CN101451144B - Fruit germplasm culture method capable of spontaneously eliminating agriculture remanet - Google Patents
Fruit germplasm culture method capable of spontaneously eliminating agriculture remanet Download PDFInfo
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- CN101451144B CN101451144B CN2008100687723A CN200810068772A CN101451144B CN 101451144 B CN101451144 B CN 101451144B CN 2008100687723 A CN2008100687723 A CN 2008100687723A CN 200810068772 A CN200810068772 A CN 200810068772A CN 101451144 B CN101451144 B CN 101451144B
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Abstract
The invention discloses a breeding method capable of spontaneously eliminating fruit germplasm of pesticide residue, which comprises the steps of: (1) cloning a fruit-specific promoter according to the prior method; (2) artificially synthesizing a gene of organophosphorus hydrolase (OPH); (3) constructing a fruit-specific OPH plant expression vector, in which an E8 amplification product and an artificially synthesized OPH gene sequence are connected to pGEM-T easy to construct pGEM-E8 and pGEM-OPH respectively, then HindIII and BamHI are used to perform double enzyme digestion on pGEM-TE8 andpSH to construct E8 to pSE8, EcoRI is used to perform single enzyme digestion on the pGEM-OPH and the pSE8 to construct OPH to pSE8OP, and a forward connection vector is screened, thereby obtaining the plant expression vector driven by the fruit-specific promoter for standby; (4) transforming soil agrobacteria and a plant explant; and (5) performing the differentiation and transplantation of a transgenic strain. The method can reduce the pesticide residue in fruits and simultaneously can not affect the efficacy of field pesticides, thereby contributing to the improvement of the yield and the quality.
Description
Technical field
The invention belongs to biological technical field, specifically relate to the method for cultivation of the plant germplasm of pesticide residue in a kind of energy specificity degraded fruit.
Background technology
Pesticidal contamination is one of global environmental problem, organophosphorus pesticide be again wherein the usage quantity maximum, use the compounds that face is the widest, toxicity is the highest.Some agricultural chemicals has " three cause " material, can reach more than the many decades in carcinogenic and latent period mutagenesis, and the agricultural chemicals that has is an environmental hormone, disturbs internal secretion after entering animal and human's body, makes the reproduction parafunction.In identified at present " environmental hormone register ", organic compound has 67 kinds, and wherein agricultural chemicals is 44 kinds, accounts for 65.7%.1,000,000 tons of agricultural chemicals need be used every year in the whole world, but be directly used in target organisms be no more than 5%, most pesticide residue are discharged into (Pimental and Levitan 1986) in the environment.The whole world is hundreds thousand of because of the annual death toll of organophosphate poisoning reaches, and (Eddleston M.2000 mainly to occur in developing country; Eddleston M, 2004).Organophosphorus pesticide residual very important in vegetables, maximum Rogor residual level reaches 1mg/kg in the Turkey tomatoes in 2003, and annual production reaches 425000 tons (Anonymous 2003).P.Aysal in 2004 etc. pass through
14C mark Rogor, it is residual to detect in the tamato fruit organophosphorus pesticide Rogor, finds in season or the end of the season residual level reaches 0.49ppm and 0.45ppm, has surpassed the 0.22ppm of the residual restriction of the maximum Rogor of European Union, wherein jam enrichment farming is residual than fruit juice height (P.Aysal, 2004).
China is a large agricultural country, also is the big country of production and consumption agricultural chemicals.In recent years, about more than 200 of the annual pesticide species of producing of China, processing shop agent kind more than 500, about 400,000 tons of the turnout of former medicine, the 2nd in the rank world, sterilant accounts for more than 70% in the agricultural chemicals, and the organic phosphorous insecticide of high poison accounts for more than 70%.Annual agricultural chemicals uses and reaches 4,500,000,000 mu times, and wherein the agricultural chemicals of 70-80% directly is scattering in the environment, and soil, surface water, underground water and agricultural-food are polluted.According to statistics, annual tens thousand of personnel injures and deaths incident of poisoning that causes, the number of poisoning accounts for 50% of the similar accident poisoning in world number.
