CN101092633A - Method for raising conversion efficiency of pollen tube chnnael method by using toxoprotein gene of Agrobacterium - Google Patents

Abstract

This invention relates to a method for increasing the conversion efficiency of pollen tube pathway method by using agrobacterium tumefaciens virulence gene. The method comprises: combining or mixing agrobacterium tumefaciens virulence gene and the target gene to be converted (green fluorescent protein gene) in vitro, packaging with liposome, and converting plants, especially cotton by using pollen tube pathway method. The method combines agrobacterium-liposome-pollen tube pathway technique, and has obviously increased protoplast conversion efficiency. Plasmids are not only protected by liposome when entering embryo sacs, but also encapsulated by the toxic protein to be expressed when entering egg cells, which can improve the stability of exogenous DAN in cell conversion process, realize precise cutting in T-DNA region, and increase the conversion efficiency.

Description

Utilize toxoprotein gene of Agrobacterium to improve the method for the transformation efficiency of pollen tube passage method

Technical field:

The present invention relates to a kind of method of utilizing toxoprotein gene of Agrobacterium to improve the transformation efficiency of pollen tube passage method.

Background technology:

Agrobacterium-mediated transformation is that present research mechanism is the clearest, most widely used transgenic method Wu Xia, Jiao Gaili etc. and cotton agrobacterium mediation converted mechanism and progress [J]. Shanxi agricultural sciences, 2001,29 (1): 27～30; Fu Yongcai etc. the gramineous crop genetic transformation new development [J] of farming sense bacterium mediation. biotechnology journal, 1999,10 (3): 1～5; Stanton B, Gelvin, Agrobacterium-Mediated Plant Ttansformation:the Biology behind the " Gene-Jockying " Tool[J] .Microbiology and Molecular Biology reviews, 2003,16-37}.Because it can be by means of the enzyme system of Agrobacterium and plant, realize high-frequency autonomous conversion, thereby be one of focus { the Michielse CB of research always, et al.Agrobacterium-Mediated Transformation of Aspergillus awamori in the Absence ofFull-Length VirD2, VirC2, or VirE2 Leads to Insert ion of Aberrant T-DNA Structures.JBacteriol.2004,186 (7): 2038-2045; K Wang, A Herrera-Estrella, and M Van Montagu.Overexpression of virD1 and virD2 genes in Agrobacterium tumefaeiens enhances T-complexformation and plant transformation.J Bacteriol, 1990,172 (8): 4432-4440; Yumin Tao etal.Expression of plant protein phosphatase 2C interferes with nuclear import of theAgrobacterium T-complex protein VirD2[J] .PNAS, 2004,101 (14): 5164-5169; G Hansen, A Das, and M D Chilton.Constitutive expression of the virulence genes improves theefficiency of plant transformation by Agrobacterium[J] .Proc Natl Acad Sci U S A, 1994,91 (16): 7603-7607; Yang Qing, Chen Min, the host plant cell factor that civilian cutting edge of a knife or a sword .T-D N A shifts. Plant Physiology Communications [J], 2004,40 (4): 521-526.}.Its T-DNA shifts and integration process is summarized as follows: in Agrobacterium, after vir district genic system activated, the expression of structural gene in the VirD operon was processed shearing to T-DNA.At first there is the active virD1 albumen of topoisomerase I to combine, makes it loose with Ti-plasmids.VirD ₂As a kind of site-specific endonuclease, in the 25bp of T-DNA border sequence, T-DNA is sheared, produce the T-DNA strand.VirD ₂5 ' the end phosphoric acid of the DNA of the tyrosine that albumen is the 29th by hydroxyl on the aromatic nucleus and incision is with the covalency chain combination, then in the mode that is similar to bacterium conjugal transfer process with T-DNA and virD ₂The mixture that albumen is formed changes vegetable cell over to.In this process, T-DNA and many VirE2 protein molecular (dna single chain binding protein, no binding specificity) combine, and form after the T chain cpd, and this mixture is at VirD ₂And VirE ₂Under two-way nuclear localization signal (NLS) guiding, with virD ₂Albumen enters the nucleus of plant for guide's transhipment.VirD ₂Albumen has external ligase enzyme activity, infer participated in T-DNA 5 ' end with the connection of plant genome DNA Tinland B.et al.TheAgrobacterium tumefaciens VirD2 Protein virulence D2 protein is responsible for preciseintegration of T-DNA into the plant genome[J] .EEO J.1995.14:3585_3595}.Shurvinton etc. Shurvinton CE et al.Nucleear localization signal and the Cterminal omega sequencein the Agrobacterium tumefaciens VirD2 endonuclease are important for tumorformation[J] .Proc Nat Acad Sci USA, 1992,89:11837-11841} finds, at VirD ₂There is a conservative region near the carboxyl terminal in district, is called the ω district.The disappearance in this zone or replace sudden change can to make the transformed plant number be that the plant number of genetic stability reduces about two orders of magnitude.Therefore, VirD ₂May play an important role in the Plant Genome integration process at T-DNA.Studies show that Zhai Wenxue, Wang Wenming etc. advance in the side. the amplification of transgenic paddy rice T-DNA flanking sequence and analysis [J]. Acta Genetica Sinica, 2001,28 (4): 345-351; Hiei Y, el aL, Efficient transformation of rice (Orzu sutivu J) mediated by Agrobacteriumand sequence analysis of the Boundaries of the T-DNA[J] .The Plant Journal 1994,6 (2): 271-282; Mysore KS.Nam J.Gelvia SB.An Arabidopsis histore H2A mutantis deficient inAgrobacterium T-DNA integration[J] .proc.Natl.Acad.Sci, 2000,97:948-953.}:T-DNA preferentially be incorporated into the transcriptionally active district, change the many form random integrations with single copy of nuclear T-DNA over to plant chromosome, it is relatively also higher that it transforms back transcriptional activity.

