CN101974540A - Application of Xanthomonas oryzae pv. oryzicola HapGxooc gene segment hpaG28-126 - Google Patents

Application of Xanthomonas oryzae pv. oryzicola HapGxooc gene segment hpaG28-126 Download PDF

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CN101974540A
CN101974540A CN 201010528319 CN201010528319A CN101974540A CN 101974540 A CN101974540 A CN 101974540A CN 201010528319 CN201010528319 CN 201010528319 CN 201010528319 A CN201010528319 A CN 201010528319A CN 101974540 A CN101974540 A CN 101974540A
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董汉松
刘昌来
王颖
龙菊英
陈蕾
钱君
邹保红
李小杰
蔡洪生
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Nanjing Agricultural University
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Abstract

The invention belongs to the field of molecular biology, disclosing an application of Xanthomonas oryzae pv. oryzicola HapGxooc gene segment hpaG28-126. The invention provides the application of Xanthomonas oryzae pv. oryzicola HapGxooc gene segment hpaG28-126 in obtaining varieties with the pest stress resistance for imported plants. The invention also provides an application of Xanthomonas oryzae pv. oryzicola HapGxooc gene segment hpaG10-42 coded by the gene in plant pest stress resistance. The invention also provides a recombinant expression vector which is composed of a recombined expression vector formed by combining an hpaG28-126 gene segment and a 44P promoter and a pCAMBIA300 vector of which the hyg gene is replaced into a manA gene. The recombinant vector can obviously improve the output of wheat and strengthens the disease-resistant capability of wheat.

Description

Paddy rice slice pinta bacterium HpaG XoocGene fragment hpaG 28-126Application
Technical field
The invention belongs to biology field, relate to paddy rice slice pinta bacterium HpaG XoocGene fragment hpaG 28-126Application.
Background technology
The Hrp gene is distributed widely in the Gram-negative phytopathogen, and wherein they are clusters, conservative and be interchangeable in some cases.Each hrp gene all contains three genoids, respectively coding constitute III type excretory system the factor, express and the factor of having secreted regulating and controlling effect by III type excretory system excretory effector molecule and to III type effector.Harpins protein is the important member of III type effector molecule, and this proteinoid is very special, the active and diverse in function of character.Harpins class exciton all has similar biochemical character: be rich in glycine, do not contain halfcystine, thermally-stabilised, to the proteolytic enzyme sensitivity, its main common biological effect is induced hypersensitivity reaction on non-host plants such as tobacco.
This laboratory early-stage Study has been found a kind of new Harpins albumen: HpaG XoocAlbumen.HpaG XoocAlbumen all contains a cysteine residues, and other harpin albumen such as HrpNEa and HrpZPss all do not contain cysteine residues.Although HpaG XoocProteic structure is different with other harpin albumen, but they have identical effect on plant.HpaG XoocNo matter external albumen is applied to plant and still carries out after the transgenosis HpaG XoocThe generation and the inducing plant defense response that can both activate different signal pathways and promote plant-growth, induce HCD.HpaG XoocExternal spraying, can increase paddy disease-resistant, promote plant-growth, and be attended by the expression (Ren et al, 2006) of stretching plain (expansin) gene OsEXP1.
HpaG XoocProtein coding gene hpaG XoocCome from xanthomonas oryzae pv. oryzicola (Xanthimonas.oryzae.Oryzicola), total length 413bp, 137 amino acid of encoding, molecular weight is about 15.18kD, two GRM structural domains that it comprises lay respectively at 72 to 77 amino acids and 107 to 112 amino acids (Liu Fang etc., " HpaG XoocIn be rich in the glycine motif and halfcystine has suppressed its a lot of effects on plant " Phytopathol96:1052-1059 (2006)); This laboratory has obtained paddy rice slice pinta bacterium HpaG by lot of experiments XoocProtein coding gene hpaG Xooc9 excalation fragment hpaG 1-315, hpaG 1-282, hpaG 1-183, hpaG 1-414, hpaG 19-138, hpaG 184-414, hpaG 28-126, hpaG 283-414, hpaG 250-282, these nine fragments are corresponding successively protein fragments HpaG respectively 1-105, HpaG 1-94, HpaG 1-61, HpaG 1-138, HpaG 7-61, HpaG 62-137, HpaG 10-42, HpaG 95-137, HpaG 84-94Studies show that HpaG 10-42Can on paddy rice and tobacco, not cause allergic reaction, but can increase the disease resistances of these crops, and strengthen the growth of paddy rice, increase the output of paddy rice.But, up to the present still have nothing to do in HpaG 10-42The report of insect-resistance function.