The using in a large number and abusing of agricultural chemicals makes that pesticide residue exceeds standard in the agricultural-food.Agricultural-food surfaces pesticide residue height not only, and, can penetrate into agricultural-food inside because many agricultural chemicals have the contact sorption, especially the plant-sourced agricultural-food can also absorb and enrichment from water body by vascular bundle, cause the agricultural byproducts pesticide residue to exceed standard, biologic chain is gone in the stepping of going forward side by side.Exceed standard as DDT in the tealeaves, contain desinsection ether in the honey, contain acephatemet in the apple, detect demeton_S_methyl, Nemacur, methiocarb (Yolanda Pic ó in the Sucus Vitis viniferae commodity beverage, 2007), contain multiple organophosphorus (Li Fengge, 2006) in the tomato-sauce, cold pork, freeze exempt from, pesticide residue exceed standard etc. in the chicken in jelly, have a strong impact on the foreign trade of China.
Utilize the gene constructed engineering bacteria of degradation of pesticide to be used for degrading enzyme production or agricultural residual reparation, carried out some researchs.Usefulness INPNC-OPH such as Shimazu navigate to intestinal bacteria (E.coli) and Mo Shi bacillus (Moraxella sp.) cell surface to agricultural chemicals lytic enzyme OPH, make its degrading organic phosphor pesticides and PNP simultaneously.Lan etc. have expressed in pETDuet simultaneously from the organophosphor hydrolytic enzyme gene opd of Flavobacterium with from the Procaine esterase gene b1 of Culex quinquefasciatus Culex pipiens, and the engineering bacteria that carries this carrier is degrading organic phosphor, carbaminate and pyrethroid three class agricultural chemicals simultaneously.Jiang Jiandong etc. are inserted into the mpd gene rDNA site of Sphingol single-cell CDS-1 bacterial strain and do not bring the external source resistance into, engineering strain CDS-1mpd and CDS-2mpd can degrade simultaneously parathion-methyl and carbofuran.Existing research mainly concentrates on water body and the soil that the degrading enzyme that uses genetic engineering bacterium or its production is repaired pesticidal contamination.
Summary of the invention
A kind of pesticide residue that can reduce fruit that the objective of the invention is to overcome above-mentioned shortcoming and provide do not influence efficacy of field pesticides simultaneously yet, thereby help improving the residual fruit germplasm culture method of the spontaneous elimination farming of a kind of energy of output and quality.
The method of cultivation of the fruit germplasm that the spontaneous elimination farming of a kind of energy of the present invention is residual comprises the following steps:
(1) clone fruit-specific promoter according to a conventional method:
Described fruit-specific promoter is fruit specific genetic expression promotors such as E8; According to the characteristics of the E8 promoter sequence that provides among the Genbank accession:AF515784.1, the design primer is that template is done pcr amplification with tomato DNA, and the primer of E8 promotor is:
P1：5’-CCCAAGCTTAGGAATTTCACGAAATCG-3’；
P2：5’-CGCGGATCCTCTTTTGCACTGTGAATG-3’；
(2) gene of synthetic organophosphor hydrolytic enzyme OPH:
Described organophosphor hydrolytic enzyme has the described aminoacid sequence of Genebank Accession:AAA24930, manually is synthesized into after translating into base sequence according to the tomato codon preference;
(3) make up fruit specific OPH plant expression vector:
The OPH gene order of E8 amplified production and synthetic is connected respectively to constructs pGEM-E8 and pGEM-OPH on the pGEM-T easy, use HindIII and BamHI double digestion pGEM-TE8 and pSH then, E8 is building up to pSE8; With EcoRI single endonuclease digestion pGEM-OPH and pSE8, OPH is building up to pSE8OP, filter out the forward connection carrier, thereby the plant expression vector that obtains the fruit-specific promoter driving is standby;
(4) transform soil Agrobacterium and plant explants
Described plant explants derives from plants such as tomato, cucumber or capsicum.
