CN1900291B - Method for cultivating transgenic sycamore plant mediated by agrobacterium - Google Patents
Method for cultivating transgenic sycamore plant mediated by agrobacterium Download PDFInfo
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- CN1900291B CN1900291B CN200610019684A CN200610019684A CN1900291B CN 1900291 B CN1900291 B CN 1900291B CN 200610019684 A CN200610019684 A CN 200610019684A CN 200610019684 A CN200610019684 A CN 200610019684A CN 1900291 B CN1900291 B CN 1900291B
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Abstract
The present invention belongs to the field of plant transgene technology, and is especially agrobacterium mediating method of cultivating transgenic plant of sycamore as one garden tree. The agrobacterium mediating method includes the steps of genetic transformation, PCR detection and Southern hybridization detection, and features that through agrobacterium mediation to transduce GUS reporter gene to acceptor sycamore, adding kanamycin as selecting agent to the basic culture medium, screening the transformed acceptor cell, PCR detection, GUS gene detection, Southern hybridization and other steps, transgenic plant of sycamore is obtained. The present invention provides technological platform for the genetic improvement of tree, especially sycamore.
Description
Technical field
The invention belongs to the ornamental trees and shrubs gene engineering technology field.Be specifically related to a kind of method of utilizing agriculture bacillus mediated cultivation transgenic sycamore plant.
Background technology
Xylophyta mostly is perennial plant greatly, for farm crop etc., biennial herb plant, and characteristics such as (gene height heterozygosis) that it generally has, and that life cycle is long, maturation is bloomed is late, genetic background is complicated.Thus, adopt conventional breeding method that it is improved to be difficult in a short time and accomplish the end in view, cause the xylophyta genetic breeding to be made slow progress.In the last few years, plant genetic engineering carry out and the genetic improvement that is applied as xylophyta in xylophyta has brought boundless vital force.
In general, because the characteristics of xylophyta self limit, its molecular biology and Study on Genetic Transformation are comparatively backward with respect to farm crop etc.Yet constantly perfect along with the continuous maturation of molecular biology research technology and plant genetic transformation technology especially in recent years for the big quantity research of xylophyta genetic transformation, causes the xylophyta genetically engineered to obtain develop rapidly.People such as Fillatti utilized the Ti-plasmids of agrobacterium tumefaciens for carrier nptII gene and antiweed glyphosate gene aroA to be imported among white poplar * canine tooth poplar (P.al6-BA x P.grandidentata) clone NC-5339 in 1987, obtain complete regenerated plant, be incorporated in the Plant Genome and obtained normal expression through Southern blot and Western hybridization proof foreign gene, this be the first be the transfer-gen plant that transformation receptor obtains with trees.In the coming years, people have obtained success again in succession in other xylophyta Study on Genetic Transformation such as apple, walnut, Lee, fully confirm genetic transformation technology Application feasibility in xylophyta, thereby accelerated the pace of progress of xylophyta genetically engineered research.Up to the present, setting up genetic conversion system (Ceng Lihui on kind of forest and the orchard fruit surplus poplar, willow, eucalyptus, loose China fir class, locust tree, sweetgum, birch, elm, Acacia, Liriodendron, rubber tree, oranges and tangerines, apple, grape, Chinese chestnut, walnut, almond, Lee, apricot, peach, Kiwifruit, papaya, cherry, persimmon, lichee, longan, coffee, mango, cowberry, Ribes nigrum L., the immature fruit of Juteleaf Raspberry etc. 40, Lv Liuxin. orchard fruit Study on Genetic Transformation progress. the fruit tree journal, 2002,19 (3): 191-198; Huang Minren, Rao Hongyu. the forest genetically engineered. see: Wang Ming extend protection chief editor. Forest Tree Genetics and Breeding. Beijing, China Forest press, 2001b, 310-335; Ahuja M R.Genetic engineering of forest trees.In:Jain S M, Minocha S C (Eds) .Molecular Biology of WoodyPlants, Volume 1.Kluwer Academic Publishers, Dordrecht, Netherlands, 1999,31-50).
In addition, people are improving xylophyta genetic transformation condition, are improving transformation efficiency, are promoting that foreign gene has carried out furtheing investigate (Hand K H at aspects such as the intravital high efficiency stable expressions of recipient plant, Ma C-P, Strauss S H.Matrix attachment regions (MARs) enhancetransformation frequency and transgene expression in poplar.Transgenic Res, 1997,6:415-420; Minocha S C.Optimization of the expression of a transgene in plants.In:Jain S M, Minocha S C (Eds) .Molecular Biology ofWoody Plants, Volume 1.Kluwer Academic Publishers, Dordrecht, Netherlands, 1999,1-30), for solid basis has been established in the widespread use of xylophyta genetically engineered improvement. however, for farm crop, the xylophyta gene transformation technology is still in all many-sided bigger problems that exists, low as the gene transformation frequency, the transgenic plant regeneration difficulty, few from the available goal gene of xylophyta own, many in the transfer-gen plant that obtains at present from conversion to sexual material, low or instability of the expression level of foreign gene in transformed plant etc., therefore to make the improvement of xylophyta genetically engineered reach practicability, the commercialization level is still needed and is done further further investigation in many aspects.
Up to now, there is no the report that relevant transgenic sycamore plant is cultivated both at home and abroad.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, obtain the method for transgenic sycamore plant, for the further improvement of afforestation trees plane tree provides technology platform by agriculture bacillus mediated genetic transforming method.
The present invention is achieved in that
A kind of method of utilizing agriculture bacillus mediated cultivation transgenosis plane tree plant, its step comprises genetic transformation, with the acceptor material of the pretreated plane tree aseptic seedling blade of aseptic mannitol solution as genetic transformation, by agriculture bacillus mediated gus reporter gene is imported acceptor, utilize kantlex the recipient cell after transforming to be screened, detect screening by PCR, gus gene expression and Southern hybridization and obtain transgenosis plane tree plant as selective agent.
Concrete grammar of the present invention is as follows:
1) with the aseptic seedling blade of aseptic mannitol solution pre-treatment plane tree, the concentration of described mannitol solution is 0.4M, plane tree aseptic seedling blade is immersed in the described mannitol solution vibration 6h under 100r/min.