Summary of the invention
The purpose of this invention is to provide paddy rice slice pinta bacterium HpaG XoocGene fragment hpaG 28-126Application.
Another object of the present invention provides the protein fragments HpaG of this genes encoding 10-42Application.
Another purpose of the present invention provides a kind of gene hpaG that comprises 28-126Recombinant expression vector and application thereof.
Technical scheme of the present invention is as follows:
Nucleotides sequence is classified the paddy rice slice pinta bacterium HpaG of SEQ ID NO.1 as XoocProtein fragments gene hpaG 28-126Obtain application in the kind that pest-resistant evil coerces importing plant.
A kind of gene hpaG 28-126The paddy rice slice pinta bacterium HpaG of coding XoocProtein fragments HpaG 10-42Application in the plant anti-insect evil is coerced, its aminoacid sequence are SEQ ID NO.2.
A kind of recombinant expression vector, this recombinant expression vector are united to form by nucleotide sequence shown in the SEQ ID NO.1 and 44P promotor and are expressed the pCAMBIA1300 carrier that combination and HYG gene be replaced by the manA gene and form.
Described transgenic plant are transgenic wheat.
The application of described recombinant expression vector in improving the transgenic crop disease resistance.
The application of described recombinant expression vector in improving transgenic crop disease resistance and output.
Beneficial effect of the present invention: by paddy rice slice pinta bacterium HpaG XoocGene fragment hpaG 28-126Encoded protein fragment HpaG 10-42Can strengthen some anti-aphid Expression of Related Genes, Arabidopis thaliana sprays HrpG 10-42With respect to spraying EVP, aphid quantity obviously reduces; And spray HrpG 10-42The food ingestion of small cabbage moth to Arabidopis thaliana can obviously be reduced with respect to spraying EVP in the back, so paddy rice slice pinta bacterium HpaG XoocProtein fragments HpaG 10-42, its encoding gene hpaG 28-126Can be in the application in the kind that importing plant acquisition disease and insect resistance is coerced.
The paddy rice slice pinta bacterium HpaG that contains of the present invention XoocGene fragment hpaG 28-126Recombinant expression vector, can obviously improve the output of wheat, strengthen the resistance against diseases of wheat.
Description of drawings
Fig. 1 hpaG 28-126The restriction enzyme mapping of gene.
Fig. 2 HpaG 10-42The purifying protein electrophorogram.
Fig. 3 sprays HrpG 10-42The anti-aphid effect in back.
Fig. 4 HrpG 10-42Influence to growth related gene.
Fig. 5 sprays HrpG 10-42The back is anti-gets the food influence to small cabbage moth.
Fig. 6 recombinant vectors transfer genetic unit structural representation,
ManA::6his: have 6 histidine-tagged phosphomannose transferase genes; The 44P:Myb44 gene promoter.
Fig. 7 pCAMBIA1300::ManA::44P carrier enzyme is cut checking,
Wherein 1300::44p is pCAMBIA1300::ManA::44P, and M is marker.
Fig. 8 pCAMBIA1300::ManA::44P::hpaG 28-126:: the restriction enzyme mapping of 6His,
Wherein 1300::hrf99 is pCAMBIA1300::ManA::44P::hpaG 28-126:: 6His, M are marker.