Pollen tube channel conversion method and its maximum different being are transferred to the DNA in the vegetable cell, do not have the nuclear localization signal guiding, can not realize autonomous integration.Owing to exist with naked form, easily by the nuclease degradation inside and outside the cell, this may be that this method transformation efficiency is low, and the changing effect poor stability is subject to the basic reason of environmental influence.Our test finds that also in the lower time of florescence temperature, the transformation efficiency of cotton obviously improves, and supposition and nuclease are under relatively lower temp, and be active low relevant.

People (1996) such as Hansen G have created " agricultural rod spear method " (Agrolistic) based on Agrobacterium-mediated Transformation mechanism in conjunction with particle bombardment, with the virD of assist in transmutation in the Agrobacterium ₁, VirD ₂Gene is together squeezed into { Hansen G in the vegetable cell with particle gun together with the goal gene on the T-DNA, Chlton M D. " Agrolistic " transformation of plant cells:integration of T-strands generatedin planta[J] .proc Natl Acad sci USA.1996,93:14978-14983; Liu Zhaoming, Liu's aim, Bai Qingwu, the application [J] of square Rong Xiang .Agroinfiltration in molecular biology of plants research. biotechnology journal, 18, (4): 411-414}.The VirD that expresses ₁, VirD ₂Can cut the DNA of conserved sequence in vegetable cell and change T-DNA over to nucleus, its frequency accounts for 20% in total transformation event.This method concentrated clean cut in the conversion method for agrobacterium to shift and characteristic such as low copy integrations and particle bombardment in the advantage that limits of no host range.Proved above-mentioned conclusion equally with the direct conversion method of corn protoplastis, and show and add the VirE2 gene in addition again, frequency { the G Hansen that can further double raising typical T-DNA integrates, et al.T-strand integration inmaize protoplasts after codelivery of a T-DNA substrate and virulence genes[J] .Proc Natl Acad Sci U SA, 1997,94 (21): 11726-11730}.

Method like the application class, people such as Ziemienowicz in external structure one by Agrobacterium toxic protein VirD ₂, virE ₂Complex body with the single stranded DNA composition, can fast and effeciently DNA be transported to { A.Ziemienowicz in the mammiferous nucleus, .Import of DNA into mammalian nuclei by proteins originating from a plantpathogenic bacterium[J], Proc Natl Acad Sci U S A.1999,96 (7): 3729-3733}.For we organically combine with the pollen tube channel conversion method agriculture bacillus mediated, a kind of rising new approaches are provided.

Agrobacterium mediation converted and pollen tube channel conversion method are the main method that present cotton foreign gene transforms.The relative merits of the two are very obvious, but complementarity is very strong.How the two is organically combined, create a kind of simple to operately, not limited by tissue and cell regeneration, the transformation frequency height, the cotton transgenic method that genetic stability is good for carrying out functional gene analysis and transgene cotton breeding, has important practical significance.

Summary of the invention:

The purpose of this invention is to provide a kind of method of utilizing toxoprotein gene of Agrobacterium to improve the transformation efficiency of pollen tube passage method, mainly utilize agriculture bacillus mediated Plant Transformation mechanism, utilize the toxoprotein gene of Agrobacterium assist in transmutation, assemble or mixing with the goal gene (green fluorescence protein gene) that needs to transform external, after the liposome packing, adopt pollen-tube pathway method, plant is transformed, and its another purpose then is to utilize above-mentioned method to plant, especially cotton is transformed.