2) blade with step 1) infects with Agrobacterium, the aseptic seedling blade of above-mentioned pretreated plane tree is hindered with graduating with cutter cause wound, places OD
600For soaking 8-10min in the 0.6-0.8 Agrobacterium bacterium liquid, the blade after will infecting is transferred on common cultivation, recovery, selection and the root media successively to be cultivated, and screening obtains the candidate transfer-gen plant;
Wherein: described step of cultivating altogether comprises, the blade that infected with Agrobacterium, insert and be total in the culture medium, under 26 ± 1 ℃ of dark conditions, cultivate 5d, described medium component is as follows altogether: MS minimum medium+4.0mg/L 6-BA+1.0mg/L IBA+100 or 200 μ mol/L Syringylethanones, additional saccharose 30g/L; Agar 7g/L, pH5.8-6.0.
Wherein: described recovery culturing step comprises, with the above-mentioned blade of cultivating altogether, insert recovery media, under 50-100lux illumination, cultivate 10-15d, described recovery media composition is as follows: MS minimum medium+6.0mg/L6-BA+0.5mg/L IBA+300 or 400mg/L Timentin, additional saccharose 30g/L; Agar 7g/L, pH5.8-6.0.
Wherein: described selection culturing step comprises, the blade of cultivating with above-mentioned recovery, insert and select substratum, evoking adventive bud under 1000-1500lux illumination, change a fresh culture until differentiation adventitious buds every 2 weeks, described selection medium component is as follows: MS+6.0mg/L6-BA+0.5mg/LIBA+300 or 400mg/L Timentin+20 or 25mg/L kantlex, additional saccharose 30g/L, agar 7g/L, pH5.8-6.0.
Wherein: described root culture step comprises, the blade that differentiation culture is crossed, insert root media, antagonism is screened by plant, thereby obtain candidate's transgenosis resistant plant, described root culture based component is as follows: 1/2MS minimum medium+0.1mg/L IBA+20 or 25mg/L kantlex, additional saccharose 30g/L, agar 7g/L, pH5.8-6.0.
3) PCR detects candidate's transfer-gen plant, and concrete grammar is as follows:
With nptII reporter gene design primer, 5 ' end primer: 5 '-CCA TCG GCT GCT CTG ATG CCG CCG T-3 '; 3 ' end primer: 5 '-AAG CGA TAG AAG GCG ATG GCT GC-3 ', pcr amplification nptII gene specific dna fragmentation, fragment length is 693bp.
4) expression of detection gus gene: the explant that transforms is blotted surface liquid with aseptic filter paper, put into the centrifuge tube of 1.5ml, (every 100ml contains the NaH of 88.9mg X-Gluc, 1ml 10%Triton-100,10g paraxin, 5ml 1M to add reaction buffer
2PO
4, 20% methyl alcohol, pH transfer to 7.5), 37 ℃ of incubated overnight; Use rifle head sucking-off reaction buffer then, add 37 ℃ of water-baths of 75% ethanol again and soak 1h to slough pigment; Under stereoscopic microscope, observe and have or not blue reaction. compare the GUS response intensity of different treatment simultaneously. the selection by 2 months is cultivated, and the budlet to kantlex performance stable resistance that has explant to differentiate carries out the staining reaction of gus gene.
5) extraction is cut with restriction enzyme EcoR I enzyme through total DNA of the plane tree plant of PCR tests positive, and the dna fragmentation that enzyme was cut is transferred to Hybond-N
+On the nylon membrane, and use
32P prepares probe, to the film that takes a turn for the better carry out molecular hybridization, wash film, compressing tablet, the screening positive plant.
More detailed technical scheme is as follows:
1) preparation of explant
Select healthy and strong plane tree aseptic seedling after expansion breeding culture medium A (seeing Table 1) upward cultivates 1 month, do not adding the MS (MurashigeT of any hormone, Skoog F.A revised medium for rapid growth and bioassays with to6-BAcco tissue cultures.Physiol Plant, 1962,15:473-497) minimum medium or root media B (seeing Table 1) go up to cultivate about 15 days, selected the explant of the blade of the about 2cm of size in the three pieces of blades in top as conversion then.
2) preparation of bacterium liquid
Get a tubule bacterium liquid before the conversion and scrape culture dish with the inoculating needle of sterilizing, line on the LB solid medium that contains the 50mg/L kantlex, be inverted for 28 ℃ and cultivate, make the bacterial classification recovery and form single bacterium colony, this coils as the bacterial classification dish.The line of picking list bacterium colony is coated with completely new LB solid medium from the bacterial classification dish, be inverted and cultivate 1～2d under 28 ℃, make Agrobacterium propagation, then with the sterilization pocket knife with microorganism collection to the AB liquid nutrient medium (Wang Guanlin etc. that contain AS (Syringylethanone), the plant genetic engineering third edition, 2002, Science Press) in, the 180-200r/min thalline that suspends is again treated to infect after bacterium liquid OD600 value reaches 0.6-0.8.
3) N.F,USP MANNITOL pre-treatment: plane tree aseptic seedling blade (hereinafter to be referred as blade) in the step 1) is immersed in the mannitol solution of 0.4M of the bacterium of going out, places 100r/min shaking table vibration 6h.
4) infect: blade in the step 3) is placed on the aseptic filter paper, after the vertical master pulse direction of blade back scratches the 3-4 cutter, place Agrobacterium bacterium liquid to soak 8-10min with knife blade.
5) cultivate altogether: inhale the bacterium liquid that goes to the surface unnecessary with blade taking-up in the step 4) and with aseptic filter paper, leaf back inserts downwards and is total among the culture medium D (seeing Table 1), and dark (0lux) cultivates 5d under 26 ± 1 ℃ of conditions.
6) recover to cultivate: blade in the step 5) is forwarded on the recovery media E (seeing Table 1), place (50-100lux) cultivation 10-15d under the corresponding low light condition.
7) select to cultivate: blade in the step 6) is changed over to select on the substratum F (seeing Table 1), (1000-1500lux) carries out inducing of indefinite bud under the high light, after this, and every changing a fresh culture 2 weeks until differentiation adventitious buds.