Expression vector pSE8OP is transformed into soil Agrobacterium LBA4404 with freeze-thaw method, and picking contains the single bacterium colony of Agrobacterium of expression vector pSE8OP, is containing 100mgL
-1Km and 20mgL
-1To be cultured to OD600 be 0.5-0.7 in 28 ℃ of concussions in the YEP substratum of Rif, and the centrifugal 5min of 6000rpm collects thalline, with the resuspended thalline of the resuspended substratum of MS; Select fresh and tender plant explants and in resuspended bacterium liquid, soak 10min, be connected on the common substratum after blotting bacterium liquid with aseptic filter paper, change over to after 2 days and take off bacterium culture medium and (contain Cef300mgL
-1) on, keep selecting to press subculture 2 times, complete until taking off bacterium;
(5) differentiation of transfer-gen plant, transplanting
To be connected on the division culture medium at the explant that takes off under surviving on the bacterium culture medium, the seedling of differentiation is transferred in the root media when growing to 2-3cm and cultivates, and treats to open bottle cap when seedling grows to 5-10cm, is transplanted in the soil behind the hardening 3-5d.
Contain 100mgL in the resuspended substratum of described MS
-1AS and 20gL
-1Sucrose.
Contain 1.0mgL in the described pre-culture medium
-16-BA, 0.1mgL
-1IAA and 20gL
-1Sucrose.
Described being total in the substratum contained 1.0mgL
-16-BA, 0.1mgL
-1IAA, 100mgL
-1AS and 20gL
-1Sucrose.
Described taking off contained 1.0mgL in the bacterium culture medium
-16-BA, 0.1mgL
-1IAA, 300mgL
-1Cef and 30gL
-1Sucrose;
Described division culture medium contains 1.0mgL
-16-BA, 0.1mgL
-1IAA, 300mgL
-1Cef, 70mgL
-1Km and 30gL
-1Sucrose;
Contain 0.1mgL in the described root media
-1IAA, 300mgL
-1Cef, 70mgL
-1Km and 20gL
-1Sucrose.
Removing root media is basic medium with 1/2MS, and all the other substratum all are basic medium with MS.
Wherein comprise in the 1L MS substratum: saltpetre (KNO
3) 1900mg, ammonium nitrate (NH
4NO
3) 1650mg, calcium chloride (CaCl
22H
2O) 440mg, sal epsom (MgSO
47H
2O) 370mg, potassium primary phosphate (KH
2PO
4) 170mg, manganous sulfate (MnSO
44H
2O) 22.3mg, zinc sulfate (ZnSO
47H
2O) 8.6mg, boric acid (H
2BO
3) 6.2mg, potassiumiodide (KI) 0.83mg, Sodium orthomolybdate (Na
2MoO
42H
2O) 0.25mg, disodium ethylene diamine tetraacetate (Na
2-EDTA.2H
2O) 37.25mg, ferrous sulfate (FeSO
47H
2O) 27.8mg, copper sulfate (CuSO
45H
2O) 0.025mg, cobalt chloride (COCl
26H
2O) 0.025mg, inositol 100mg, nicotinic acid 0.5mg, vitamin (dimension B
1) 0.1mg, pyridoxine hydrochloride (dimension B
6) 0.5mg, glycine 2mg, pH value 5.80; 1L YEP cultivates to concentrate and comprises: 5.0g peptone, 5.0g yeast extract, 2.5gNaCl, pH value 7.20.
As from the foregoing, method of the present invention compared with prior art, the present invention transforms plant explants with the agrobacterium-mediated transformation handle by fruit-specific promoter E8 and the gene constructed plant expression vector pE8OP of organophosphor hydrolytic enzyme OPH, make it obtain the residual ability of fruit specific degrading organic phosphor pesticides, do not influence the effect of whole plant spraying pesticide yet, strengthened the food safety of fruits and vegetables disease control.Below advance beneficial effect of the present invention a bit is described by embodiment.
Description of drawings
The clone of Fig. 1 E8 promotor;
The base sequence and the aminoacid sequence of Fig. 2 synthetic organophosphor hydrolytic enzyme OPH gene;
The structure synoptic diagram of Fig. 3 plant expression vector pE8OP;
Fig. 4 tomato GUS dyeing and enzyme analysis chart alive;
Embodiment
Embodiment 1: the residual tomato germplasm of spontaneous elimination farming is cultivated
(1) clone fruit-specific promoter according to a conventional method:
Extract genomic dna from tomato leaf, with PCR method clone 1.1KbE8 promotor (see figure 1), according to the E8 promoter sequence design PCR primer that provides among the Genbank ID:AF515784.1, synthetic by Shanghai JaRa bio-engineering corporation, sequence is:
P1：5’-CCCAAGCTTAGGAATTTCACGAAATCG-3’；
P2：5’-CGCGGATCCTCTTTTGCACTGTGAATG-3’；
Upstream primer is introduced the HindIII restriction enzyme site, and downstream primer is introduced the BamHI restriction enzyme site.According to plasmid pGEM-T easy operation instruction, to clone product and be connected to Transformed E .coli TG1 on the plasmid pGEM-T easy, obtain positive colony by blue hickie screening, send the order-checking of Beijing three rich polygala root biotech firms, to obtain correct E8 promoter gene sequence.