Embodiment
Embodiment 1hpaG 28-126The preparation method of gene
Paddy rice streak germ extracting genome DNA: the paddy rice streak germ culture of getting the 1ml incubated overnight is in the 1.5mlEP pipe, the centrifugal 5min of room temperature 8000rpm, abandon supernatant, precipitation is suspended among the 1ml TE (pH8.0) again, the N,O-Diacetylmuramidase that adds 6 μ l 50mg/ml (is given birth to emerging, China), 37 ℃ of effect 2h.Add 2mol/L NaCl 50 μ l again, 10%SDS110 μ l, the Proteinase K of 20mg/ml (living emerging, China) 3 μ l, 50 ℃ of effect 3h (this moment, bacterium liquid should be transparent thick liquid).Bacterium liquid is all assigned to two 1.5ml EP pipes, adds isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), and mixing, room temperature is placed 5-10min.The centrifugal 10min of 12000rpm carries out extracting, and extracting twice adds the Virahol of 0.6 times of volume, mixing, and room temperature is placed 10min.The centrifugal 10min of 12000rpm precipitates the washing with alcohol with 75%, after draining, is dissolved in 50 μ l ddH 2Making pcr template among the O uses.
Pcr amplification hpaG 28-126The gene primer sequence is: upstream primer: SEQ ID NO.3, downstream primer: SEQ IDNO.4; 5 ' end at upstream primer adds the EcoRI restriction enzyme site, and 5 ' end adding HindIII restriction enzyme site at downstream primer adds two successive terminator codons (CTA and TTA) in downstream primer.Utilize above-mentioned primer to carry out PCR, 25 μ L reaction systems are: 10 * Ex taq buffer, 2.5 μ L, dNTP 2.0 μ L, Mg 2+1.5 μ L, each 1 μ L of upstream and downstream primer, Ex taq 0.2 μ L, paddy rice streak germ genomic dna 1 μ L, ddH 2O15.8 μ L.Response procedures is: 95 ℃ of 3min, 95 ℃ of 50sec, 58 ℃ of 50sec, 72 ℃ of 1min, 72 ℃ of 10min.After the purified recovery of PCR product, be connected to pMD TM19-T Simple Vector carrier (Takara, Japan) on, use EcoRI and HindIII to carry out double digestion, obtain restriction enzyme mapping such as Fig. 1, (Takara Japan), serves the order-checking of extra large invitrogen company to recombinant vectors transformed into escherichia coli competent cell DH5 α.
Embodiment 2 paddy rice slice pinta bacterium HpaG XoocProtein fragments HpaG 10-42Preparation method
Use EcoRI and HindIII insertion hpaG to making up among the embodiment 1 28-126The pMD of gene fragment TM19-TSimple Vector carrier carries out double digestion, obtains hpaG 28-126Gene fragment is inserted in pET30a (+) (invitrogen USA) carrier with correct direction, and recombinant vectors transformed into escherichia coli BL21 (Takara, Japan).Thalline is inoculated in the LB liquid nutrient medium, and under 28 ℃ of conditions, 200rpm shaking culture 18-20 hour is used the spectrophotometric determination bacterial concentration, requires OD620 〉=2.0.It is overnight to add the IPTG that final concentration is 0.4mM (giving birth to emerging China) inducing culture this moment, carries out microorganism collection.Same method transforms pET30a (+) empty carrier, and the inactive protein solution (EVP) of generation in contrast.Albumen extracts (Bauer et al, 1995 according to ordinary method; Liu et al, 2006).5,000 * g was resuspended in the imidazoles binding buffer liquid of 1/20th or 1/10th volumes to collect thalline in centrifugal 15 minutes.Add final concentration 1 μ l/ml Ready-Lyse (Epicenter Biotech., Rockford, IL, USA) and the proteinase inhibitor phenylmethylsulfonyl fluoride of 100 μ g/ml (Phenylmethyl Sulfonyl Fluoride PMSF), placed 10 minutes on ice.Bacterium liquid carries out fragmentation with ultrasonic wave, and broken bacterium liquid is to clear under the condition of ice bath.Use 4 ℃ again, 10,000g went precipitation in centrifugal 20 minutes.Supernatant liquor boiled in boiling water bath 10 minutes, 12,000 * g, and centrifugal 20 minutes, the supernatant liquor of removing the precipitation gained was in-20 ℃ of freeze overnight, and the back centrifugal (12,000 * g, 20 minutes) that thaws obtains new supernatant liquor and is not celliferous protein Preparation liquid.Albumen uses HisTrap HP Kit, and (NJ, pillar USA) carry out nickel post affinity chromatography purification for Amersham Biosci Corp, Piscataway.The nickel post notices that with the combination of 10mL imidazoles binding buffer liquid elder generation concentration is consistent with proteolytic imidazoles binding buffer liquid.Use the proteinic molecular weight of DANStar software prediction, utilize polyacrylamide gel electrophoresis technology conclusive evidence protein molecular size then as Fig. 2.