Purpose of the present invention mainly realizes by following process:

(1) structure of plant enhanced green fluorescence protein gene (EGFP) plant expression vector;

(2) virD ₁, virD ₂And virE ₂Gene clone, prokaryotic expression analysis;

Construction of prokaryotic expression vector and expression analysis: with virD ₁, virD ₂And virE ₂Gene downcuts from cloning vector; It is fused to the secreting type prokaryotic expression carrier; The downstream of colibacillus periplasm protein phoA or adventitia ompA protein signal guiding peptide nucleic acid sequence; Be built into the secreting type prokaryotic expression carrier; Be transformed in the Escherichia coli of L-type Cell Wall Deficient; Product directly is secreted in the culture medium; Extract purifying (alternative scheme adopts the method for conventional renaturing inclusion bodies); Albumen with purifying adds Freunds adjuvant immunity NZw; Extracting serum; The preparation primary antibodie is also measured and is tired

(3) virD ₁, virD ₂And virE ₂The structure of gene plant transient expression carrier;

The structure of plant transient expression carrier: utilize the sequence map and the restriction enzyme mapping of Agrobacterium Ti-plasmids, at virD ₁, virD ₂And virE ₂Gene coding region both sides design Auele Specific Primer, and on primer, add the restriction enzyme site that is used to clone, carry out corresponding gene amplification by the high-fidelity round pcr, product is directly implemented on the plant expression vector that has anti-kantlex marker gene, the complete expression cassette that will have strong plant promoter-toxoprotein gene-terminator downcuts, be building up on the multiple clone site of high-efficient cloning carrier pUC18, obtain not plant transient expression carrier with left and right sides border sequence;

(4) virD1, virD2 and virE2 gene or albumen and reporter gene (GFP) are studied transient expression and conversion integrating frequency that tobacco protoplast transforms through the liposome packing;

The assembled in vitro of T-DNA albumen composition: (this project is used green fluorescent protein to the goal gene that has that will need to transform, and GFP) pBin-EGFP plant expression vector joins in the vegetable cell extracting solution, and is sequentially added into and extracts and the virD of purifying ₁, virD ₂Albumen, then add dna polymerase i, produce corresponding T-DNA strand by nick translation, add virE2 albumen and hatch, use nuclease complete digestion mixture then, the gel blocking electrophoretic analysis, phenol chloroform extracting albumen meanwhile, ethanol sedimentation, purify DNA finally pass through Auele Specific Primer, corresponding T-DNA fragment is carried out the fluorescent quantitation amplification, assessment virD ₁, virD ₂And virE ₂Protein-active, best assembling mode of research T-DNA protein complexes and concentration proportioning;

Liposome packing: with T-DNA albumen composition or virD ₁, virD ₂And virE ₂Gene (having complete expression of plants framework) with contain goal gene GFP plasmid vector and mix by a certain percentage, pack processing with cationic-liposome (commercialization), and tobacco protoplast transformed, with fluorescent microscope transformant is carried out transient expression research, utilize microbiotic simultaneously, transformation of tobacco somatocyte system is carried out many generation screenings, regeneration plant also detects its integration efficiency, infer indirectly that with this toxalbumin is active in the intravital assembling of plant, on this basis liposome optimum conversion condition and damping fluid composition are studied;

(5) virD1, virD2 and virE2 gene can be in plant materials normal expression and having on the active basis, utilize the mechanism of Agrobacterium-mediated Transformation plant, liposome conversion and pollen-tube pathway method are combined, cotton transformation efficiency and genetic stability are studied and estimated;

(6) evaluation of improvement pollen-tube pathway method system: on the basis of (4) (5) research, utilize the mechanism of Agrobacterium-mediated Transformation plant, liposome conversion and pollen-tube pathway method are combined, the field transforms specific cotton, the offspring utilizes microbiotic and long-wave ultra violet lamp to screen in seedling stage, carry out the molecule checking simultaneously, determine that its transformation frequency carries out the tracking investigation of continuous multi-generation pedigree formula simultaneously to transgenic progeny, study its genetic stability.

Above-mentioned process is by per 1 * 10 ⁵～5 * 10 ⁶Individual protoplasm somatocyte adds the plasmid that concentration is 5～25 μ L pUC-pBin-GFP respectively, and concentration is 5～15 μ L pUC-pBin-VirD ₁, pUC-pBin-VirD ₂And pUC-pBin-VirE ₂The assistance gene assemble.

Above-mentioned optimization assembling is: pUC-pBin-VirD ₁+ UC-pBin-VirD ₂+ pUC-pBin-VirE ₂+ pUC-pBin-GFP, pUC-pBin-GFP plasmid concentration assist the concentration of gene little to the transformation efficiency influence with 15～20 μ L the bests.