8) screening of resistant buds: the blade of the budlet that regenerates in the step 7) is taken out from substratum, downcut stem with bud, change over to have and carry out the resistant buds screening among 25mg/LKan+300 or the 400mg/LTimentin expansion breeding culture medium A (seeing Table 1).
9) expansion of resistance seedling numerous with take root: expand numerous with carrying out subculture through the tissue cultured seedling of screening in the step 8) to forming a large amount of tissue cultured seedling, get tissue cultured seedling that height of seedling surpasses 2cm and downcut to change among the root media B (seeing Table 1) that contains kantlex 20mg/L and carry out root culture, thereby obtain candidate's transgenosis resistant plant.
Above-mentioned substratum is according to conventional reported method, and 121 ℃ sterilization 20min is standby down in high pressure steam.
10) PCR of resistant plant detects: resistant plant in the step 9) is carried out PCR detect, (specific procedure is edited referring to Liang Guodong, up-to-date molecular biology experiment technology to utilize PCR Molecular Identification technology, Science Press, 2001), with reference to (Xu QH, Zhang XL ﹠amp such as Xu; Nie YC (2002) Genetic diversity evaluation of cultivars (G.hirsutum L) resistant to Fusarium wilt by RAPD markers.Sci.Agricult.Sin.35:272-276) report, according to the sequence data of reporter gene (nptII) design primer: 5 ' end primer: 5 '-CCA TCG GCTGCT CTG ATG CCG CCG T-3 '; 3 ' end primer: 5 '-AAG CGA TAG AAG GCG ATG GCT GC-3 '.PCR circulating reaction parameter: 94 ℃ of sex change 4min, then successively at 94 ℃ of sex change 1min, 57 ℃ of renaturation 1min, 72 ℃ are extended 1min, circulates to be incubated 10min, last 4 ℃ of preservations at 72 ℃ after 35 times.Get 10 μ L amplified productions and carry out electrophoresis detection containing on 0.8% sepharose of ethidium bromide, observations and taking a picture under the Ultraluminescence.
11) the Southern hybridization of the positive strain of PCR system detects: extraction step 10) through the total DNA of the plant of PCR tests positive, get 10-20 μ g transgenosis plane tree plant DNA, number is prepared the endonuclease reaction mixed solution per sample: each reaction system contains 10 * Buffer, 5 μ L, restriction enzyme EcoR I 2 μ L contain (20u), water 28 μ L, mixing, divide and be filled in the centrifuge tube of 0.5mL, every pipe 35 μ l, add total DNA 15 μ L, the enzyme system of cutting is 50 μ L, and careful bullet is even, instantaneous centrifugal, place 37 ℃ of water-baths to spend the night (12-18h), the next morning gets 3 μ L electrophoresis detection enzymes and cuts effect, and enzyme is cut abundant back adding 5 μ L tetrabromophenol sulfonphthalein damping fluids termination enzyme and cut, place 4 ℃, until electrophoresis.
With 0.7% agarose (Sigma company product) gel electrophoresis, 0.8-1.0V/cm electrophoresis 18-24h.Glue is cut into film equally size and shape; With 0.2mol/L HCl sex change 10min, placing changes on the film platform, as transfering buffering liquid, dna fragmentation is transferred to Hybond-N with 0.4mol/L NaOH
+On the nylon membrane, shift about 24h.The film that takes a turn for the better is with 2 * SSC rinsing twice, each 5min; With the filter paper pads folder, more than 80 ℃ of vacuum baking film 2h; Take out cooling, it is standby to place 4 ℃ of refrigerators to preserve.
32The preparation of p probe, molecular hybridization, the operation of washing film, compressing tablet are undertaken by the described method of molecular cloning test guide (third edition) (2002, Science Press) that Sa nurse Brooker, Russell write.
Table 1, enumerated the prescription that is used for all kinds of substratum that transgenic sycamore plant cultivates involved in the present invention:
The substratum table look-up that table 1 the present invention adopts
Remarks: used substratum in the experiment, equal additional saccharose 30g/L, agar 7g/L, pH5.8-6.0
Positively effect of the present invention is:
The present invention obtains transgenic sycamore plant at home and abroad first, for the genetic improvement of ornamental trees and shrubs such as plane tree from now on provides new technology platform as utilizing transgenic technology to cultivate no cone plane tree.
Description of drawings
Fig. 1. the technology of the present invention route map
Fig. 2. be the physical map of the plasmid pCAMBIA2301 of a report using of the present invention
Fig. 3. the PCR detected result of plane tree gus gene resistant plant
Fig. 4. be the Southern hybridization detected result of the positive strain of PCR system in the embodiments of the invention
Fig. 5. the kantlex of different concns is to the influence of plane tree aseptic seedling leaf regeneration
Fig. 6. the fungistat of different sorts and concentration is to the influence of plane tree aseptic seedling leaf regeneration
Fig. 7. the susceptibility that the blade of different physiological statuss infects for Agrobacterium and to the influence of differentiation adventitious buds
Fig. 8 .GUS moment detection of expression
Fig. 9. the GUS stably express of resistant buds detects
Figure 10. breaking up and resistant buds Cheng Miao of explant through selection
Figure 11. the PCR of kalamycin resistance bud detects the blade size infects plane tree to Agrobacterium influence
Figure 12. height oozes the influence to the moment expression rate of treatment time and mode
Figure 13. incubation time is to the influence of GUS moment expression rate altogether
Figure 14. bacterial concentration is to the influence of moment expression rate
Figure 15. time of infection is to the influence of moment expression rate
Embodiment
Following examples further define the present invention.According to above description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.
Embodiment 1: the method for cultivation of transgenic sycamore plant
1, transformation receptor material and cultural method
Gathering the plane tree seed in Chinese Wuhan City, Hubei Province Hua Zhong Agriculture University campus is induced material, and inducing the test-tube plantlet of generation through hypocotyl is experiment material.Test-tube plantlet propagation, take root and the leaf regeneration cultural method with reference to Liu Guofeng and Bao Manzhu (Liu G, Bao M, Adventitious shootregeneration from vitro cultured leaves of London plane tree (Platanus acerifolia Willd.) Plant Cell Rep, 2003,21:640-44) reported method.