(2) gene of synthetic organophosphor hydrolytic enzyme OPH:
Announce organophosphor hydrolytic enzyme aminoacid sequence and tomato codon preference according to Genebank gi:148713, design OPH gene order (see figure 2), synthetic by Shanghai JaRa bio-engineering corporation.
(3) make up fruit specific OPH plant expression vector:
The OPH gene order of E8 amplified production and synthetic is connected respectively to constructs pGEM-E8 and pGEM-OPH on the pGEM-T easy, use HindIII and BamHI double digestion pGEM-TE8 and pSH then, the E8 promotor is replaced pSH go up 35S promoter, E8 is building up to pSE8; With EcoRI single endonuclease digestion pGEM-OPH and pSE8, connect by the T4DNA ligase enzyme, OPH is building up to pSE8OP, then by HindIII and KpnI double digestion screening forward plant expression vector, thereby obtain the plant expression vector that fruit-specific promoter drives, Transformed E .coli TG1 preserves the standby (see figure 3) of bacterial classification.
Plasmid pGEM-T easy is available from Promega company, and all host bacterium E.coli TG1 and Agrobacterium tumefaciens LBA4404 are preserved by this chamber.The LA.Taq archaeal dna polymerase, and various restriction enzyme BamHI, KpnI, HindIII and EcoRI, T4 dna ligase be the TaKaRa product, all the other reagent are analytical pure.It is the LB substratum that bacterial multiplication is cultivated used substratum, and containing and adding kantlex concentration in the substratum of plasmid thalline is 100mg/l;
(4) transform soil Agrobacterium and tomato explant
Expression vector pSE8OP is transformed into soil Agrobacterium LBA4404 with freeze-thaw method.Picking contains the single bacterium colony of Agrobacterium of expression vector pSE8OP, is containing 100mgL
-1Km and 20mgL
-1To be cultured to OD600 be 0.5-0.7 in 28 ℃ of concussions in the YEP substratum of Rif, and the centrifugal 5min of 6000rpm collects thalline, is used to infect the tomato explant with the resuspended thalline of the resuspended substratum of MS.
Little queen's cherry tomato seed is bought from vegetable or flower institute of the Chinese Academy of Agricultural Sciences.Sophisticated tomato seeds is with 75% alcohol-pickled 45s, soak 60S with 0.05%HgCl2 again, use sterile water wash 5 times, suck dry moisture is connected on the MS substratum, when seed germination has 5cm height and cotyledon fully to stretch, cut hypocotyl and cotyledon piece and on pre-culture medium, cultivated 2 days.The genetic transformation of tomato adopts agrobacterium-mediated transformation.Pre-incubated tomato hypocotyl and cotyledon piece are soaked 10min in resuspended bacterium liquid, be connected on the common substratum after blotting bacterium liquid with aseptic filter paper, change over to after 2 days and take off bacterium culture medium and (contain Cef 300mgL
-1) on, keep selecting to press subculture 2 times, complete until taking off bacterium;
(5) differentiation of transfer-gen plant, transplanting
To take off bacterium completely explant be connected on the division culture medium illumination and cultivate, light intensity is 4000lux, 14h/d.Per 20 days subcultures once, the seedling of differentiation is transferred in the root media when growing to 3-5cm and cultivates, and treats to open bottle cap when seedling grows to 5-10cm, is transplanted in the soil behind the hardening 3-5d.