During above-mentioned process liposome packing, utilize the toxoprotein gene of Agrobacterium assist in transmutation, external with goal gene (green fluorescence protein gene) assembling that needs to transform or mix, with plasmid concentration: [GFP]=3～6ug/ul; [VirD1]=3～5ug/ul; [VirD2]=3～5ug/ul; [VirE2]=3～5ug/ul, by 1: 1: 1: 5 assemblings, after the liposome packing, use pollen-tube pathway method, cotton is transformed, the effect comparison illumination that transforms shows raising, raise the efficiency and reach more than 4 times, after packing with liposome simultaneously, transform, the transformation efficiency comparison uses the assembling that contains among the present invention also can transform the other plant material according to improving more than 4.5 times.

The application of the method for transformation of above-mentioned pollen tube channel in cotton planting, the cotton bud of selecting be about to open in second day is clamped the bud top with clip, carry out selfing, select first and second fruits of each fruit branch to save the object of the flower of position as transgeneic procedure, bloom and transform about the 18～24h of back, before the conversion, need not shell flower and separate bract, directly use single-edge blade in the crosscut of calyx top, with style and the excision of tangent plane top petal, cut out the tangent plane of 0.3～0.7cm diameter simultaneously at the ovary top, drip dna solution with microsyringe then, amount 5～the 10uL that drips, DNA concentration 2～5ug/uL.

Compared with prior art, the present invention organically combines Agrobacterium-liposome-pollen-tube pathway method, utilizes the toxoprotein gene (virD of Agrobacterium assist in transmutation ₁, virD ₂And virE ₂), mix according to a certain percentage with the goal gene (green fluorescence protein gene) that needs to transform external, after the liposome packing, adopt pollen-tube pathway method to change plant over to, this is the improved route that a kind of utmost point has development potentiality for the transformation efficiency and the genetic stability that improve pollen tube channel.At gene VirD ₁, VirD ₂, VirE ₂Acting in conjunction under, the transformation efficiency of protoplastis has reached 60～66% apparently higher than other processing.

Method for transformation after the improvement utilizes liposome easily to go into by cytolemma picked-up characteristic, helps exogenous plasmid to enter cell cytoplasm from blastular, then, has the toxalbumin of nuclear localization signal, carries goal gene and independently passes nucleopore and enter nucleus.Plasmid is not only being gone into the blastular stage; be subjected to liposome protection; and after entering ovum; the toxalbumin bag quilt that can be expressed; improved the stability of foreign DNA in cell conversion process; can insert genomic mode and be mostly single copy accurately in the processing cutting of T-DNA district, and toxalbumin virD ₁Have the T-DNA of promotion and genome conformity, improve the function of transformation efficiency.

Description of drawings:

Fig. 1 is clone and the prokaryotic expression figure of green fluorescence protein gene GFP:

Fig. 2 is the clone of VirD1 gene and the technological line schema that prokaryotic expression carrier makes up:

Fig. 3 is the structure schema of VirD2 and prokaryotic expression carrier pET30a:

Fig. 4 is the clone and the prokaryotic expression schema of VirE2 gene:

Fig. 5 is the techniqueflow chart of the structure of virD1, virD2 and virE2 gene plant transient expression carrier:

Fig. 6 is microscopically GFP transient expression figure as a result in protoplastis:

Fig. 7 is the cell mass expression of results figure of microscopically GFP when cultivating the 28th day in protoplastis:

Fig. 8 is the PCR detected result figure of the protoplastis of conversion:

Fig. 9 cotton pollen tube passage method importing time experiment PCR detected result figure:

Among Fig. 8:

1: positive control, 2,3: negative control, 4,6: sedimentary PCR result in the liquid that cell is handled, 5,7: the PCR result of supernatant in the liquid that cell is handled, 8:DNA Marker DL2000.

Among Fig. 9:

Imported in 1～3:18 hour, imported in 4～6:22 hour, imported 10 in 7～9:48 hour: negative control, 11:DNA Marker, 12,13:24 hour imports, 14,15:36 hour imports 16～18: etiolated seedling negative control, 19: positive control.

Embodiment:

Embodiment 1:

The structure (referring to Fig. 1) of plant enhanced green fluorescence protein gene (EGFP) plant expression vector.

Embodiment 2:

VirD ₁, virD ₂And virE ₂Gene clone

1, the clone (referring to Fig. 2) of crown gall soil Agrobacterium Ti-plasmids toxicity district VirD1 gene

Crown gall soil Agrobacterium C58 VirD1 protein gene complete sequence according to U.Washington delivers designs a pair of primer voluntarily, and is as follows:

P1 is:＜210〉1,

5 '-CG GATATCATGTCGCAAGGCAGTAGG-3 ' Eco V (underscore) 26bp;

P2 is:＜210〉2,

5 '-CG AAGCTTTTACAAGGCGTCTTTCAGCA-3 ' HindIII (underscore) 28bp,

Article two, stride width of cloth 459bp between the primer, enzyme-added clip size of cutting after increasing with the protection base in the site is 474bp.

