CN1948480B - Agricultural bacillus introduced eucommia trangenic method - Google Patents
Agricultural bacillus introduced eucommia trangenic method Download PDFInfo
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- CN1948480B CN1948480B CN2006101145658A CN200610114565A CN1948480B CN 1948480 B CN1948480 B CN 1948480B CN 2006101145658 A CN2006101145658 A CN 2006101145658A CN 200610114565 A CN200610114565 A CN 200610114565A CN 1948480 B CN1948480 B CN 1948480B
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Abstract
This invention relates to a eucommia bark transgenic preparation of agrobacterium mediating. A eucommia bark transgenic preparation of agrobacterium mediating, includes following steps: a. eucommia bark seed is inoculated in MS nutrient medium, then embryo is striped out, then inoculated in MS nutrient medium of supervening gibberellic acid; b. agrobacterium bacterium liquid is prepared by agrobacterium thallus which is collected by re-hanging centrifugalization of 1/2MS re-hanging nutrient medium; c. Embryo hypocotyl explantation is inoculated in induced nutrient medium in dark to cultivate, thrown into agrobacterium bacterium liquid, making explantation and agrobacterium sufficiently contact, explantation is taken out, transferred in co-nutrient medium to cultivante, then transferred in screening nutrient medium to induce regeneration of adventitious bud, at last materials are inoculated in root media to induce producing root. This invention founds eucommia bark genetic transformation system of agrobacterium mediating, establishing base for using transgenic technology to deeply research and unearth medicine usage, plastic usage of eucommia bark. genetic transformation rate of embryonic axis is about 2.8% by using this preparation.
Description
Technical field
Technical field under the present invention is a biological technical field, further, the present invention relates to a kind of agriculture bacillus mediated bark of eucommia transgenic method.
Background technology
Bark of eucommia Eucommia ulmoides Oliv. autogenus, list genus section, being China's endemic tree, is that the Eucommiaceae Eucommiaceae bark of eucommia belongs to the Relict Plant of only depositing, and now is put into " Chinese Plants Red Data Book---rare threatened plant " first roll as rare plant.The bark of eucommia is one of valuable Chinese medicine commonly used, also is the glue source plant that the temperate zone has exploitation prospect most.Yan Ruifang etc. have widened the range of application of gutta-percha greatly by sulfuration gutta-percha technology.Several key enzymes in the gutta-percha building-up process are cloned, the focus that the research of aspects such as separation of bark of eucommia medicinal ingredients and genes involved clone has become.Utilize transgenic technology with the external source functional gene genetic transformation bark of eucommia, thereby further study the biosynthesis mechanism of Chinese gutta percha and medicinal ingredients thereof, and improvement bark of eucommia quality is significant.
The bark of eucommia is comparatively a kind of xylophyta of difficulty of tissue culture.The research of at present relevant its regeneration system is less, and mostly is the somatocyte embryogenesis path, and directly organogenetic formal report is domestic does not see.Because tissue culture is difficulty comparatively, do not see the report of relevant bark of eucommia genetic transformation aspect so far as yet.
Summary of the invention
At the blank in the above-mentioned field, the invention provides a kind of agriculture bacillus mediated bark of eucommia transgenic method, for medicinal, glue by the transgenic technology further investigation and the excavation bark of eucommia are laid a good foundation with being worth.
A kind of agriculture bacillus mediated bark of eucommia transgenic method comprises the following steps:
A, eucommia bark seed is inoculated in the MS substratum, when treating that embryo is prominent and breaking in the seed coat the 0.4-0.7cm left and right sides, embryo is stripped out, be inoculated in the MS substratum of additional 1.0-2.0mg/L Plant hormones regulators,gibberellins;
B, the single bacterium colony of Agrobacterium is added the YEP liquid nutrient medium cultivate, reach 0.4～0.7 up to the OD value, centrifugal collection thalline is with the resuspended Agrobacterium bacterium liquid that makes of the resuspended substratum of 1/2MS;
C, the embryo plumular axis is cut into explant about 0.4-0.7cm, be inoculated into inducing culture based on after cultivating in the dark, drop in the step b Agrobacterium bacterium liquid, explant is fully contacted with Agrobacterium, take out explant, change on the common substratum and cultivate, change the regeneration of evoking adventive bud in the screening culture medium 1 then over to, in screening culture medium 2, carry out strong sprout again, at last material is inoculated into root induction in the root media.
Described being total in the substratum contained 50mg/L Syringylethanone and 20g/L sucrose, contains 20g/L sucrose in the resuspended substratum of 1/2MS.
