importing has improved the winter resistance of transgenic line, thereby provides important evidence for cultivating cold-resistant kind.

Accompanying drawing explanation

Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described, to better understand the present invention.

Fig. 1 is transgenosis photinia glabra PCR detection figure (description of symbols in figure: 1:DNA Marker in the embodiment of the present invention 1; 4: plasmid DNA, positive control; 8: unconverted plant, negative control; 2,3,5,6,7,12,13: transformed plant).

Fig. 2 is Southern detection figure (description of symbols in figure: M:DNAmarker, the DL2000 of transgenosis photinia glabra in the embodiment of the present invention 1; CK1: positive control, plasmid DNA; CK2: negative control, water; CK3: negative control, unconverted plant; R6, R7, R8 and R10: four different transgenic lines).

Embodiment

Embodiment 1:

Before particle gun bombardment, will cut little callus at following two kinds of pre-culture mediums (pre-bombardment culture media) the upper cultivation of PBCM I-II 3d:

PBCMI：MS+6-BA5mg／L+NAA0.5mg／L

PBCM?II：MS+6-BA5mg／L+NAA0.5mg／L+CaCl24.49／L

Comprise directly with tungsten powder bombardment and the tungsten powder bombardment that is enclosed with plasmid DNA (Plasmid Type is identical with the later plasmid by Agrobacterium-mediated Transformation) for micro-bullet of particle gun bombardment.

Get the tungsten powder that 60mg diameter is 1 μ m, add 1ml dehydrated alcohol, 95 ℃ of incubation 1h, supersound process 3 times under peak power, each 5min (or ultrasonic hot to feel with ultrasonic grinder), the centrifugal supernatant of removing; Add 1ml dehydrated alcohol, vortex concussion 3-5min, the centrifugal supernatant of removing; Add 1ml sterilized water, vortex concussion, centrifugal, abandon supernatant, repeat 3 times; With the sterilized water suspension containing 50% glycerine, making its concentration is 60mg/ml.

Get above-mentioned suspension 50 μ l, add 6 μ l plasmid DNA (1 μ g/ μ l), 20 μ l spermidines (0.1M), 50 μ lCaCl2 (2.5M).Concussion 1min, leaves standstill 1min, centrifugal, abandons supernatant; Add 70% ethanol to 600 μ l, supersound process 4-6 times under 200W power, each 1 second, centrifugal, abandon supernatant; Add 150 μ l dehydrated alcohols, do not destroy precipitation, leave standstill 1min, abandon supernatant; Add 60 μ l dehydrated alcohols, concussion, each bombardment amount 8-10 μ l.According to above condition, micro-bullet concentration of every rifle is 1 μ g/500 μ g (DNA/ tungsten powder).

The bombarding conditions of particle gun is: vacuum tightness-0.095MPa, and nitrogen pressure 7MPa, range 5cm, bombards after an bombards and carries out immediately Agrobacterium-mediated Transformation processing.Choosing length is the stem section of 1-1.5cm, at least retains a bud, by the shallow stem section differentiation culture primary surface that is embedded in.Substratum thickness 1-2cm, every bottle of approximately 15-20ml of culture volume, every bottle of substratum can be put 6-8 stem sections.The stem section that embeds substratum is placed in to the dark 2-6d of cultivation under dark surrounds, 25 ℃ of left and right of culture temperature.

First the Agrobacterium LBA4404 bacterium liquid preparing and MS mother liquor are mixed by 1/3-1/2 volume ratio, mixed solution is poured into empty sterile culture flask and regulated pH6.2-6.7; Pre-incubated acceptor material stem section is taken out again, be placed in aseptic empty culture dish, soak acceptor stem section immediately with the liquid that infects preparing, carry out afterwards suitably carving wound under aseptic condition, control material is carved injury reason simultaneously equally.Carve after injury reason, acceptor material be transferred to fill in the culturing bottle that infects liquid and infect 15-20min, infect liquid long-pending the material of not infecting to be advisable, during infecting, shake intermittence and to infect bottle with hand so that material with infect liquid and fully contact.

By the acceptor stem section after infecting, blot surface liquid with aseptic filter paper, turn in surface and add on the Double-Medium of the wetting aseptic filter paper of lid layer MS mother liquor, still place by 6-8 stem sections of every bottle of substratum.The culturing bottle of sealing mouthful is placed in and under dark condition, cultivates altogether 2-4d.