Each stage culture condition of plant tissue culture is: induce, be converted into dark cultivation; Take off bacterium, break up, take root and cultivate for light, light intensity is 4000lux, 14h/d.Contain 100mgL in the resuspended substratum of MS
-1AS and 20gL
-1Sucrose; Contain 1.0mgL in the pre-culture medium
-16-BA, 0.1mgL
-1IAA and 20gL
-1Sucrose; Contain 1.0mgL in the substratum altogether
-16-BA, 0.1mgL
-1IAA, 100mgL
-1AS and 20gL
-1Sucrose; Take off and contain 1.0mgL in the bacterium culture medium
-16-BA, 0.1mgL
-1IAA, 300mgL
-1Cef and 30gL
-1Sucrose; Division culture medium contains 1.0mgL
-16-BA, 0.1mgL
-1IAA, 300mgL
-1Cef, 70mgL
-1Km and 30gL
-1Sucrose; Contain 0.1mgL in the root media
-1IAA, 300mgL
-1Cef, 70mgL
-1Km and 20gL
-1Sucrose.Removing root media in the experiment is basic medium with 1/2MS, and all the other substratum all are basic medium with MS.
Wherein comprise in the 1L MS substratum: saltpetre (KNO
3) 1900mg, ammonium nitrate (NH
4NO
3) 1650mg, calcium chloride (CaCl
22H
2O) 440mg, sal epsom (MgSO
47H
2O) 370mg, potassium primary phosphate (KH
2PO
4) 170mg, manganous sulfate (MnSO
44H
2O) 22.3mg, zinc sulfate (ZnSO
47H
2O) 8.6mg, boric acid (H
2BO
3) 6.2mg, potassiumiodide (KI) 0.83mg, Sodium orthomolybdate (Na
2MoO
42H
2O) 0.25mg, disodium ethylene diamine tetraacetate (Na
2-EDTA.2H
2O) 37.25mg, ferrous sulfate (FeSO
47H
2O) 27.8mg, copper sulfate (CuSO
45H
2O) 0.025mg, cobalt chloride (COCl
26H
2O) 0.025mg, inositol 100mg, nicotinic acid 0.5mg, vitamin (dimension B
1) 0.1mg, pyridoxine hydrochloride (dimension B
6) 0.5mg, glycine 2mg, pH value 5.80; 1L YEP cultivates to concentrate and comprises: 5.0g peptone, 5.0g yeast extract, 2.5gNaCl, pH value 7.20.
Carry out rapid detection by the following method:
The instantaneous conversion of a, OPH gene pairs tomato
The Agrobacterium LBA4404 of preparation expression vector pE8OP is being contained 100mgL
-1Km and 20mgL
-1When 28 ℃ of concussions were cultured to OD600 and are about 0.5-0.7 in the YEP substratum of Rif, 5min collected thalline under 6000rpm, was about 2.0 with the resuspended thalline of the resuspended substratum of MS to OD600.It is dark to insert 3-4mm with the 1ml syringe from Chinese olive phase cherry tomato base portion, the light and slow injection tomato pulp of the resuspended bacterium liquid in 6ml left and right sides tissue, analyzes behind 28 ℃ of cultivation 3d.
B, the GUS dyeing that transforms tomato and enzyme biopsy survey:
Cultivate 3d after the conversion, cut thin slice and carry out GUS dyeing, cut successful tomato, the remainder of dyeing and carry out enzyme analysis alive.After the tomato of GUS stained positive carried out surface sterilization, grind pulping fast, add 50mM Tris-Hcl (pH 8.4) damping fluid of 2-3 times of volume with liquid nitrogen flash freezer, continue to be ground under the ice bath transparent, 12000rpm then, 15min gets supernatant as crude enzyme liquid.Get 50mM Tris-Hcl (pH 7.4) damping fluid that 200 μ l contain 0.1mM Coumaphos, and add 20 μ l crude enzyme liquids in reaction system, after 28 ℃ of camera bellows left standstill 30min, 340nm UV observed fluorescence production (see figure 4) down.
In same reaction system, by contrast A (the conversion fruit crude enzyme liquid of deactivation) and B (transforming the fruit crude enzyme liquid), and fluorescence production between the C (unconverted fruit crude enzyme liquid), the result shows through the tomato of instantaneous conversion can express GUS and organophosphor hydrolytic enzyme OPH, this enzyme can generate the coumarins derivative with the degraded of organophosphorus pesticide Coumaphos, thereby discharges fluorescence under UV excites.Because the intervening sequences on the expression vector behind the fruit promoters makes Agrobacterium itself not express lytic enzyme OPH, so the enzyme work that reflects is the situation that tamato fruit is expressed, effectively degrading organic phosphor pesticides.