2, the clone (referring to Fig. 3) of crown gall soil Agrobacterium Ti-plasmids toxicity district VirD2 gene

The crown gall soil Agrobacterium C58 VirD that delivers according to U.Washington ₂The gene complete sequence designs a pair of primer voluntarily, gives birth to worker biotech firm by Shanghai and synthesizes, and its forward primer gene order P1 is:＜210〉3,

5’-CGCCATGGATGCCCGATCGAGCTCAAG-3’；

Reverse primer P2 is:＜210〉4,

5’-CGGTCGACTGCCATCACTCGGTCCTTC-3’。

Primer P1, the width of cloth of striding of P2 is 1380bp, and wherein P1 includes initiator codon ATG and Nco I (the following setting-out place) restriction enzyme site of the VirD1 that encodes, and P2 comprises SalI (following setting-out place) restriction enzyme site.

3, the clone (referring to Fig. 4) of crown gall soil Agrobacterium Ti-plasmids toxicity district VirE2 gene

Crown gall soil Agrobacterium C58 VIRE2 gene complete sequence according to U.Washington delivers designs a pair of primer voluntarily, gives birth to worker biotech firm by Shanghai and synthesizes, and its forward primer gene order P1 is:＜210〉5,

5’-CG AAGCTTATGGATCCGAAGGCCGA-3’；

Reverse primer P2 is:＜210〉6,

5’-CG CTCGAGTGACGCGGTATCAGGAA-3’。

Primer P1, the width of cloth of striding of P2 is 1690bp, wherein P1 includes coding VirE ₂Initiator codon ATG and HindIII (following setting-out place) restriction enzyme site, P2 comprises Xho I (following setting-out place) restriction enzyme site.

Embodiment 3:

VirD ₁, virD ₂And virE ₂Gene plant transient expression carrier pUC-pBin-VirD ₁, pUC-pBin-VirD ₂, pUC-pBin-VirE ₂And the structure of pUC-pBin-GFP.Technological line is seen Fig. 5.

Embodiment 4:

The transient expression of tobacco protoplast

1 extracts plasmid

Extract pUC-pBin-VirD in a small amount with alkaline lysis ₁, pUC-pBin-VirD ₂, pUC-pBin-VirE ₂With pUC-pBin-GFP, pBin-GFP, pUC-pBin plasmid, with the membrane filtration sterilization of 0.22 μ m, the back is standby in-20 ℃ of preservations.

2 transient expressions

2.1 following several processing is set:

1: negative control does not add plasmid;

2: add carrier pUC-pBin plasmid;

3: positive control adds the pUC-pBin-GFP plasmid;

4: load physique grain pUC-pBin and pUC-pBin-GFP plasmid;

5: add pUC-pBin-VirD ₁, pUC-pBin-VirD ₂, pUC-pBin-VirE ₂With the pUC-pBin-GFP plasmid;

6: add pUC-pBin-VirD ₂With the pUC-pBin-GFP plasmid;

7: add pUC-pBin-VirE ₂With the pUC-pBin-GFP plasmid;

8: add pUC-pBin-VirD ₁With the pUC-pBin-GFP plasmid;

2.2 following several plasmid concentration is set to be handled

Add plasmid by above-mentioned design.Usually with alkaline lysis carry plasmid concentration be approximately 0.1mg/mL, by per 10 ⁵

Individual protoplasm somatocyte adds pUC-pBin-GFP plasmid 5 μ L respectively, 10 μ L, and 15 μ L, 20 μ L, several different concentration gradients of 25 μ L are assisted gene pUC-pBin-VirD ₁, pUC-pBin-VirD ₂And pUC-pBin-VirE ₂With 5 μ L, 10 μ L, several different concentration gradients of 15 μ L, mixing gently after the adding leaves standstill jointly protoplastis and plasmid and secretly cultivates under room temperature; After cultivating 30min, begin under fluorescent microscope, to observe.

Embodiment 5:

The preparation of tobacco protoplast and purifying

(1) callus is moved in the aseptic culture dish, and be cut into small pieces, add enzyme liquid A, every 2g tissue adds 10mL enzyme liquid;

(2) culture dish is sealed with sealing film, the 7-9hr that slowly vibrates under 28 ℃ of conditions checks under inverted microscope, up to producing enough protoplastiss;

(3) the protoplastis suspension behind the enzymolysis is filtered with 80 order stainless steel mesh screens (or double-deck lens wiping paper), organize completely to remove not enzymolysis;

(4) filtrate is divided in the graduated centrifuge tube, the centrifugal 5min of 4000rpm precipitates protoplastis;

(5) remove supernatant with the transfer pipet suction, sedimentary protoplastis is suspended in the CaCl of 2/3 volume 0.2M ₂In;

(6) slowly inject 20% sucrose solution 0.5mL, the centrifugal 5min of 4000rpm with syringe to the centrifuge tube bottom; (after this step finishes, the purified complete protoplastis band of one deck will occur between the two phase liquid interface, impurity and fragment will sink to the pipe end.)