Acceptor material is transferred and in the screening culture medium that contains Kanamycin (kantlex) and ablastins Cefradine (cephamycin), continued to cultivate, and point 3 stages are carried out (pH6.2-7.0).First stage Kan15mg/L, Cef400mg/L, cultivates 8d; Subordinate phase Kan45mg/L, Cef350mg/L, cultivates 21d; Phase III card Kan50mg/L, Cef200mg/L, cultivates 30d.

Fig. 1 is transgenosis photinia glabra PCR detection figure (description of symbols in figure: 1:DNAMarker in the embodiment of the present invention; 4: plasmid DNA, positive control; 8: unconverted plant, negative control; 2,3,5,6,7,12,13: transformed plant).

Fig. 2 is Southern detection figure (description of symbols in figure: M:DNAmarker, the DL2000 of transgenosis photinia glabra in the embodiment of the present invention; CK1: positive control, plasmid DNA; CK2: negative control, water; CK3: negative control, unconverted plant; R6, R7, R8 and R10: four different transgenic lines).

Embodiment 2:

Before particle gun bombardment, will cut little callus at following two kinds of pre-culture mediums (pre-bombardment culture media):

PBCM I-II) the upper 3d that cultivates:

PBCMI：MS+6-BA5mg／L+NAA0.5mg／L+PVP16g／L

PBCMII：MS+6-BA5mg／L+NAA0.5mg／L+CaCl24.4g／L+PVP16g／L

Get the tungsten powder that 60mg diameter is 1 μ m, add 1ml dehydrated alcohol, 95 ℃ of incubation 1h, supersound process 3 times under peak power, each 5min (or ultrasonic hot to feel with ultrasonic grinder), the centrifugal supernatant of removing; Add 1ml dehydrated alcohol, vortex concussion 3-5min, the centrifugal supernatant of removing; Add 1ml sterilized water, vortex concussion, centrifugal, abandon supernatant, repeat 3 times; With the sterilized water suspension containing 50% glycerine, making its concentration is 60mg/ml..

Get above-mentioned suspension 50 μ l, add 6 μ l plasmid DNA (1 μ g/ μ l), 20 μ l spermidines (0.1M), 50 μ lCaCl2 (2.5M).Concussion 1min, leaves standstill 1min, centrifugal, abandons supernatant; Add 70% ethanol to 600 μ l, supersound process 4-6 time under 200W power, each 1 second, centrifugal, abandon supernatant; Add 150 μ l dehydrated alcohols, do not destroy precipitation, leave standstill 1min, abandon supernatant; Add 60 μ l dehydrated alcohols, concussion, each bombardment amount 8-10 μ l.According to above condition, micro-bullet concentration of every rifle is 1 μ g/500 μ g (DNA/ tungsten powder).

The bombarding conditions of particle gun is: vacuum tightness-0.095MPa, and nitrogen pressure 7MPa, range 10cm, bombards after an bombards and carries out immediately Agrobacterium-mediated Transformation processing.Choosing length is the stem section of 1-1.5cm, at least retains a bud, by the shallow stem section differentiation culture primary surface that is embedded in.Substratum thickness 1-2cm, every bottle of approximately 15-20ml of culture volume, every bottle of substratum can be put 6-8 stem sections.The stem section that embeds substratum is placed in to the dark 2-6d of cultivation under dark surrounds, 25 ℃ of left and right of culture temperature.

First the Agrobacterium LBA4404 bacterium liquid preparing and MS mother liquor are mixed by 1/4-1/3 volume ratio, mixed solution is poured into empty sterile culture flask and regulated pH6.2-6.7; Pre-incubated acceptor material stem section is taken out again, be placed in aseptic empty culture dish, soak acceptor stem section immediately with the liquid that infects preparing, carry out afterwards suitably carving wound under aseptic condition, control material is carved injury reason simultaneously equally.Carve after injury reason, acceptor material be transferred to fill in the culturing bottle that infects liquid and infect 20-25min, infect liquid long-pending the material of not infecting to be advisable, during infecting, shake intermittence and to infect bottle with hand so that material with infect liquid and fully contact.

By the acceptor stem section after infecting, blot surface liquid with aseptic filter paper, turn in surface and add on the Double-Medium of the wetting aseptic filter paper of lid layer MS mother liquor, still place by 6-8 stem sections of every bottle of substratum.The culturing bottle of sealing mouthful is placed in and under dark condition, cultivates altogether 2-4d.