Embodiment 3HrpG 10-42Protein fragments is to the resistance of aphid
HrpG 10-42After albumen uses nickel post affinity chromatography purification, according to (Dong, H., Delaney, T.P., Bauer, D.W., and Beer, S.V.1999.Harpin induces disease resistance in Arabidopsis through thesystemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene.PlantJ.20:207-215.) method is determined concentration, albumen is mixed with the concentration of 12 μ g/ml, and add 0.03% tensio-active agent Silwet-77 (Momentive Performance Materials Inc., China Branch Beijing) sprays 30 days Arabidopis thaliana of growth, does contrast to spray EVP.Every strain Arabidopis thaliana inoculate 1 age 15 of aphids in lobus cardiacus, the inoculation back uses guard to overlap, and writes down the quantity of blade aphid every day, in 1 week of continuous recording, 10 repetitions are done in each experiment.As Fig. 3, spray HrpG as can be seen from Figure 3 10-42Obviously reduce with respect to the aphid quantity that sprays EVP.
Embodiment 4HrpG 10-42Protein fragments is induced anti-aphid related gene expression
Arabidopis thaliana was cultivated 30 days, sprayed 12 μ g/ml HrpG then 10-42(embodiment 3 preparations) and EVP are after 1 day, and the inoculation aphid respectively at 0 day, was taken a sample in 3 days, got the complete unfolded of plant top tender leaf 100mg, and liquid nitrogen freezing grinds; Homogenate is transferred in the 2mL Eppendorf pipe, adds Trizol (Takara, Japan) solvent 1mL, mixing, room temperature placement 5 minutes; Add chloroform 0.2mL, vortex vibrator 15 seconds; Room temperature was placed 2-15 minute; 4 ℃, centrifugal 15 minutes of 12000rpm; The water that the upper strata is colourless is transferred in the new Eppendorf pipe; Add the 0.5mL Virahol, put upside down mixing several times; Room temperature was placed 5-10 minute, made the RNA precipitation; 4 ℃, 12, centrifugal 15 minutes of 000rpm; Add 1mL 75% alcohol flushing or preservation; 4 ℃, 12, centrifugal 15 minutes of 000rpm abandons supernatant; Drying at room temperature is removed unnecessary alcohol; Add the no RNase ultrapure water that an amount of DEPC handles; The DNase that in RNA solution, adds no RNAase, to final concentration be 0.1U/ μ L, 37 ℃ of processing 0.5 hour; Phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting; 4 ℃, 12, centrifugal 15 minutes of 000rpm; Get the dehydrated alcohol that upper water is added to 2 times of volumes, mixing gently ,-20 ℃ are spent the night; 4 ℃, 12, centrifugal 20 minutes of 000rpm abandons supernatant; 70% ice ethanol is washed precipitation, the ultrapure water dissolving RNA that handled with DEPC after the drying at room temperature.The RT-PCR anti-aphid genes involved that increases from the total RNA that obtains: AtEXP1, AtEXP2, Hel1, PDF1.2, EF1 α, the PCR reaction system is that 25 μ L reaction systems are: 10 * Ex taq buffer, 2.5 μ L, dNTP 2.0 μ L, Mg 2+1.5 μ L, each 1 μ L of upstream and downstream primer, Ex taq 0.2 μ L, genomic dna 1 μ L, ddH 2O15.8 μ L.Response procedures: 95 ℃ of 3min, 95 ℃ of 50s, 58 ℃ of 50s, 72 ℃ of 50s, 72 ℃ of 10min, 30cycles.The pcr amplification upstream and downstream primer sequence of gene A tEXP1 is respectively SEQ ID NO.5, SEQ ID NO.6; The pcr amplification upstream and downstream primer sequence of gene A tEXP2 is respectively SEQ ID