Except that specifying, medium component of the present invention is as shown in table 1.The pH value transfers to 5.8-6.0, according to a conventional method sterilization.
2, conversion carrier material and cultural method
Comprise report plasmid pCAMBIA2301 and (see Genebank, gene accession number: gi:7638149, the physical map of this plasmid is referring to accompanying drawing 2) the agrobacterium tumefaciens bacterial strain be that EHA105 is so kind as to give by Institute of Botany, Chinese Academy of Sciences, this plasmid carries gus reporter gene and nptII selects gene, wherein gus gene contains an intron, guaranteeing that gus gene only expresses in vegetable cell, and in Agrobacterium, do not express.Make substratum with LB (Wang Guanlin and the Fang Hong skin of bamboo, 1998), take by weighing peptone 10g, yeast extract 5g respectively, sodium-chlor 10g is settled to 1L, and pH transfers to 7.0-7.2, adds the 13g/L agar powder during solid culture, presses the preceding method sterilization.
(1) activation of bacterial strain
Take out the bacterial classification of being preserved from-70 ℃ of refrigerators, draw with transfering loop and get directly stroke flat board of back, flat board is the LB substratum, cultivates additional kantlex (Kan) 50mg/L of Agrobacterium.Flat board is put into 28 ℃ of incubators, is cultured to single bacterium colony and produces.
(2) preservation of bacterial strain
Flat board is put into 4 ℃ of refrigerators preserve, every two weeks activation once.If will preserve down at-70 ℃, then choose single bacterium colony to answer the liquid LB substratum of 100mg/L Kan in additional phase from flat board, put into that 28 ℃ of 150rpm/min shake bacterium to OD on the constant temperature shaking table
600=1.0, in sterilized 1.5mLEppendorf pipe, add 0.85mL Agrobacterium bacterium liquid and 0.15mL glycerine, put into the refrigerator prolonged preservation.
3, agriculture bacillus mediated genetic transforming method
1) N.F,USP MANNITOL pre-treatment: select the blade of the about 2cm of size in three at the top of plane tree aseptic seedling to be immersed in the mannitol solution of 0.4M of the bacterium of going out, place the 100r/min 6h that vibrates.
2) infect: blade in the step 1) is placed on the aseptic filter paper, behind knife blade scuffing blade 3-4 cutter (preferable methods is to hinder with graduating with cutter in the vertical master pulse direction of blade back), place OD
600Soak 8-10min in the Agrobacterium bacterium liquid for 0.6-0.8.
3) cultivate altogether: step 2) in blade take out and inhale the bacterium liquid that goes to the surface unnecessary with aseptic filter paper, leaf back inserts downwards altogether among the culture medium D (seeing Table 1), dark culturing 5d under 26 ± 1 ℃ of conditions.
4) recover to cultivate: blade in the step 3) is forwarded on the recovery media E (seeing Table 1), place (50-100lux) cultivation 10-15d under the corresponding low light condition.
5) select to cultivate: blade in the step 4) is changed over to select on the division culture medium F (seeing Table 1), (1000-1500lux) carries out inducing of indefinite bud under the high light.After this, every changing fresh culture 2 weeks one time.
6) screening of resistant buds: the blade of the budlet that regenerates in the step 5) is taken out from substratum, downcut band bud fragment, change over to have and carry out the resistant buds screening among 25mg/LKan+300 or the 400mg/L Timentin expansion breeding culture medium A (seeing Table 1).
Establish blade that inoculation infects without Agrobacterium in the conversion and infect but indiscriminate two kinds of processing compare through Agrobacterium.
3, the histochemical method of gus gene moment expression detects
The blade of cultivating altogether, blot surface liquid with aseptic filter paper after, put into the centrifuge tube of 1.5ml, (every 100ml contains the NaH of 88.9mg X-Gluc, 1ml 10%Triton-100,10g paraxin, 5ml 1M to add reaction buffer
2PO
4, 20% methyl alcohol, pH transfer to 7.5), 37 ℃ of incubated overnight.The sucking-off reaction buffer adds 37 ℃ of water-baths immersions of 75% ethanol 1h and sloughs pigment then.Observation has or not blue reaction under the Nikon-AFX-II stereoscopic microscope, compares the GUS response intensity of different treatment simultaneously.10 explants of every processing observation, statistics produces Bluepoint (or locus coeruleus) number that occurs on the blue explant number that reacts and the every explant.
Explant differentiates resistant buds through after selecting cultivation, gets 1-2 sheet young leaflet tablet or part young shoot, puts into the centrifuge tube of 1.5ml, adds reaction buffer (the same), 37 ℃ of incubated overnight.The sucking-off reaction buffer adds 37 ℃ of water-baths of 75% ethanol and soaks 1h to slough pigment then, observes to have or not blue reaction.
More than 5 repetition are all established in test, and at least 10 blades of repeated using at every turn carry out statistical study with ANOVA, the horizontal P=0.05 of least significant difference after the percentage ratio result is converted into inverse sine.
The moment expression of gus gene is seen Fig. 8, and Fig. 8 shows that the GUS expressive site of partial blade concentrates on the vein place, and especially the wound is expressed stronger.And the leaf regeneration position of plane tree also mainly is to sprout in blade master pulse wound, and therefore, the result who expresses from GUS moment it seems that plane tree regenerative cell position is consistent with transformed competence colibacillus cell position.The processing of various combination, GUS expresses strong and weak different, and coloration result shows, with the N.F,USP MANNITOL pre-treatment 6h of 0.4mol/L, be that the bacterium liquid of 0.6-0.8 infects 10min with the OD value, cultivate the processing of 5d altogether, after GUS dyeing, dye locus coeruleus that blue leaf expression goes out at most, the brightest (seeing Fig. 8-1).
4, the screening of taking root of resistant buds
Learnt from else's experience and selected to cultivate the resistance budlet of individual month leaf explant differentiation of 2-3, insert in the plane tree proliferated culture medium of additional 300mg/L Timentin and 20mg/L Kan, change additional 300mg/L Timentin and 25mg/L Kan root media after two months over to. find through observing, the false bud that transforms of part begins to occur yellow leaf in the process of breeding and taking root, stop growing, until last browning, dead (seeing Figure 10-6). as seen in selecting the process of cultivating, still have the appearance of false positive resistant buds, therefore, in the propagation in later stage and rooting process, continue to apply selective pressure and be very important, can screen the false resistant buds of a part.