Acceptor material is transferred and in the screening culture medium that contains Kanamyein (kantlex) and ablastins Cefradine (cephamycin), continued to cultivate, and point 3 stages are carried out (pH6.2-7.0).First stage Kan10mg/L, Cef350mg/L, cultivates 5d; Subordinate phase Kan40mg/L, Cef300mg/L, cultivates 14d; Phase III card Kan50mg/L, Cef200mg/L, cultivates 20d.

Embodiment 3: the winter resistance of transgenosis photinia glabra detects:

By expanding numerous subculture plant that obtains sufficient amount by cutting bud cuttage, to transplant on proliferated culture medium (formula is: MS+BA1.0mg/L+NAA0.05mg/L), every bottle of 4 strains, continue to cultivate 20d.Pre-treatment 24h under 4 ℃ of conditions, then respectively at 0 ℃ and-6 ℃ of freezing treatment 6,12,24 and 36h.After freezing treatment finishes, at 4 ℃, substratum 3h thaws under dark condition, then be placed at normal growth temperature recovery and cultivate that (temperature is 24 ℃, light application time 12h/d) after 48h, observe its phenotype, blade frostbite situation, adds up the plant survival rate (surviving total strain number × 100% of strain number/confession examination after recovery) in various processing.To its effect after two transgenic lines (R6 and R7) and not genetically modified adjoining tree freezing treatment be: process in experiment at 0 ℃, two transgenic lines, until process 24h, all do not show obvious injury; But not genetically modified adjoining tree, processing after 12h, shows obvious injury; Process after 24h plant mortality; Process after 36h, all dead.Process in experiment at-6 ℃, two transgenic lines until all do not show obvious injury after processing 12h, are being processed after 24h, and plant shows obvious injury, and plant mortality is being processed after 36h in the death of part plant.And not genetically modified adjoining tree shows obvious injury after processing 6h, the death of part plant, processes after 12h, and plant is all dead.The above results shows that the winter resistance of transgenic line R6 and R7 is significantly improved, and the AmGS gene of importing has improved the winter resistance of transgenic line.

The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.

Claims (1)

1. the method that photinia glabra high efficiency gene transforms, is characterized in that: what specifically adopt is the gene transformation method of particle bombardment and agrobacterium-mediated transformation combination, and its step comprises: the extraction of (1) plasmid DNA; (2) preparation of acceptor material; (3) tungsten powder pre-treatment; (4) micro-bullet preparation; (5) particle gun bombardment; (6) preculture of acceptor material; (7) Agrobacterium is infected; (8) cultivate altogether; (9) antibacterial and kantlex screening and culturing; (10) transformed plant PCR and Southern Blotting detect;

Above steps is specific as follows:

The extraction of described step (1) plasmid DNA: the agrobacterium strains LBA4404 that contains goal gene used is the binary vector pCAMBIA2300-35S-OCS that contains 35S promoter, goal gene, terminator NOS and marker gene NPTII, the cultivation of the thalline inoculation YEP solid medium that picking is preserved on Bechtop, bacterium plate is placed in constant incubator, cultivate 2～3d for 28 ℃, to single bacterium colony generation; The cultivation of picking list colony inoculation YEP liquid nutrient medium, on constant-temperature table, 28 ℃, 200～210r/min incubated overnight (24h left and right is to the logarithmic growth later stage), be to can be used for the extraction of plasmid DNA at 0.5～0.8 o'clock until OD600 value; From connect Agrobacterium cell the liquid YEP substratum containing 20mg/l Kanamycin and 15mg/lGentamycin5-8ml, then cultivate 24h at 28 ℃, 200-220rpm; Then carry out plasmid extraction; Fresh preparation SolII solution (880 μ l H20,100 μ lSDS10%, 20 μ l10N NaOH), and 200 μ l SolI (P1) solution that comprise 4mg/ml N,O-Diacetylmuramidase Lysozyme; Under 3600rpm speed, carry out centrifugal 12min to cultivating circadian Falcon test tube, abandon supernatant; With the resuspended agrobatcerium cell of 200 μ l5M NaCl, utilize vortex that agrobatcerium cell is mixed, transfer in Eppendorf pipe; Add 20 μ l10%N-Lauroylsarcosine sodium solution, turned upside down mixes (being inverted from different directions) for 6-8 times; The lower centrifugal 2min of top speed (13000rpm), loses supernatant liquor; Sol I (P1) (Sol I:50mM glucose with 200 μ l containing 4mg/ml Lysozyme, 25mM Tris-Cl (8.0), 10M EDTA (8.0)) resuspended precipitation, vortex is until mix, be placed on ice, add 400 μ l Sol II (P2) (Sol II:880 μ lH2O, 100 μ l SDS10%, 20 μ l10N NaOH), turned upside down mixes (being inverted from different directions) for 6-8 time, place 5min on ice, add 300 μ l Sol III (P3), vibrate with hand; Place 10min on ice; SolIII=Buffer P3, the lower centrifugal 12min of Neutrilization solution. top speed (12000rpm), by supernatant liquor, (700 μ l) transfer in new Eppendorf pipe, with 1 × phenol/chloroform (Phenol/Chloroforum:350 μ l Phenol+350 μ lChlororum, " Choloforum ": 24 times of volume chloroforum and 1 times of volume i soamylalcohol) extracting; In stink cupboard, in Eppendorf pipe, add phenol and chloroform; Vortex 15s, centrifugal 2min under 12000rpm; By upper strata phase, (700 μ l) carefully move in new Eppendorf pipe; Tilt to draw, prevent from sucking the phenol/chloroform of lower floor; By 700 μ lisopropanel precipitations, under room temperature, place 5min; Centrifugal 15min under 12000rpmg, carefully removes supernatant; First remove most of supernatant with large transfer pipet, residue 50 μ l left and right centrifugal 5min again; With 70% alcohol washing; Add 1ml70% alcohol, centrifugal 5min under 12000rpm, carefully removes supernatant; Under room temperature, dry; The resuspended precipitation of 20 μ lTE+10mg/ml RNase A; Cold house place diel, after store in-20 ℃ of refrigerators; By 100 times of DNA extraction liquid dilutions, measure its absorption value under wavelength 260nm and 280nm wavelength with UV-2450 ultraviolet-visible pectrophotometer, OD260/OD280=1.8～1.9 are high purity DNA, and being greater than 1.9 has RNA to pollute, and being less than 1.8 explanations has protein contamination; DNA concentration is calculated as follows: DNA concentration (ng/ μ L)=OD260 × 50 × extension rate; Be 200 μ g/mL, 500 μ g/mL, tri-kinds of concentration of 800 μ g/mL by plasmid DNA with 0.1%TE solution dilution, point install be housed in-20 ℃ of refrigerators for subsequent use;

The preparation of described step (2) acceptor material: photinia glabra stem segment length is 1-1.5em, do the use of particle gun bombardment through 0.4mol/L osmotic pressure substratum (MS substratum adds 0.2mol/L N.F,USP MANNITOL, 0.2mol/L sorbyl alcohol) processing 4-6h;

Described step (3) tungsten powder pre-treatment: get the tungsten powder that 60mg diameter is 1 μ m, add 1ml dehydrated alcohol, 95 ℃ of incubation 1h, supersound process 3 times under peak power, each 5min (, the centrifugal supernatant of removing; Add 1ml dehydrated alcohol, vortex concussion 3-5min, the centrifugal supernatant of removing; Add 1ml sterilized water, vortex concussion, centrifugal, abandon supernatant, repeat 3 times; With the sterilized water suspension containing 50% glycerine, making its concentration is 60mg/ml;

The micro-bullet of described step (4) preparation: get step (3) suspension 50 μ l, add 6 μ l plasmid DNA (1 μ g/ μ l), 20 μ l spermidines (0.1M), 50 μ lCaCl2 (2.5M); Concussion 1min, leaves standstill 1min, centrifugal, abandons supernatant; Add 70% ethanol to 600 μ l, supersound process 4-6 times under 200W power, each 1 second, centrifugal, abandon supernatant; Add 150 μ l dehydrated alcohols, do not destroy precipitation, leave standstill 1min, abandon supernatant; Add 60 μ l dehydrated alcohols, concussion, each bombardment amount 5-10 μ l;

Described step (5) particle gun bombardment: particle gun is Bio-Rad PDS1000/He type, gets bronze suspension (60mg, the bronze of diameter 1.5-3.0 μ m, sterilize with dehydrated alcohol, be suspended in 1ml sterilized water) 25 μ l, add 5 μ gAmGS plasmid DNA, 50 μ l2.5mol/LCacl2,20 μ l0.1mol/L spermidines, fully mix, centrifugal, dehydrated alcohol precipitation, again be suspended from 60 μ l dehydrated alcohols, for subsequent use; Can split film and select 1550psi, can split film, carrier film and use in advance for 70% alcohol immersion half an hour, dry at Bechtop; Each 10 μ lDNA-bronze complex bodys of drawing are coated carrier film, fully dry; Choose vigorous, the of the same size callus of growth conditions and bombard, target material distance can be split film 5-10cm, every ware acceptor material bombardment 1 time; Callus after bombardment forwards in time on MS solid medium and to recover growth 4-7 days, screens, and the good stem section of choosing growth conditions is carried out Agrobacterium and infected conversion;