Embodiment 2: the residual cucumber germplasm of spontaneous elimination farming is cultivated
(1) clone fruit-specific promoter according to a conventional method:
With embodiment 1;
(2) gene of synthetic organophosphor hydrolytic enzyme OPH:
With embodiment 1;
(3) make up fruit specific OPH plant expression vector:
With embodiment 1;
(4) transform soil Agrobacterium and cucumber explant
Transform soil Agrobacterium with embodiment 1;
No. 4 cucumber seeds are ground available from the academy of agricultural sciences, Tianjin in Tianjin.Sophisticated cucumber seeds warm water soaking 1h, with 75% alcohol-pickled 45s, use 0.1%HgCl again after the peeling
2Soak 5min, use sterile water wash 5 times, the sterilizing paper suck dry moisture is connected on the MS substratum, and when seed germination had 5cm height and cotyledon fully to stretch, the following half that cuts cotyledon band handle was cultivated on pre-culture medium 2 days.When Agrobacterium LBA4404 28 ℃ of concussions in containing the YEP substratum of 100mgL-1Km and 20mgL-1Rif of preparation expression vector pE8OP were cultured to OD600 and are about 0.5-0.7,5min collected thalline under 6000rpm, with the resuspended thalline of the resuspended substratum of MS.Select the pre-incubated cotyledon piece of deep green and in resuspended bacterium liquid, soak, constantly shake therebetween, take out behind the 10min and blot bacterium liquid with aseptic filter paper and be connected on the common substratum and cultivate.Explant after cultivating 3 days is altogether transferred in taking off bacterium culture medium, and it is complete to keep selecting pressure to guarantee to take off bacterium in 7 days.
(5) differentiation of transfer-gen plant, transplanting
With embodiment 1.
Carry out rapid detection by the following method:
The instantaneous conversion of a, OPH gene pairs cucumber
The Agrobacterium LBA4404 of preparation expression vector pE8OP is being contained 100mgL
-1Km and 20mgL
-1When 28 ℃ of concussions were cultured to OD600 and are about 0.5-0.7 in the YEP substratum of Rif, 5min collected thalline under 6000rpm, was about 2.0 with the resuspended thalline of the resuspended substratum of MS to OD600.Under vacuum 70kpa condition, soak into cucumber fruits thin slice 10min with resuspended bacterium liquid, repeat to soak into 10min behind the snap-out release, carry out enzyme analysis alive behind 28 ℃ of cultivation 3d.
The GUS dyeing and the enzyme biopsy of b, transformation of cucumber are surveyed
After 3d are cultivated in 28 ℃ of transformation of cucumber fruit sections, cut part and carry out GUS dyeing, cut the successful cucumber remainder of dyeing and carry out the enzyme analysis of living.Cucumber to the GUS stained positive is ground pulping fast with liquid nitrogen flash freezer, adds 50mM Tris-Hcl (pH 8.4) damping fluid of 2-3 times of volume, continue to be ground under the ice bath transparent, 12000rpm then, 15min gets supernatant as crude enzyme liquid.Get 50mM Tris-Hcl (pH 7.4) damping fluid that 200 μ l contain 0.1mM Coumaphos, and add 20 μ l crude enzyme liquids in reaction system, after 28 ℃ of camera bellows left standstill 30min, 340nm UV observed the fluorescence production down.The result shows through the cucumber of instantaneous conversion can express GUS and organophosphor hydrolytic enzyme OPH.