Contain 0.8mg/L 6-benzyl aminopurine, 0.1mg/L naphthylacetic acid, 50mg/L kantlex, 100mg/L Timentine and 30g/L sucrose in the described screening culture medium 1, contain 1.0mg/L 6-benzyl aminopurine, 0.1mg/L naphthylacetic acid, 50mg/L kantlex, 100mg/L Timentine and 30g/L sucrose in the screening culture medium 2.
Contain 1.5mg/L indolebutyric acid, 1g/L gac, 50mg/L kantlex, 100mg/LTimentine and 20g/L sucrose in the described root media.
Described eucommia bark seed was through 4 ℃ of deepfreezes at least 3 days.
Described Agrobacterium bacterium liquid OD value is 0.3-0.4.
Described step c embryo is 20 day age.
Described explant is 48-72 hour at inducing culture and the incubation time that is total on the substratum.
Described explant before dropping into Agrobacterium bacterium liquid, mark laterally or/vertically be deep to the wound of epidermis.
In each stage of plant tissue culture, except that cultivating altogether and pre-cultivation stage does not need the illumination, culture condition is that intensity of illumination is 2000lx, illumination every day 16 hours, culture temperature (26 scholar 2) ℃.
The resuspended substratum of root media and 1/2MS is the 1/2MS substratum in the experiment, and all the other substratum are the conventional substratum of MS.
Bark of eucommia embryo contains more not differentiation and the more shallow meristematic cell of differentiation degree, has very strong regeneration activity, and under suitable growth hormone and cytokinin concentration, its wound cortex can be differentiated to form bud initial body and the direct regenerated adventitious bud of high frequency.It is explant that bark of eucommia embryo is chosen in this experiment, undertaken adventitious bud inducing by directly generation approach of meristematic cell organ, and the situation of inducing is seen " Fig. 2 ", has the inductivity height, and stability is high, and it is low to make a variation, and is beneficial to the advantage of carrying out agriculture bacillus mediated genetic transformation.Adopting present method plumular axis genetic transformation rate is about 2.8%.
Owing to lack bark of eucommia tissue culture regeneration system efficiently, do not see the report of relevant bark of eucommia genetic transformation aspect as yet.It is explant that bark of eucommia plumular axis is adopted in this research, and directly evoking adventive bud takes place, perfect bark of eucommia adventitious bud inducing regeneration system.On the basis of above-mentioned research, filter out reasonably genetic transformation scheme, set up agriculture bacillus mediated bark of eucommia genetic conversion system, for by transgenic technology, further investigate and excavate medicinal, the glue of the bark of eucommia and lay a good foundation with being worth.
Description of drawings
Fig. 1. screening 25d bark of eucommia plumular axis adventitious bud inducing situation (whole ware)
Wherein, a is transgenic line (a whole ware), and b is non-transgenic contrast (a whole ware).
Fig. 2. by the meristematic cell organ approach takes place directly and bark of eucommia plumular axis is carried out adventitious bud inducing situation " a figure " (this figure is " Fig. 1; a for screening 25d bark of eucommia plumular axis indefinite bud " in the enlarged view of single explant), " b figure " is screening 45d bark of eucommia plumular axis indefinite bud.
Fig. 3. the strong bud of bark of eucommia transgenic line, and the rooting tube plantlet that obtains
Fig. 4. the GUS dyeing detected result of transfer-gen plant
Wherein, a is a rotaring gene plant blade dyeing situation, and b is a non-transgenic adjoining tree blade dyeing situation.