The preculture of described step (6) acceptor material: recover the stem section after screening in selecting step (3), length is 1-1.5cm, retains at least one bud, by the shallow stem section differentiation culture primary surface that is embedded in; Substratum thickness 1-2cm, every bottle of approximately 15-20ml of culture volume, every bottle of substratum can be put 6-8 stem sections; The stem section that embeds substratum is placed in to the dark 2-6d of cultivation under dark surrounds, 25 ℃ of left and right of culture temperature;

In described step (7), Agrobacterium is infected: first the Agrobacterium LBA4404 bacterium liquid preparing and MS mother liquor are mixed by 1/3-1/2 volume ratio, mixed solution is poured into empty sterile culture flask and regulated pH6.2-6.7; Pre-incubated acceptor material stem section is taken out again, be placed in aseptic empty culture dish, soak acceptor stem section immediately with the liquid that infects preparing, carry out afterwards suitably carving wound under aseptic condition, control material is carved injury reason simultaneously equally; Carve after injury reason, acceptor material be transferred to fill in the culturing bottle that infects liquid and infect 15-20min, infect liquid long-pending the material of not infecting to be advisable, during infecting, shake intermittence and to infect bottle with hand so that material with infect liquid and fully contact;

Described step (8) is cultivated altogether: by the acceptor stem section after infecting, blot surface liquid with aseptic filter paper, turn in surface and add on the Double-Medium of the wetting aseptic filter paper of lid layer MS mother liquor, still place by 6-8 stem sections of every bottle of substratum; The culturing bottle of sealing mouthful is placed in and under dark condition, cultivates altogether 2-4d;

Antibacterial and kantlex screening and culturing in described step (9): acceptor material is transferred and continued to cultivate in the screening culture medium that contains Kanamycin (kantlex) and ablastins Cefradine (cephamycin), and point 3 stages are carried out (pH6.2-7.0); First stage Kan10-15mg/L, Cef350-400mg/L, cultivates 5-8d; Subordinate phase Kan40-45mg/L, Cef300-350mg/L, cultivates 14-21d; Phase III card Kan45-50mg/L, Cef150-200mg/L, cultivates 20-30d;

In described step (10), transformed plant PCR and Southern Blotting detect: through antibacterial and screening and culturing, acceptor stem segment table reveals obvious separation growth conditions, exceed 1/4 stem section occur by chrysanthemum to withered or variation, approximately 3/4 stem section sprouts green resistance sprouting, and CTAB method is extracted the genomic dna performing PCR augmentation detection of going forward side by side; Goal gene primer sequence is as follows: AmGS-F:TCA TGG CAC CTG ATA TCA CCA CCG CT; AmGS-R:TAT TAG GCA GCG GAT GGG GCG GGA A; Reaction system: ddH2O (38.5 μ L); Buffer (6 μ L); DNTP (2 μ L); Primerl (1 μ L); Primer2 (1 μ L); Template DNA (1 μ L); TaqE (0.5 μ L); Total (50 μ L); Amplification program: response procedures: denaturation, 94 ℃, 5min; Sex change, 94 ℃, 30sec; Renaturation, 55 ℃, 40sec; Amplification, 72 ℃, 1Min, 30cycle; Extend, 72 ℃, 10Min; Transfer-gen plant to the PCR detected result positive and not genetically modified adjoining tree adopt PCR labelling method to carry out Southern detection; Concrete grammar is: with NotI and SacI double digestion pATCAmGS plasmid DNA, DIG-dUTP mark (digoxin hybridization check test kit), the fragment that obtains 800bp is label probe; Prehybridization: get the efficient hybridization solution of 10.0mlHyb (Hyb-100), add in hybrid pipe, prehybridization 2h in 60 ℃ of hybrid heaters; Probe sex change: probe 100 ℃ of sex change 10 minutes in PCR instrument, put immediately the cooling 5min of ice-water bath; Hybridization: drain prehybridization solution, add the good probe of the new sex change of 4.0 μ l at the efficient hybridization solution of 10.0mlHyb (Hyb-100), mix, hybridize and spend the night in 60 ℃ of hybridization instruments; Wash film, signal detection.

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