NO.7, SEQ ID NO.8; The pcr amplification upstream and downstream primer sequence of gene Hel1 is respectively SEQ ID NO.9, SEQ ID NO.10; The pcr amplification upstream and downstream primer sequence of gene PDF1.2 is respectively SEQ ID NO.11, SEQ ID NO.12; The pcr amplification upstream and downstream primer sequence of gene EF1 α is respectively SEQ ID NO.13, SEQ ID NO.14.The laggard row agarose gel electrophoresis that increases is spraying HrpG as shown in Figure 4 10-42Following AtEXP1, AtEXP2, Hel1, these three genes of PDF1.2 obviously strengthen expression, show HrpG 10-42Can strengthen some anti-aphid Expression of Related Genes.
Embodiment 5HrpG10-42 protein fragments is got the food influence to small cabbage moth
Use EVP and 12 μ g/ml hrpG respectively 10-42(embodiment 3 preparation) handled Arabidopis thaliana after 3 days, and (in 2 ages, 2-3mm) to the Arabidopis thaliana young leaves, every day, the quilt of observed and recorded Arabidopis thaliana was got the food situation to inoculate 2 diamondback moth larvaes of hatching 72h.As shown in Figure 5, obviously reduced the food ingestion of small cabbage moth with respect to spraying EVP after spraying HrpG10-42 to Arabidopis thaliana.
The unitary structure of embodiment 6 transgenosiss
Use pNOV2820 (Syngenta, Research Triangle Park, North Carolina) the HYG gene replaced on pCAMBIA1300 (available from the CAMBIA company) carrier of PMI (ManA) gene on the carrier becomes pCAMBIA1300::ManA (this process has Jin Site company to finish).
The acquisition of 44P promotor: the 1) acquisition of arabidopsis thaliana genomic dna: liquid nitrogen grinding 0.1g blade adds 1mlCTAB (traditional Chinese medicines China) (2%), 65 ℃ of water-bath 1h, every 10-20min, the mixing sample once adds equal-volume phenol, mixing, room temperature, 7000rpm, 10min gets supernatant, adds equal-volume chloroform/Virahol (24: 1), mixing, 4 ℃, 10000rpm, 10min gets supernatant, repeats the step, extracting once more, to two liquid level inclusion-frees, get supernatant, add the 3M NaAC (pH7.0) of 1/10 volume precooling and the cold dehydrated alcohol of 2 times of volumes, slowly shake up ,-20 ℃ are spent the night.4 ℃, 12000rpm, 15min abandons supernatant 12000rpm, and 15min uses 75% and 95% cold absolute ethanol washing respectively once, afterwards, air-dry DNA.Add 100 μ LddH 2O and 0.5 μ LRNase (10 μ g/ml), 37 ℃, 30min1% glue detects ,-20 ℃ of preservations.
2) pcr amplification 44P: design upstream primer SEQ ID NO.15 and downstream primer SEQ ID NO.16, at 5 ' end adding EcoRI restriction enzyme site of upstream primer, at 5 ' end adding SacI restriction enzyme site of downstream primer.Carry out PCR with above-mentioned primer, 25 μ L reaction systems are: 10 * Ex taq buffer, 2.5 μ L, dNTP 2.0 μ L, Mg2+1.5 μ L, each 1 μ L of upstream and downstream primer, Ex taq 0.2 μ L, arabidopsis thaliana genomic dna 1 μ L, ddH2O15.8 μ L.Response procedures the following is 95 ℃ of 3min, 95 ℃ of 50sec, 58 ℃ of 50sec, 72 ℃ of 1min, 72 ℃ of 10min.After the purified recovery of PCR product, carry out the EcoRI/SacI double digestion and obtain the 44P promotor, be inserted into pCAMBIA1300::ManA, become pCAMBIA1300::ManA::44P.Enzyme is cut checking as Fig. 7, and wherein 1300::44P is pCAMBIA1300::ManA::44P, and M is marker.Serve the order-checking of extra large invitrogen company.