Fig. 5. transgenosis bark of eucommia PCR detected result
Wherein, the 1st road is Marker (λ DNA is through BamH I, and the HindIII double digestion obtains), and the 2nd road is the non-transgenic contrast, the positive plasmid in the 3rd road, and the 4th, 5,6 roads are bark of eucommia transgenic line.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
Substratum and culture condition: contain the 50mg/L Syringylethanone in the substratum altogether, and 20g/L sucrose, contain 20g/L sucrose in the resuspended substratum of 1/2MS, contain the 0.8mg/L 6-benzyl aminopurine in the screening culture medium 1,0.1mg/L naphthylacetic acid, the 50mg/L kantlex, 100mg/L Tinmentine and 30g/L sucrose, contain the 1.0mg/L 6-benzyl aminopurine in the screening culture medium 2,0.1mg/L naphthylacetic acid, the 50mg/L kantlex, 100mg/L Tinmentine and 30g/L sucrose, contain the 1.5mg/L indolebutyric acid in the root media, the 1g/L gac, the 50mg/L kantlex, 100mg/L Tinmentine and 20g/L sucrose, the resuspended substratum of root media and 1/2MS is the 1/2MS substratum in the experiment, and all the other substratum are the conventional substratum of MS.In each stage of plant tissue culture, except that cultivating altogether and pre-cultivation stage does not need the illumination, culture condition is that intensity of illumination is 2000lx, illumination every day 16 hours, culture temperature (26 scholar 2) ℃.Wherein comprise in the 1L MS substratum: saltpetre (KNO
3) 1900 milligrams, ammonium nitrate (NH
4NO
3) 1650 milligrams, calcium chloride (CaCl
22H
2O) 440 milligrams, sal epsom (MgSO
47H
2O) 370 milligrams, potassium primary phosphate (KH
2PO
4) 170 milligrams, manganous sulfate (MnSO
44H
2O) 22.3 milligrams, zinc sulfate (ZnSO
47H
2O) 8.6 milligrams, boric acid (H
2BO
3) 6.2 milligrams, 0.83 milligram of potassiumiodide (KI), Sodium orthomolybdate (Na
2MoO
42H
2O) 0.25 milligram, disodium ethylene diamine tetraacetate (Na
2-EDTA2H
2O) 37.25 milligrams, ferrous sulfate (FeSO
47H
2O) 27.8 milligrams, copper sulfate (CuSO
45H
2O) 0.025 milligram, cobalt chloride (COCl
26H
2O) 0.025 milligram, 100 milligrams of inositols, 0.5 milligram in nicotinic acid, vitamin (dimension B
1) 0.1 milligram, pyridoxine hydrochloride (dimension B
6) 0.5 milligram, 2 milligrams of glycine, pH value 5.80.1LYEP cultivates to concentrate and comprises: 5.0g peptone, 5.0g yeast extract, 2.5gNaCl, pH value 7.20.
1, bark of eucommia plumular axis antibiotics sensitivity experiment
The structure that is used for bark of eucommia genetic transformation in this experiment contains kalamycin resistance gene, therefore adopts kantlex that transfer-gen plant is screened, and this experiment adopts Tinmentine as the Agrobacterium inhibitor of bark of eucommia material in the middle of group training process in addition.In bark of eucommia inducing culture, additional respectively 0mg/L kantlex, 0mg/L kantlex, 100mg/LTinmentine, 25mg/L kantlex, 50mg/L kantlex and 100mg/L kantlex.With the plumular axis segment about 0.5cm, be inoculated in the above-mentioned substratum differentiation of statistical material and survival condition behind the 25d.The result shows that the Tinmentine of 100mg/L is little to the differentiation influence of bark of eucommia plumular axis and cotyledon, so we adopt the Tinmentine of 100mg/L antibacterial when screening.Bark of eucommia plumular axis is relatively responsive to transgenic line, and when kantlex concentration was 25mg/L, the explant mortality ratio was 45.3%, and the differentiation of bud seedling appears in individual material, but the serious vitrifying of bud that produces, can not normal growth; When kantlex concentration was 50mg/L, explant yellow, vitrifying were serious, and mortality ratio 65.6% does not have the differentiation of indefinite bud; When kantlex concentration reaches 100mg/L, most explant mortality ratio.The screening that we select is pressed and is kantlex 50mg/L, and additional 100mg/L Tinmentine suppresses growth of Agrobacterium.
2, agriculture bacillus mediated foreign gene genetic transformation bark of eucommia plumular axis
2.1 material and culture condition: get eucommia bark seed (pick up from Guizhou University and spend campus, Xi Nan school district), peel off crust, place 4 ℃ of refrigerator deepfreezes at least 3 days stand-by.Intensity of illumination is 2000lx, illumination every day 16h, culture temperature (26 scholar 2) ℃.
2.2 materials disinfection method: get the seed of 4 ℃ of preservations, wash 30min, in Bechtop, soak 15min, soak 30min, rinsed with sterile water 5 times, soaked overnight with changing in the 0.1% mercuric chloride water behind the 75% alcohol disinfecting 60s again with sterilized water with tap water.Seed after the sterilization is inoculated in the MS substratum.