Embodiment 3: the residual capsicum germplasm of spontaneous elimination farming is cultivated
(1) clone fruit-specific promoter according to a conventional method:
With embodiment 1;
(2) gene of synthetic organophosphor hydrolytic enzyme OPH:
With embodiment 1;
(3) make up fruit specific OPH plant expression vector:
With embodiment 1;
(4) transform soil Agrobacterium and capsicum explant:
Transform soil Agrobacterium with embodiment 1;
Pepper seed of it green pepper is available from Beijing Jin Tianli company.Seed-coat with 95% alcohol-pickled 45s, is soaked 10min with 50% SYNTHETIC OPTICAL WHITNER again, use sterile water wash 5 times, the sterilizing paper suck dry moisture is connected on the MS substratum, 25 ℃ of illumination cultivation when the seed germination cotyledon fully stretches, cut hypocotyl and cultivated on pre-culture medium 2 days.When Agrobacterium LBA4404 28 ℃ of concussions in containing the YEP substratum of 100mgL-1Km and 20mgL-1Rif of preparation expression vector pE8OP were cultured to OD600 and are about 0.5-0.7,5min collected thalline under 6000rpm, with the resuspended thalline of the resuspended substratum of MS.Hypocotyl section after pre-the cultivation is soaked in resuspended bacterium liquid, constantly shakes therebetween, takes out behind the 10min, blots bacterium liquid with aseptic filter paper and is connected on the common substratum and cultivates.Explant after cultivating 3 days is altogether transferred in taking off bacterium culture medium, and it is complete to keep selecting pressure to guarantee to take off bacterium in 7 days.
(5) differentiation of transfer-gen plant, transplanting
With embodiment 1.
Carry out rapid detection by the following method:
The instantaneous conversion of a, OPH gene pairs capsicum
The Agrobacterium LBA4404 of preparation expression vector pE8OP is being contained 100mgL
-1Km and 20mgL
-1When 28 ℃ of concussions were cultured to OD600 and are about 0.5-0.7 in the YEP substratum of Rif, 5min collected thalline under 6000rpm, was about 2.0 with the resuspended thalline of the resuspended substratum of MS to OD600.Under vacuum 70kpa condition, soak into capsicum pericarp 20min with resuspended bacterium liquid, repeat to soak into 20min behind the snap-out release, carry out enzyme analysis alive behind 28 ℃ of cultivation 3d.
B, the GUS dyeing that transforms capsicum and enzyme biopsy survey
Grind pulping transforming the capsicum pericarp fast with liquid nitrogen flash freezer, add 50mMTris-Hcl (pH 8.4) damping fluid of 2-3 times of volume, continue to be ground under the ice bath transparent, 12000rpm then, 15min gets supernatant as crude enzyme liquid.Get 50mM Tris-Hcl (pH7.4) damping fluid that 200 μ l contain 0.1mM Coumaphos, and add 20 μ l crude enzyme liquids in reaction system, after 28 ℃ of camera bellows left standstill 30min, 340nm UV observed the fluorescence production down.The result shows through the capsicum of instantaneous conversion can express GUS and organophosphor hydrolytic enzyme OPH.
Claims (2)
1. the method for cultivation of the residual tamato fruit germplasm of the spontaneous elimination organophosphorus farming of an energy comprises the following steps:
(1) clone fruit-specific promoter according to a conventional method:
Described fruit-specific promoter is an E8 fruit specific genetic expression promotor; According to the characteristics of the E8 promoter sequence that provides among the GenbankAccession:AF515784.1, the design primer is that template is done pcr amplification with tomato DNA, and the primer of E8 promotor is:
P1：5’-CCCAAGCTTAGGAATTTCACGAAATCG-3’；
P2：5’-CGCGGATCCTCTTTTGCACTGTGAATG-3’；
(2) gene of synthetic organophosphor hydrolytic enzyme OPH:
Described organophosphor hydrolytic enzyme is the described aminoacid sequence of Genbank Accession:AAA24930, manually is synthesized into after translating into base sequence according to the tomato codon preference;
(3) make up fruit specific OPH plant expression vector:
The OPH gene order of E8 amplified production and synthetic is connected respectively to constructs pGEM-TE8 and pGEM-OPH on the pGEM-T easy, use HindIII and BamHI double digestion pGEM-TE8 and pSH then, E8 is building up to pSH obtains pSE8; With EcoRI single endonuclease digestion pGEM-OPH and pSE8, OPH is building up to pSE8 obtains pSE8OP, filter out the forward connection carrier, thereby the plant expression vector pSE8OP that obtains the fruit-specific promoter driving is standby;