In addition, compare with adjoining tree, resistant buds is taken root and is taken about late 15-20 days, and root system do not contrast sturdyly, and secondary root also obviously reduces.
5, the histological chemistry of gus gene stably express is detected
Selection by 2 months is cultivated, the budlet to kantlex performance stable resistance that has explant to differentiate carries out the staining reaction (see figure 9) of gus gene, found that, the colour developing position difference of resistant buds, the resistant buds integral body that has presents light blueness, have plenty of spire and show light bluely, and very bright locus coeruleus appears in certain the bud point of having only on whole resistant buds that also has.
6, the PCR of transfer-gen plant detects
With reference to (Xu QH, Zhang XL ﹠amp such as Xu; Nie YC (2002) Genetic diversity evaluation of cultivars (G.hirsutum L.) resistant to Fusarium wilt by RAPD markers.Sci.Agricult.Sin.35:272-276) reported method, according to the sequence data design primer of reporter gene (nptII), primer sequence is as follows:
5 ' end primer: 5 '-CCA TCG GCT GCT CTG ATG CCG CCG T-3 '
3 ' end primer: 5 '-AAG CGA TAG AAG GCG ATG GCT GC-3 '.
Reaction system is:
Deionized water 12.75 μ l
dNTP(2mM) 2μl
Primer1(10mM) 1μl
Primer2(10mM) 1μl
IOXBuffer 2μl
Taq(5U/L) 0.25μl
Template DNA (20ng/ μ l) 1 μ l
Cumulative volume 20 μ l
PCR circulating reaction parameter: 94 ℃ of sex change 4min, then successively at 94 ℃ of sex change 1min, 57 ℃ of renaturation 1min, 72 ℃ are extended 1min, circulates to be incubated 10min, last 4 ℃ of preservations at 72 ℃ after 35 times.Get 10 μ L amplified productions and carry out electrophoresis detection containing on 0.8% sepharose of ethidium bromide, observations and taking a picture under the Ultraluminescence.
According to the preliminary transformation system of setting up, 236 explants are transformed, obtain 12 strain resistant plants, extract plant genome DNA as template from blade, with the Agrobacterium plasmid DNA as positive control, with unconverted aseptic seedling DNA as negative control, carry out pcr amplification, the result as shown in Figure 3, PCR specific amplified fragment does not appear in unconverted plant genome, genetically modified plant obtains the fragment of a 693bp the same with the expection length scale through pcr amplification.12 strain resistant plants are carried out PCR detect, 8 strains be positive (Fig. 3) are wherein arranged.
7, the Southern of transfer-gen plant hybridization detects
Get 10-20 μ g transgenosis plane tree plant DNA, number is prepared the endonuclease reaction mixed solution per sample: each reaction system contains 10 * Buffer5 μ L, restriction enzyme EcoR I 2 μ L contain (20u), water 28 μ L, mixing, divide to be filled in the centrifuge tube of 0.5mL, every pipe 35 μ 1 add total DNA 15 μ L, the enzyme system of cutting is 50 μ L, careful bullet is even, and is instantaneous centrifugal, places 37 ℃ of water-baths spend the night (12-18h), the next morning gets 3 μ L electrophoresis detection enzymes and cuts effect, enzyme is cut abundant back adding 5 μ L tetrabromophenol sulfonphthalein damping fluids termination enzyme and is cut, and places 4 ℃, until electrophoresis.
With 0.7% agarose (available from Sigma company) gel electrophoresis, 0.8-1.0V/cm electrophoresis 18-24h.Glue is cut into film equally size and shape; With 0.2mol/L HCl sex change 10min, placing changes on the film platform, as transfering buffering liquid, dna fragmentation is transferred to Hybond-N with 0.4mol/L NaOH
+On the nylon membrane, shift about 24h.The film that takes a turn for the better is with 2 * SSC rinsing twice, each 5min; With the filter paper pads folder, 80 ℃ (more than the vacuum baking film 2h; Take out cooling, it is standby to place 4 ℃ of refrigerators to preserve.
32P prepares probe, molecular hybridization, washes film, the operation of compressing tablet is undertaken by the described method of molecular cloning test guide (third edition) (2002, Science Press) that Sa nurse Brooker, Russell write.
In 8 strain systems of PCR male, 62.5% (5 strain systems) Southern hybridization is positive, and transformation efficiency is 2.12% (5/236).The Southern results of hybridization shows that the npt-II gene inserts at random, and copy number is 1-2, and wherein 60% is single copy, and 40% is two copies.Other 3 PCR are positive, and fail to stablize in the insertion plane tree genome but the negative strain system of Southern hybridization illustrates gus gene, may lose in the subculture process (Fig. 4).
Embodiment 2: simultaneous test embodiment
1, the effectively experiment of selective pressure of kantlex (Kan)
With plane tree aseptic seedling blade is explant, the selective pressure of corresponding kantlex (Kan) when determining its regeneration.Respectively with the plane tree blade inoculation that scratches on the division culture medium C (seeing Table 1) of additional different concns Kan, relatively the different concns microbiotic is to the restraining effect of explant evoking adventive bud differentiation, to determine that suitable screening concentration is used for genetic transformation.According to the preliminary experiment result, the plane tree leaf explant is established 0,12.5,20,25, five kinds of Kan concentration gradients of 50mg/L.Changed once new substratum in per 15 days, the brownization rate and the differentiation rate of statistics explant after 60 days.
In the plant genetic conversion process, especially with the conversion system of adventitious organogenesis regeneration plant, tend to produce more mosaic or false transformant, therefore, the selective agent of using proper concn when transforming seedling rooting has vital role for the further screening of transfer-gen plant.The test-tube plantlet of getting band 3-4 sheet leaf is inoculated in respectively and adds different concns Kan (0,12.5,20,25, among root media B 50mg/L) (seeing Table 1), observe the influence of the selective agent of different concns to rooting of vitro seedling and growth, screening transforms the suitable selective pressure of seedling when determining to take root.5 bottles of every concentration of treatment inoculations, 5 explants of every bottle graft.After 2 months, rooting rate, mean elements and the root of statistics test-tube plantlet are long.