The pMD of the insertion hpaG28-126 gene fragment that makes up with embodiment 1 TM19-T Simple Vector carrier is a template, utilizes upstream primer SEQ ID NO.17, the downstream primer SEQ ID NO.18 hpaG that increases 28-126Gene fragment at 5 ' end adding SacI restriction enzyme site of upstream primer, at 5 ' end adding BamHI restriction enzyme site of downstream primer, and in 6 GTG sites of downstream primer introducing, obtains containing the hpaG of 6 His marks 28-126Gene fragment.The PCR reaction system is according to embodiment 1.The gene hpaG that amplification is obtained 28-126Be connected to pMD TM19-T Simple Vector carrier (Takara, Japan), with this recombinant vectors called after T::hrf 28-126, recombinant vectors T::hrf 28-126Change in the bacillus coli DH 5 alpha, after order-checking is correct, then carrier pCAMBIA1300::ManA::44P is cut with the SacI/BamHI enzyme, carrier T::hrf 28-126Also cut and obtain hpaG with the SacI/BamHI enzyme 28-126Fragment, hpaG 28-126Fragment is connected with the pCAMBIA1300::ManA::44P carrier, obtains expression vector pCAMBIA1300::ManA::44P::hpaG 28-126:: 6His, change carrier over to DH5 α, Fig. 6 is pCAMBIA1300::ManA::44P::hpaG 28-126:: 6His expression vector collection of illustrative plates; Be the restriction enzyme mapping of pCAMBIA1300::ManA::44P among Fig. 7, wherein 1300::44p is pCAMBIA1300::ManA::44P; Fig. 8 is pCAMBIA1300::ManA::44P::hpaG 28-126:: the restriction enzyme mapping of 6His.Wherein 1300::hrf99 is pCAMBIA1300::ManA::44P::hpaG 28-126:: 6His, M are marker.
The preparation of embodiment 7 particle gun particulate bullets
Extract the transgene carrier unit pCAMBIA1300::ManA::44P::hpaG among the embodiment 6 28-126:: 6His and empty carrier pCAMBIA1300::ManA::44P.
Claim bronze 30mg (the BIA-RAD company U.S.), be dissolved in 500 μ l raw spirits, centrifugal 2 times, add 500 μ lddH 20, bronze concentration is 60 μ g/ μ l, and it is standby to put-20 ℃ of refrigerators.Be equipped with 2.5M Cacl2, take by weighing 5.549gCacl2 → 20mlddH 2O, suction filtration or autoclaving, it is standby to put-20 ℃ of refrigerators.Be equipped with the 0.1M spermidine, take by weighing the 145.2mg spermidine and add 20mlddH2O, suction filtration or autoclaving, it is standby to put-20 ℃ of refrigerators.
Get 15 μ l bronzes at the 1.5ml centrifuge tube, centrifugal 30 seconds of 1400rpm removes supernatant liquor, adds the 1ml sterilized water, and centrifugal 5 minutes, remove supernatant liquor, add the carrier pCAMBIA1300::ManA::44P::hpaG that makes up among the embodiment 6 respectively 28-126:: 6His carrier and empty carrier pCAMBIA1300::ManA::44P 25 μ l (concentration is 1 μ g/ μ l)
+220μl?ddH 2O
+250μl?2.5M?CaCl 2(sigma?USA)
+ 50 μ l 0.1M spermidines (sigma USA)
Vortex 10 minutes (being preferably in-4 ℃), centrifugal 5 minutes, remove supernatant liquor, add 600 μ l raw spirits, centrifugal 1 minute, remove alcohol, add 250 μ l alcohol and suspend.