(4) transform soil Agrobacterium and plant explants
Expression vector pSE8OP is transformed into soil Agrobacterium LBA4404 with freeze-thaw method, and picking contains the single bacterium colony of Agrobacterium of expression vector pSE8OP, is containing 100mgL
-1Km and 20mgL
-128 ℃ of concussions are cultured to OD in the YEP substratum of Rif
600Be 0.5-0.7, the centrifugal 5min of 6000rpm collects thalline, with the resuspended thalline of the resuspended substratum of MS; Select fresh and tender plant explants and in resuspended bacterium liquid, soak 10min, be connected on the common substratum after blotting bacterium liquid with aseptic filter paper, change over to after 2 days and contain Cef300mgL
-1Take off on the bacterium culture medium, keep the selective pressure subculture 2 times, complete until taking off bacterium;
(5) differentiation of transfer-gen plant, transplanting
To be connected on the division culture medium at the explant that takes off under surviving on the bacterium culture medium, the seedling of differentiation is transferred in the root media when growing to 2-3cm and cultivates, and treats to open bottle cap when seedling grows to 5-10cm, is transplanted to behind the hardening 3-5d in the soil;
Wherein: plant explants derives from tomato;
The prescription of described substratum is as follows: root media is basic medium with 1/2MS, and all the other substratum are basic medium with MS all, in basic medium, add following material and constitute described substratum,
Add 100mgL in the resuspended substratum of MS
-1AS and 20gL
-1Sucrose;
Add 1.0mgL in the substratum altogether
-16-BA, 0.1mgL
-1IAA, 100mgL
-1AS and 20gL
-1Sucrose;
Take off and add 1.0mgL in the bacterium culture medium
-16-BA, 0.1mgL
-1IAA, 300mgL
-1Cef and 30gL
-1Sucrose;
Division culture medium adds 1.0mgL
-16-BA, 0.1mgL
-1IAA, 300mgL
-1Cef, 70mgL
-1Km and 30gL
-1Sucrose;
Add 0.1mgL in the root media
-1IAA, 300mgL
-1Cef, 70mgL
-1Km and 20gL
-1Sucrose.
2. according to claim 1 can be spontaneous the method for cultivation of the residual tamato fruit germplasm of elimination organophosphorus farming, wherein: comprise in the 1L MS substratum: KNO
3, 1900mg; NH
4NO
3, 1650mg; CaCl
22H
2O, 440mg; MgSO
47H
2O, 370mg; KH
2PO
4, 170mg; MnSO
44H
2O, 22.3mg; ZnSO
47H
2O, 8.6mg; H
2BO
3, 6.2mg; KI, 0.83mg; Na
2MoO
42H
2O, 0.25mg; Na
2-EDTA.2H
2O, 37.25mg; FeSO
47H
2O, 27.8mg; CuSO
45H
2O, 0.025mg; CoCl
26H
2O, 0.025mg; Inositol, 100mg; Nicotinic acid, 0.5mg; Vitamin, 0.1mg; Pyridoxine hydrochloride, 0.5mg; Glycine, 2mg; PH value 5.80;
Comprise in the 1L YEP substratum: peptone, 5.0g; Yeast extract, 5.0g; NaCl, 2.5g; PH value, 7.20.
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| CN101451144B true CN101451144B (en) | 2011-06-01 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109280654A (en) * | 2018-09-12 | 2019-01-29 | 天津大学 | Organophosphor hydrolytic enzyme and purposes |
| CN114276954B (en) * | 2021-12-07 | 2023-09-26 | 广西大学 | Agrobacterium rhizogenes strain AT13 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302896A (en) * | 2001-01-17 | 2001-07-11 | 南京农业大学 | Methyltiophos hydrolase gene |
| WO2003056904A2 (en) * | 2002-01-08 | 2003-07-17 | Raab Michael R | Transgenic plants expressing civps or intein modified proteins and related method |
| WO2008036061A2 (en) * | 2005-04-06 | 2008-03-27 | Verenium Corporation | Enzymes and formulations for broad-specificity decontamination of chemical and biological warfare agents |
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2008
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302896A (en) * | 2001-01-17 | 2001-07-11 | 南京农业大学 | Methyltiophos hydrolase gene |
| WO2003056904A2 (en) * | 2002-01-08 | 2003-07-17 | Raab Michael R | Transgenic plants expressing civps or intein modified proteins and related method |
| WO2008036061A2 (en) * | 2005-04-06 | 2008-03-27 | Verenium Corporation | Enzymes and formulations for broad-specificity decontamination of chemical and biological warfare agents |
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