The used plasmid of present embodiment is pCAMBIA2301, and it is nptII that this plasmid carries marker gene, is transformed explant to show as kalamycin resistance.Cell transformed goes up at the selection substratum F (seeing Table 1) of additional certain density kantlex can normal growth, differentiation and obtain regeneration plant.The present invention inserts unconverted plane tree aseptic seedling blade, test-tube plantlet respectively in the substratum of the kantlex that contains different concns, observe and add up of the influence of the selective agent of different concns, to determine different explants corresponding concentration of selecting in the different choice agent to the different explants growth.
As can be seen from Table 2, the blade of plane tree is very responsive to kantlex. from the explant of experiment, observe the blade of plane tree itself than being easier to brownization, in the time of on the substratum that does not add kantlex, just there is higher brownization rate the wound, cultivate after 40 days, brownization of blade rate in the control medium also reaches 16.0%. when blade inoculation during to the division culture medium C that contains the 12.5mg/L kantlex (seeing Table 1), brownization rate reaches 24.0%, though most blades still keep oyster, obviously do not expand but have, substantially do not form callus, bud ratio also only is 0.2%; When kantlex concentration is increased to 20mg/L, suppressed the formation of callus and the differentiation of bud fully, and in the kantlex more than 25mg/L, the blade majority of plane tree becomes chocolate, final dead (see figure 5).
Therefore the optimal concentration of kantlex is 20mg/L in the selection substratum of the present invention.
The kantlex of table 2 different concns (Kan) is to the influence of plane tree leaf regeneration
Annotate: data are mean value ± standard error in the table, the significant difference (P=0.05) that exists between the upper right target The English alphabet registration certificate of same column of figure.
The test-tube plantlet of outstanding bell art changed over to respectively contain 0,12.5,20,25, the root media B of 50mg/L kantlex (seeing Table 1), as can be seen from Table 3, in the root media that contains the 12.5mg/L kantlex, the rooting rate of test-tube plantlet drops to 52%; And when kantlex was 20mg/L, the rootability of test-tube plantlet then dropped to 12% immediately, and growth of seedlings also obviously slows down; When kantlex is 25mg/L when above, test-tube plantlet can not be taken root fully.And along with the increase of Kan concentration, seedling stops growing gradually, and yellow, browning appear in blade.When plane tree conversion seedling was taken root screening, the kantlex preferred concentration was 25mg/L.
The kantlex of table 3 different concns (Kan) is to the influence of plane tree rooting of vitro seedling
Annotate: data are mean value ± standard error in the table, the significant difference (P=0.05) that exists between the upper right target The English alphabet registration certificate of same column of figure.
2, different fungistat susceptibility experiments
In explant surface after cultivating altogether and the shallow layer tissue a large amount of symbiotic Agrobacteriums are arranged, as not controlling and suppress the growth of bacterium timely and effectively, can have a strong impact on the growth and the differentiation of explant, therefore must take off bacterium cultivates. and different fungistat is for the difference that influences of different plant explants generations. and the applicant has selected two kinds of fungistats to compare test in the present embodiment, therefrom select the suitableeest fungistat. after the blade scuffing with plane tree sterile test tube seedling, be inoculated in different concns (0 respectively, 100,300,500mg/L) cephamycin or (0,100,300,500mg/L) among the division culture medium C of Timentin (seeing Table 1), the cephamycin of observation different concns or Timentin add up its differentiation rate to the influence of blade adventitious bud inducing after 60 days.
Test-results shows that cephamycin has the obvious suppression effect to the regeneration of plane tree blade.As shown in table 4, when the blade inoculation of plane tree was in the substratum that contains the different concns cephamycin, the differentiation of its indefinite bud all was subjected to influence in various degree.Wherein, the cephamycin of 100mg/L just with the induction frequency of indefinite bud by being reduced to more than 70% about 55%, continuous increase along with cephamycin concentration, the inductivity of indefinite bud descends gradually, the also even more serious (see figure 6) of brownization of explant, when cephamycin is increased to 500mg/L, the differentiation frequency of blade has only about 10%: when the blade of plane tree is incubated at the substratum that contains more than the cephamycin 300mg/L, the indefinite bud clump of its differentiation is the jade-green noble cells group that comprises most bud points more, is difficult to direct Cheng Miao (seeing Fig. 6-8).
Timentin is (available from U.S. Glaxo SmithKline company commodity, the Research Triangle Park of CompanyAddress, NC27709)) be the mixture of ticarcillin (Ticarcillin) and Clavulanic Potassium, usually effectively Mlc is 300mg/L, from this experimental result as can be seen, when Timentin concentration arrives 300mg/L, regeneration for the plane tree blade does not only have detrimentally affect, also has promoter action, from table 4, see, when Timentin reached 300mg/L, differentiation rate did not only descend, and exceeds 64% of contrast on the contrary.And in the substratum of additional Timentin100mg/L and 300mg/L, the bud ratio of every leaf all is higher than the combination of contrast, particularly 300mg/L, and bud ratio is near 2 times of contrast.
Therefore applicant's suggestion should not adopt cephamycin as the growth of Agrobacterium inhibitor in the genetic transformation of plane tree, and Timentin does not only have too big influence for the regeneration of plane tree blade, even also has certain promotion during for 300mg/L when concentration.
The fungistat of table 4 different sorts different concns is to the influence of plane tree leaf regeneration
Annotate: data are mean value ± standard error in the table, the significant difference (P=0.05) that exists between the upper right target The English alphabet registration certificate of same column of figure.
3, different blade physiological statuss influence that Agrobacterium is infected
Get different sizes (0.5,1,2, blade 3cm) carries out Agrobacterium and infects test as explant under the same conversion condition, cultivate through cultivating, take off bacterium altogether, observe the tolerance after blade infects for Agrobacterium and recover to cultivate back regenerated capacity variation, take off bacterium and cultivate back its brownization rate of statistics, changed in per 15 days and once to take off bacterium culture medium, add up differentiation rate after 2 months.