(7) inhale impurity and the sucrose solution of bottom and the CaCl on top that goes to manage the end with syringe ₂Solution; (only stay purified protoplastis in the centrifuge tube this moment)

(8) CaCl of usefulness 0.2M ₂Suspend, the centrifugal 5min of 4000rpm inhales and removes supernatant, uses DPD liquid nutrient medium repeated washing 2-3 time;

(9) protoplastis of collecting is suspended in an amount of DPD liquid nutrient medium, packing, each culture dish 2mL, the dull and stereotyped density of protoplastis is about 1 * 10 ⁵～5 * 10 ⁶Individual/mL;

(10) seal the dark 16～20hr that cultivates under 26 ℃ of conditions with sealing film.

Embodiment 6:

The result's statistics and the analysis of tobacco protoplast transient expression

Present embodiment has designed 8 kinds of processing (referring to embodiment 4:2.1), (referring to Fig. 6, Fig. 7) carried out counting statistics to fluorescigenic cell under fluorescent microscope: 5 repetitions are established in every kind of processing, each repeats to select at random 10 visuals field, 5 points are selected in each visual field, count fluorescigenic number of cells in 200 cells of each point, then each group data is on average drawn following result.Owing to handle 1 and add in the processing 2 of carrier pUC-pBin plasmid and do not have green fluorescence to produce under the ultraviolet excitation of wavelength 365nm, and protoplastis is black, and statistic data is 0, so do not list following table in.Other is handled the green fluorescence that all has in various degree and produces under ultraviolet excitation.Below be goal gene and assist gene fluorescigenic number of cells in every continuous 200 cells under various concentration combination:

Transformation efficiency cartogram under the table different treatment

From cartogram as can be seen, GFP is as broad as long to the transformation efficiency of protoplastis under the situation that vector plasmid pUC-pBin exists, the transformation efficiency of statistical result showed protoplastis is minimum, about 0.3%, illustrates that vector plasmid pUC-pBin is inoperative when GFP transforms protoplastis.

At VirD ₁, VirD ₂, VirE ₂In Jie Dao the conversion, transformation efficiency is compared the raising that has in various degree, wherein VirD with positive control respectively ₂, VirE ₂Jie Dao conversion is apparently higher than by VirD respectively ₁The conversion of mediation has reached about 7～13%, and VirD ₁The transformation efficiency of mediation is about 3～10%.

At gene VirD ₁, VirD ₂, VirE ₂Acting in conjunction under, the transformation efficiency of protoplastis has reached 60～66% apparently higher than other processing.

Embodiment 7:

The PCR of the protoplastis that transforms detects (referring to Fig. 9)

To use pUC-pBin-VirD ₁, pUC-pBin-VirD ₂, pUC-pBin-VirE ₂The protoplastis that transforms with the pBin-GFP plasmid is when cultivating 42 days, with the cell cultures mixed solution with the centrifugal 5min of 4000rpm, abandon supernatant, precipitation is suspended in TE (pH=8.0) solution, in high-temperature boiling 5min, the genome of tobacco is discharged, the above respectively cleerer and more peaceful template that is precipitated as, carry out PCR and detect (establishing two repetitions), the agarose electrophoresis with 1.0% detects PCR result.As shown in Figure 8,4 and 6 for not have the purpose band with the PCR that is precipitated as template, because precipitation may be dead cell and tissue; 5 and 7 for being the PCR result of template with the supernatant, conforms to expected results, illustrates that GFP has been incorporated in the tobacco gene group.

Embodiment 8:

Cotton pollen tube passage conversion method

1. the method for transformation of pollen tube channel

Selected second day to be about to open bud, and carried out selfing with plastic peg folder bud top.Select first and second fruits of each fruit branch to save the object of the flower of position as transgeneic procedure.Bloom and transform about the 18-24h of back.Before the conversion, need not shell flower, separately bract directly uses single-edge blade in the crosscut of calyx top, with style and the excision of tangent plane top petal, cuts out the tangent plane of 0.5cm diameter simultaneously at the ovary top.Drip dna solution with microsyringe then.Amount 5～the 10uL that drips, DNA concentration 2～5ug/uL.