2.3 inoculation: when treating that embryo is prominent and breaking in the seed coat the 0.5cm left and right sides, get growing way preferably material embryo is stripped out, with conventional aseptic technique method, be inoculated in the MS substratum that adds 1.0-2.0mg/L Plant hormones regulators,gibberellins, make its quick elongation.3 all left and right sides embryos are extending to the 3cm (owing to removed the constraint of planting skin, the growth of material is more consistent)
2.4 the preparation of Agrobacterium: dip in inoculating needle and to get-80 ℃ and frozen contain the pBin19 recombinant plasmid Agrobacterium of (containing beta-glucosiduronatase gene and recombinase gene flp in the recombinant plasmid), on the YEP solid medium, draw dull and stereotyped, about 28 ℃ of constant temperature culture 24h, up to the bacterial plaque that grows suitable size.Join in the 5mlYEP liquid nutrient medium with the single bacterium colony of toothpick picking Agrobacterium, 24h is cultivated in 28 ℃ of concussions of 200rpm, gets 500ul and add in the 50ml YEP liquid nutrient medium in 5ml bacterium liquid, and 200rpm28 ℃ of concussion cultivated, and reaches 0.4～0.7 up to the OD value.Bacterium liquid is transferred in the 50ml centrifuge tube, 6000rpm, 4 ℃ of centrifugal 10min collect thalline.With the Agrobacterium of the resuspended centrifugal collection of the resuspended substratum of 1/2MS, regulate bacterium liquid OD value between 0.3～0.4.
2.5 the Agrobacterium of vegetable material is infected: get 20d embryo in age, plumular axis is cut into segment about 0.5cm, be inoculated into and cultivate 48-72d in the inducing culture in the dark, on pre-incubated explant, mark several horizontal, vertical wounds (degree of depth is to epidermis) to enlarge injured area, drop in the preprepared Agrobacterium bacterium liquid, constantly shake, explant is fully contacted with Agrobacterium, behind 6～8min, take out explant, place to inhale on the dry aseptic filter paper and remove unnecessary bacterium liquid, change on the common substratum, cultivate 48h-72h.Change the regeneration of evoking adventive bud in the screening culture medium 1 at once over to.The screening of transgenic plant and contrast, and the indefinite bud situation of directly inducing is seen Fig. 1 and Fig. 2 respectively.In the screening culture medium that contains the 50mg/L kantlex, owing to imported kalamycin resistance gene, so transgenic line can decompose the kantlex in the substratum, so can survive; And the death of non-transgenic control material.
2.6 differentiation and strong sprout: material changes screening culture medium 1 back visible budlet of 4-5 week over to and sprouts (the adventitious bud inducing situation that inoculate transgenic line after 25 days, reaches non-transgenic control material plumular axis is seen Fig. 1), the indefinite bud that induces advances subculture 2 times in screening culture medium 2, in about 4-5 week, it is high that seedling can grow to the 3-4 centimetre.(see Fig. 2, b)
2.7 the acquisition of whole plant: seedling is gone to root induction in the root media.Material lower end, 2 week back grows white rootlet, continues to cultivate promptly to obtain bark of eucommia transfer-gen plant test-tube plantlet.(see figure 3)
3, the GUS of transfer-gen plant and PCR detect
(1) activity of gus gene detects: the structure that is used for bark of eucommia genetic transformation in this experiment contains beta-glucosiduronatase gene, can carry out GUS dyeing.After cleaning, the blade of getting material to be detected vacuumizes, in 5-bromo-4-chloro-3-indoles-β-D-glucuronide staining fluid (X-Gluc 0.5mg/mL, 100mmol/L phosphoric acid buffer ph=7.0,0.1%Triton X100, the 1mmol/L Tripotassium iron hexacyanide, the 1mmol/L yellow prussiate of potash) in 37 ℃ of following incubated overnight, use 10% respectively then, 25%, 50%, 75%, 95% ethanol decolorization, the ethanol of the every kind of concentration 5min that decolours respectively, tissue continues to be placed in 95% the ethanolic soln and soaks at last, decorporates up to green.
Detected result is seen " Fig. 4 ".Through GUS dyeing, the blade that changes basic plant is because of being blueness, and that the blade of adjoining tree does not have is painted, is white in color.
(2) PCR of transgenosis bark of eucommia plant detects: the structure that is used for bark of eucommia genetic transformation in this experiment contains the flp gene, therefore adopts the flp primer that transfer-gen plant is carried out PCR and detects.The CTAB method is extracted bark of eucommia genomic dna, is that masterplate utilizes the flp primer to carry out pcr amplification with the genomic dna, and fragment length to be amplified is 1270bp.
(25ul) pcr amplification system: 2.5ul 10X PCR buffer, 1.0ul 25mmol MgCL
2, 0.5ul 10mmol dNTP, 1.0ulDNA masterplate, each 1.0ul 20mmol flp primer, 0.5ul 5u/ul tag enzyme), reaction conditions is: 94 ℃ of 7min, (94 ℃ of 30s, 63 ℃ of 1min, 72 ℃ of 1min) 35cycle, 72 ℃ of 7min.