The explant that is used to transform is also tackled in Agrobacterium, is selected microbiotic and fungistat that certain tolerance is arranged, with this assurance as the follow-up cultivation stage of conversion except the basis of regeneration rate as conversion that high frequency will be arranged.
Based on the former study experience, the young more tender blade differentiation rate of the high-efficiency regeneration system of the employed plane tree blade of present embodiment is high more, wherein the regeneration frequency of first piece of blade of aseptic seedling in MS+6.0mg/L 6-BA+0.5mg/L IBA be up to more than 90%, but the tolerance of young leaflet tablet is common also lower.
In the present embodiment blade is divided into below the 0.5cm, 1cm, 2cm, four grades of 3cm, with the OD value be 0.8 agrobacterium tumefaciens bacterium liquid carry out infect test, after cultivating 5 days altogether, clean surperficial thalline with sterile distilled water, blot, insert and take off in the bacterium culture medium with aseptic filter paper, experimental result shows, the obvious difference of big and small blade tolerance.Brownization is that influence transforms successful important factor, generalized case, transformant only forms in the wound, if this moment, brownization can cause transformant death, therefore explant that we can say brownization of wound is difficult to produce transformant, so the height of brownization rate also is the important indicator of a gene transformation system quality of measurement, as seen from Figure 11, vanelets is starkly lower than other blades for the tolerance of Agrobacterium, after infecting, Agrobacterium will soon turn to be yellow, browning is cultivated back brownization rate up to 83.3% through taking off bacterium, does not produce callus basically, having a strong impact on differentiation rate, only is 13.3%.From Fig. 7, analyze as can be known, the blade about 2cm and 3cm, brownization rate is low to be respectively 23.3% and 13.3%, and the blade differentiation rate of 2cm is the highest, reaches 60.0%, and the above blade of 3cm may be because its degree of lignification be higher, the blade differentiation rate obviously reduces.Reach a conclusion thus, the blade about 2cm is best suited for the explant of Agrobacterium-mediated Transformation.
4, height oozes the influence of processing to GUS moment expression rate
Get the equal size that is fit to infect and the blade of leaf age, divide two kinds to carry out the pre-treatment experiment, the one group of blade that will get directly is soaked in 0.4mol/L and went out in the mannitol solution of bacterium; Scalpel with sterilization when another group is taken off blade scratches at blade back, soaks with the went out mannitol solution of bacterium of 0.4mol/L again.Two groups are all placed on the shaking table, the 100r/min vibration, be provided with the different treatment times (0,2,4,6,8,10,12h); Carry out Agrobacterium then under the same conditions and infect test, observe and infect the variation of rear blade vitality, add up its transient expression rate through cultivating the back altogether.
Height oozes the method that improves transformation efficiency in the genetic transformation that is used to plants gramineous such as wheat of handling, repeatedly reports in the literature, but for different plant species, different explants, the treatment time and the mode of different transform modes also are not quite similar.Two kinds of processing mode combinations of this experimental design, under all identical situation of other condition, divide two groups of N.F,USP MANNITOL with 0.4mol/L to carry out osmotic treated: one group is that intact leaf directly soaks, and height oozes and carries out Agrobacterium bacterium liquid with the sterile razor blade scuffing again after pre-treatment finishes and infect; After another group is taken off blade from aseptic seedling, scratch at leaf back with sterile razor blade immediately, be dipped in the mannitol solution and handle, processing finishes the back and infects with Agrobacterium bacterium liquid.
From experimental result, see, blade without treatment with mannitol, detect transient expression fully less than gus gene, and with the above combination of treatment with mannitol 4h, the moment that all detects gus gene expresses, through the treatment time of 8h, the moment expression rate of two kinds of processing modes all reaches the highest, and find after continuing to prolong the treatment time, moment, expression rate obviously descended, tracing it to its cause, may to be long high osmotic pressure have caused the plasmolysis of irrecoverable property to the cell of explant, produces grievous injury (Figure 12).In the treatment time of 4-8h, contrast two kinds of processing modes finds all to be higher than the scuffing blade without the blade that scratches its expression rate moment, this may be because plane tree blade itself is thinner, and paperyization, the cell of wound circumference is dead at short notice easily, therefore, and after handling several hrs, infect the difficult vitality of recovering of the cell of wound again with Agrobacterium.In follow-up culturing process, we also find the explant handled through 8h, and obviously not as handling 6h and 4h, blade is softening usually, is taking off poor growth on the bacterium culture medium for vitality, and brownization is serious.In sum, the applicant thinks the key factor that whether successfully transforms plane tree with the pre-treatment of N.F,USP MANNITOL before transforming, and determines that the pretreated Best Times of plane tree blade is 6h.
5, the influence of different Agrobacterium bacterial concentration GUS moment expression rates
The OD value is set is respectively 0.2,0.4,0.6,0.8,1.0 the Agrobacterium bacterium liquid of five kinds of different concns. transform under the identical situation of parameter at other, by cultivating the physiological status and the detected GUS moment expression rate of blade later, the optimum concn of selecting optimal OD value to infect the most relatively altogether.
The conversion of vegetable cell at first needs the Agrobacterium of some amount promptly adherent with contacting of cell walls, so bacterial concentration has certain influence to transformation frequency.Bacterium liquid with different concns in the present embodiment infects the aseptic seedling blade of plane tree under the same conditions, and the time is 8min, and incubation time is 5 days altogether.Experimental result shows, along with the raising of bacterial concentration, moment expression rate slightly change, when the OD value is lower than 0.4, moment expression rate be 0, when the OD value is between 0.6-1.0, moment expression rate higher, and do not have notable difference between three kinds of different concns.Generally speaking, high bacterial concentration for moment expression rate raising certain effect is arranged.But bacterial concentration is too high, easily causes the explant blade by agroinfection, through repeatedly cleaning dirty, and final lethal situation.Must express through the moment that GUS appears in cultivating one's ability altogether more than 4 days owing to the result who cultivates altogether determines plane tree, therefore, we think that the bacterial concentration of selection 0.6-0.8 is suitable (Figure 13) comparatively in agriculture bacillus mediated plane tree genetic transformation experiment.