2. the preliminary screening of transgene cotton

The seed of results is handled with 95% vitriol oil lint.Kantlex is mixed with the seed soaking aqueous solution of 300～500ppm concentration, soaks seed, seed soaking time 24h is a germinating bed with the fine sand of washing, after planting keeps germinateing 28 ℃, and humidity keeps 75～80%; Through about 7 days, cotton young plant leaf is open and flat fully, and cotton seedling is counted, and presses macula lutea rate preliminary evaluation transgenosis resistance individual plant.Extract total DNA of the plant of tool kalamycin resistance, with the cotyledon jaundice or from plant and do not have plants transformed to do negative control respectively, carry out PCR and identify.

3. improve the research of cotton pollen tube passage transformation efficiency

Cotton pollination back different time sections imports data statistics

The importing time (h)	15	18	22	24	36	48
							Take off bell rate (%)	23.45	10.25	0.76	1.48	1.91	5.38
Seedling greening-rate (%)	0	0.83％	0.57％	0.41％	0.05％	0.08％
							Green seedling PCR test positive rate (%)	0	100％	100％	100％	100％	100％

24h is as the criterion with self-pollination, and the importing time, more early it was high more to take off the bell rate.Along with the prolongation of self-pollination time, become the bell rate high more.Represent the transgenic positive rate with seedling greening-rate, better with 18～24h.All the other time transformation efficiencies are relatively poor.

The different importing made up the pollen tube passage method data statistics

Plasmid (ug/ul)	The actual bell number that imports	The actual bell number of receiving	Bolling rate (%)	The number of setting seeds	Green seedling number	Kantlex seedling greening-rate %
							GFP	1025	272	26.53	4532	24	0.52
1ug/ul GFP+ 1ug/ul VirD1+ 1ug/ul VirD2+ 5ug/ul VirE2	1053	436	41.41	6541	116	1.77
							1ug/ul GFP+ 1ug/ul VirD1+ 1ug/ul VirD2+ 5ug/ul VirE2+ liposome	534	331	61.98	5619	124	2.21
Agrobacterium directly drips	587	42	7.40	786	2	0.25

Plasmid concentration: [GFP]=5.23ug/ul; [VirD1]=3.48ug/ul; [VirD2]=4.92ug/ul; [VirE2]=4.33ug/ul

See in the table, show with the auxiliary effect comparison illumination that transforms of toxoprotein gene of Agrobacterium and improve that raise the efficiency and reach more than 3.4 times, if after pack with liposome simultaneously, transform, the transformation efficiency comparison is according to 4.25 times of raisings.The changing effect that Agrobacterium directly drips is not obvious.

Sequence table

＜110〉Shihezi Univ

＜120〉utilize toxoprotein gene of Agrobacterium to improve the method for the transformation efficiency of pollen tube passage method

<160>6

<210>1

<211>18

<212>DNA

＜213〉artificial sequence

<400>1

atgtcgcaaggcagtagg

<210>2

<211>20

<212>DNA

＜213〉artificial sequence

<400>2

ttacaaggcgtctttcagca

<210>3

<211>27

<212>DNA

＜213〉artificial sequence

<400>3

cgccatggatgcccgatcgagctcaag

<210>4

<211>27

<212>DNA

＜213〉artificial sequence

<400>4

cggtcgactgccatcactcggtccttc

<210>5

<211>17

<212>DNA

＜213〉artificial sequence

<400>5

atggatccgaaggccga

<210>6

<211>17

<212>DNA

＜213〉artificial sequence

<400>6

tgacgcggtatcaggaa

Claims (5)

1, a kind of method of utilizing toxoprotein gene of Agrobacterium to improve the transformation efficiency of pollen tube passage method is characterized in that realizing by following process:

(1) structure of plant enhanced green fluorescence protein gene plant expression vector;

(2) virD ₁, virD ₂And virE ₂Gene clone, prokaryotic expression analysis;

Construction of prokaryotic expression vector and expression analysis: with virD ₁, virD ₂And virE ₂Gene downcuts from cloning vector, it is fused to the secretor type prokaryotic expression carrier, the downstream of colibacillus periplasm protein phoA or adventitia ompA protein signal guiding peptide nucleic acid sequence is built into the secretor type prokaryotic expression carrier, is transformed in the intestinal bacteria of L type cell walls defective, the product direct secretion is in substratum, extract purifying, add freund's adjuvant immunity New Zealand white rabbit, extracting serum with the albumen of purifying, preparation one resists and measures and tire

(3) virD ₁, virD ₂And virE ₂The structure of gene plant transient expression carrier;

The structure of plant transient expression carrier: utilize the sequence map and the restriction enzyme mapping of Agrobacterium Ti-plasmids, at virD ₁, virD ₂And virE ₂Gene coding region both sides design Auele Specific Primer, and on primer, add the restriction enzyme site that is used to clone, carry out corresponding gene amplification by the high-fidelity round pcr, product is directly implemented on the plant expression vector that has anti-kantlex marker gene, the complete expression cassette that will have strong plant promoter-toxoprotein gene-terminator downcuts, be building up on the multiple clone site of high-efficient cloning carrier pUC18, obtain not plant transient expression carrier with left and right sides border sequence;