Flp primer sequence: flp-Ks-F, 5 ' ggg gta ccc aag tcg acc aat gcc aca att tgg tat att atg 3 '
flp-x-R，5’ccg?atc?gag?tta?tat?gcg?tct?att?tat?gta?gg?3’
Detected result is seen " Fig. 5 ".4th, the bark of eucommia transgenic line in 5,6 roads has amplified the band of 1270bp, and the result is consistent with positive plasmid, and the non-transgenic control material does not amplify band.
Claims (3)
1. an agriculture bacillus mediated bark of eucommia transgenic method comprises the following steps:
A, eucommia bark seed are inoculated into the eucommia bark seed handled in MS substratum through 4 ℃ of deepfreezes 3 days at least, when treating that embryo is prominent and breaking in the seed coat the 0.4-0.7cm left and right sides, embryo are stripped out, and are inoculated in the MS substratum of additional 1.0-2.0mg/L Plant hormones regulators,gibberellins;
B, will contain the single bacterium colony of Agrobacterium of pBin19 recombinant plasmid, use the toothpick picking, join and cultivate the concussion cultivation in the YEP liquid nutrient medium, reach 0.4～0.7 up to the OD value, centrifugal collection thalline, with the resuspended Agrobacterium bacterium liquid that makes of the resuspended substratum of 1/2MS, Agrobacterium bacterium liquid OD value is 0.3-0.4, contains beta-glucosiduronatase gene and recombinase gene flp in the described recombinant plasmid;
C, with 20 day age the embryo plumular axis be cut into explant about 0.4-0.7cm, be inoculated into inducing culture based on after cultivating in advance in the dark, in the Agrobacterium bacterium liquid that input step b makes, explant is fully contacted with Agrobacterium, take out explant, change on the common substratum and cultivate, change the regeneration of evoking adventive bud in the screening culture medium 1 then over to, in screening culture medium 2, carry out strong sprout again, at last material is inoculated into root induction in the root media; Described explant is 48-72 hour at inducing culture and the incubation time that is total on the substratum;
Described being total in the substratum contained 50mg/L Syringylethanone and 20g/L sucrose, contains 20g/L sucrose in the resuspended substratum of 1/2MS,
Contain 0.8mg/L 6-benzyl aminopurine, 0.1mg/L naphthylacetic acid, 50mg/L kantlex, 100mg/L Tinmentine and 30g/L sucrose in the described screening culture medium 1, contain 1.0mg/L 6-benzyl aminopurine, 0.1mg/L naphthylacetic acid, 50mg/L kantlex, 100mg/L Tinmentine and 30g/L sucrose in the screening culture medium 2
Contain 1.5mg/L indolebutyric acid, 1g/L gac, 50mg/L kantlex, 100mg/LTinmentine and 20g/L sucrose in the described root media.
2. agriculture bacillus mediated bark of eucommia transgenic method according to claim 1, described explant marked the wound that laterally or vertically is deep to epidermis before the Agrobacterium bacterium liquid that input step b makes.
3. agriculture bacillus mediated bark of eucommia transgenic method according to claim 1, each stage of described plant tissue culture does not need the illumination except that being total to cultivation and pre-cultivation stage, and culture condition is that intensity of illumination is 2000 lx, illumination every day 16 hours, 26 ± 2 ℃ of culture temperature.
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| CN103583372B (en) * | 2013-11-29 | 2015-05-13 | 陕西理工学院 | Eucommia ulmoides Oliv. in-vitro rapid-propagation method |
| CN105861543B (en) * | 2016-06-29 | 2019-08-23 | 云南纳博生物科技有限公司 | A method of establishing mediated by agriculture bacillus fourleaf peperomia herb rotaring redyeing system |
| CN111066506B (en) * | 2019-12-12 | 2021-06-18 | 贵州大学 | Micro-grafting propagation method for transgenic resistant buds of eucommia ulmoides |
| CN112501211B (en) * | 2020-12-21 | 2022-11-01 | 贵州大学 | Method for genetic transformation of eucommia ulmoides by injection |
| CN112931227B (en) * | 2021-04-26 | 2022-07-08 | 北京林业大学 | Method for whole plant induction plant regeneration of each part of eucommia ulmoides and construction of transgenic plant regeneration system |
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| Title |
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| .发根农杆菌LBA9402 Bin19转化红豆草及再生转基因植株(英文).实验生物学报33 1.2000,33(1),63-68. |
| 徐子勤 马洪军 郝建国 贾敬芬.发根农杆菌LBA9402 Bin19转化红豆草及再生转基因植株(英文).实验生物学报33 1.2000,33(1),63-68. * |
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