6, the influence of different time of infection GUS moment expression rates
To infect through the Agrobacterium bacterium liquid of pretreated blade with same concentrations, time of infection is arranged to different gradients, is respectively 2,4,6,8,10,20min.Observe of the vitality influence of different times of infection, statistics GUS moment expression rate to leaf explant.
In conversion process, blade time of infection in bacterium liquid has certain influence to transformation efficiency, when time of infection is lower than 6min, moment expression rate be 0, can not realize transforming purpose; When time of infection surpasses 10min, though moment, expression rate was also higher, but brownization of blade is serious, and in follow-up culturing process, still there are a large amount of thalline to surround plane tree aseptic seedling leaf growth through repeatedly cleaning, even the covering one-piece blade, cause the explant blade finally dead, have a strong impact on the differentiation rate of explant.Therefore, we determine that 8-10min is the time of infection that suits, moment expression rate can reach 63.6% and 70.0%, through after 5d cultivates altogether, the outer planting physical efficiency is cleaned, and follow-up cultivation stage is not caused too big influence (Figure 14).
7, different incubation times altogether are to the influence of GUS moment expression rate
Will be through the leaf explant of the Agrobacterium-mediated Transformation of the same terms, respectively through 2,3,4,5, the detection of GUS moment expression rate is carried out in the common cultivation of 6d, statistics moment expression rate.
In the present embodiment, the best is the transient expression assay method of determining main employing gus gene of incubation time altogether, and promptly be total to and cultivate the gus gene color reaction that explant is regularly measured in the back, statistical presentation rate and expression area, with the expression rate height, the expression area is greatly excellent.Also to consider the degree that is hurt of explant simultaneously, be advisable with the vigorous growth that guarantees explant.
Experimental result shows, cultivate altogether and be lower than 4d, can't detect the moment expression rate, when cultivating 5d altogether, moment expression rate promptly be significantly improved, reach 66.7%, the moment expression rate of cultivating 6d altogether is the highest, but the explant that process 6d cultivates altogether, brownization degree improves, and thalline is through repeatedly cleaning dirty, has a significant impact for normal growth, the differentiation of explant.Take all factors into consideration above factor, the applicant thinks that 5d is the Best Times (Figure 15) that agriculture bacillus mediated plane tree blade transforms.
Some chemistry that this specification sheets is used or the abbreviation of biochemical are shown in following contraction table:
The chemistry that table 5 the present invention uses or the contraction table of biochemical
Claims (2)
1. method of utilizing agriculture bacillus mediated cultivation transgenosis plane tree plant, its step comprises genetic transformation, it is characterized in that, adopt plane tree aseptic seedling blade that aseptic mannitol solution handled acceptor material as genetic transformation, infect, cultivate altogether, recover cultivation, selection and root culture through Agrobacterium, gus reporter gene is imported acceptor, utilize kantlex the recipient cell after transforming to be screened, detect screening by PCR, gus gene expression and Southern hybridization and obtain transgenosis plane tree plant as selective agent;
Step is as follows:
1) the aseptic seedling blade with plane tree is immersed in the mannitol solution of 0.4mol/L, vibration pre-treatment 6h under 100r/min;
2) step 1) is pretreated blade is hindered with graduating with cutter, places OD
600Be to soak 8～10min in 0.6～0.8 the Agrobacterium bacterium liquid; The blade that will infect inserts and is total in the culture medium, cultivates 5d under 26 ± 1 ℃ of dark conditions; Change the blade after cultivating altogether over to recovery media, under 50～100lux illumination condition, cultivate 10～15d; Change the blade of cultivating in the recovery media over to the selection substratum again, evoking adventive bud under 1000～1500lux illumination is changed a fresh culture every 2 weeks, until differentiation adventitious buds; Change regenerated indefinite bud in the division culture medium over to root media at last, the screening resistant plant obtains candidate's transfer-gen plant;
Wherein, in above-mentioned recovery and selection culturing process, employing 300 or 400mg/L Timentin are as fungistat, to improve the regeneration rate of indefinite bud;
3) adopt Southern hybridization to detect the positive strain of PCR system, obtain transfer-gen plant;
Described medium component is as follows:
Be total to culture medium: MS minimum medium+4.0mg/L 6-BA+1.0mg/L IBA+100 or 200 μ mol/L Syringylethanones, additional saccharose 30g/L, agar 7g/L, pH5.8-6.0;
Recovery media: MS minimum medium+6.0mg/L 6-BA+0.5mg/L IBA+300 or 400mg/L Timentin, additional saccharose 30g/L, agar 7g/L, pH5.8-6.0;
Select culture medium: MS+6.0mg/L6-BA+0.5mg/LIBA+300 or 400mg/L Timentin+20 or 25mg/L kantlex, additional saccharose 30g/L, agar 7g/L, pH5.8-6.0;
Root media: 1/2MS minimum medium+0.1mg/L IBA+20 or 25mg/L kantlex, additional saccharose 30g/L, agar 7g/L, pH5.8-6.0.
2. the described method of claim 1 is in the application of cultivating on the transgenic sycamore plant.
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| CN101144091B (en) * | 2007-09-04 | 2011-10-26 | 西北农林科技大学 | Method for obtaining switchgrass transgene plant |
| CN101731148B (en) * | 2009-12-03 | 2012-04-25 | 江苏省农业科学院 | Chinar tissue culture method suitable for Agrobacterium rhizogenes mediation |
| CN102586317B (en) * | 2011-01-10 | 2013-08-07 | 华中农业大学 | Method for transforming and cultivating citrus transgenic plant by agrobacterium-mediated leaf |
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| CN106367436B (en) * | 2016-08-29 | 2020-01-14 | 东北林业大学 | Tamarix hispida instantaneous conversion method |
| CN106857259B (en) * | 2017-02-27 | 2020-01-10 | 华中农业大学 | Method for constructing high-frequency regeneration system of cotyledon of plane barberry and application of high-frequency regeneration system |
| CN107860774B (en) * | 2017-11-10 | 2020-01-07 | 青岛市农业科学研究院 | Screening method of sensitive genotype of agrobacterium tumefaciens of cucumber |
| CN114875064B (en) * | 2022-06-22 | 2023-07-04 | 北京林业大学 | Hybrid sweetgum gene editing vector, construction method and application thereof |
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