(4) virD1, virD2 and virE2 gene or albumen and reporter gene are packed through liposome, and transient expression and conversion integrating frequency that tobacco protoplast transforms are studied;

The assembled in vitro of T-DNA albumen composition: what will need to transform has a goal gene pBin-EGFP plant expression vector, joins in the vegetable cell extracting solution, and is sequentially added into and extracts and the virD of purifying ₁, virD ₂Albumen, then add dna polymerase i, produce corresponding T-DNA strand by nick translation, add virE2 albumen and hatch, use nuclease complete digestion mixture then, the gel blocking electrophoretic analysis, phenol chloroform extracting albumen meanwhile, ethanol sedimentation, purify DNA finally pass through Auele Specific Primer, corresponding T-DNA fragment is carried out the fluorescent quantitation amplification, assessment virD ₁, virD ₂And virE ₂Protein-active, best assembling mode of research T-DNA protein complexes and concentration proportioning;

Liposome packing: with T-DNA albumen composition or virD ₁, virD ₂And virE ₂Gene with contain goal gene GFP plasmid vector and mix by a certain percentage, pack processing with cationic-liposome, and tobacco protoplast transformed, with fluorescent microscope transformant is carried out transient expression research, utilize microbiotic simultaneously, transformation of tobacco somatocyte system is carried out many generation screenings, regeneration plant also detects its integration efficiency, infer indirectly that with this toxalbumin is active in the intravital assembling of plant, on this basis liposome optimum conversion condition and damping fluid composition are studied;

(5) virD1, virD2 and virE2 gene can be in plant materials normal expression and having on the active basis, utilize the mechanism of Agrobacterium-mediated Transformation plant, liposome conversion and pollen-tube pathway method are combined, cotton transformation efficiency and genetic stability are studied and estimated;

(6) evaluation of improvement pollen-tube pathway method system: on the basis of (4) (5) research, utilize the mechanism of Agrobacterium-mediated Transformation plant, liposome conversion and pollen-tube pathway method are combined, the field transforms specific cotton, the offspring utilizes microbiotic and long-wave ultra violet lamp to screen in seedling stage, carry out the molecule checking simultaneously, determine that its transformation frequency carries out the tracking investigation of continuous multi-generation pedigree formula simultaneously to transgenic progeny, study its genetic stability.

2, the method for utilizing toxoprotein gene of Agrobacterium to improve the transformation efficiency of pollen tube passage method as claimed in claim 1 is characterized in that in the described process, by per 1 * 10 ⁵～5 * 10 ⁶It is 5～15 μ L pUC-pBin-VirD that individual protoplasm somatocyte adds plasmid, the concentration that concentration is 5～25 μ LpUC-pBin-GFP respectively ₁, 5～15 μ L pUC-pBin-VirD ₂With 5～15 μ LpUC-pBin-VirE ₂The assistance gene assemble.

3, the method for utilizing toxoprotein gene of Agrobacterium to improve the transformation efficiency of pollen tube passage method as claimed in claim 2 is characterized in that in the described process, its assembling is:

PUC-pBin-VirD ₁+ UC-pBin-VirD ₂+ pUC-pBin-VirE ₂+ pUC-pBin-GFP, pUC-pBin-GFP plasmid concentration are 15～20 μ L.

4, as claim 1 or the 2 or 3 described methods of utilizing toxoprotein gene of Agrobacterium to improve the transformation efficiency of pollen tube passage method, when it is characterized in that described process liposome is packed, utilize the toxoprotein gene of Agrobacterium assist in transmutation, external with goal gene (green fluorescence protein gene) assembling that need to transform or mix, with plasmid concentration: [GFP]=3～6ug/ul; [VirD1]=3～5ug/ul; [VirD2]=3～5ug/ul; [VirE2]=3～5ug/ul, by 1: 1: 1: 5 assemblings, after the liposome packing, use pollen-tube pathway method, cotton is transformed.

5, a kind of application of method in cotton planting that utilizes toxoprotein gene of Agrobacterium to improve the transformation efficiency of pollen tube passage method, it is characterized in that selecting second day soon open cotton bud to clamp the bud top with clip, carry out selfing, select first and second fruits of each fruit branch to save the object of the flower of position as transgeneic procedure, bloom and transform about the 18-24h of back, before the conversion, need not shell flower and separate bract, directly use single-edge blade in the crosscut of calyx top, with style and the excision of tangent plane top petal, cut out the tangent plane of 0.3～0.7cm diameter simultaneously at the ovary top, drip dna solution with microsyringe then, amount 5～the 10uL that drips, DNA concentration 2～5ug/uL.

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