Embodiment 8HpaG 10-42The transgenic wheat production process
Collection is bloomed 12-14 days the immature seed in back (under the greenhouse experiment, change to some extent with the variation of temperature time, the about 1mm of rataria diameter was in from transparent to translucent transition period), 70% ethanol disinfection 2 minutes, 0.1% mercuric chloride sterilization 15-20 minute, aseptic water washing 4-5 time takes out rataria with scalper on Bechtop, scultellum is inoculated in MSY callus of induce substratum (SD2 up, modified MS medium can), 25 ℃ of dark culturing.
Cultivated 6-7 days, and selected fine quality rataria callus, concentrate on the culture dish center that contains 0.4M osmotic pressure substratum (0.2M N.F,USP MANNITOL+0.2M sorbyl alcohol), diameter is 2.5cm, about 50 ratarias, osmotic pressure pre-treatment 4 hours.
Through the pretreated rataria callus of osmotic pressure, use two kinds of particulate bullets that make among the embodiment 7, the PDS 1000/He particle gun that uses BIA-RAD company to produce bombards, the aftertreatment 16 hours on the osmotic pressure substratum of the callus after (psi1100, Hg 27.5) bombardment.
Callus is transferred to MSY substratum (SD2 or modified MS medium) to be recovered to cultivate for 2 weeks.Callus after recovering to cultivate is transferred to the additional NAA1mg/L of 1/2MS, the differentiation screening culture medium of KT1mg/L (or ZT 5mg/L), and the concentration of selective agent seminose (West Asia reagent China) is 10g/L, 24-26 ℃ of illumination cultivation.
Treat that transferring to 1/2MS behind the callus differentiation seedling does not have on the growth screening culture medium of hormone, the concentration of selective agent seminose is 10g/L, 24-26 ℃ of illumination cultivation.If it is more to transform seedling quantity, can screen once again.
Transfer to strong sprout on the strong seedling culture base of additional IAA0.5mg/L of 1/2MS (giving birth to emerging China) and MET (multiple-effect short) (giving birth to emerging China) through the conversion seedling of 2-3 screening, treat the conversion seedling preparation transplanting of height of seedling 7-8cm and well developed root system.
In order to improve transplanting survival rate, the wheat tissue cultured seedling is transplanted needs a low temperature to preserve moisture the stage, earlier little transplantation of seedlings in the paper nutrition pot, be placed in illumination box or the controlled greenhouse, temperature is controlled at about 15 ℃, and plastic covering cloth is preserved moisture, note suitably ventilation in order to avoid mildew, move on to the greenhouse after going back seedling.
Embodiment 9HpaG 10-42Transgenic wheat and commentaries on classics empty carrier transgenic lines rate ratio are
Each gathers 1000 of sophisticated wheat seeds, and relatively its weight repeats 5 times, finds HpaG 10-42Transgenic wheat thousand seed weight is higher than empty carrier transgenic lines 10%.
Embodiment 10HpaG 10-42Transgenic wheat and empty carrier transgenic lines anti gibberellic disease are relatively
With inoculating needle choose the gibberella mycelia a little, place 25-28 ℃ of shaking culture 4-7 days detectable level of sweet mung bean soup nutrient solution.Take a morsel spore suspension in an aseptic bottle, the concentration of red mould spore is adjusted to sterilized water under the visual field of 10 (eyepiece) * 20 (object lens) and is advisable about 20-30 spore.To the early flowering season, the wheat head that selection is not bloomed as yet uses the spore liquid for preparing at the wheat full heading time, and pin is annotated inoculation.Calculate the infected seed number after 21 days.Find HpaG 10-42Transgenic wheat has obviously few infected seed number than the empty carrier transgenic lines.
Figure IDA0000030477230000011
Figure IDA0000030477230000031
Figure IDA0000030477230000041
Figure IDA0000030477230000051

Claims (7)

1. nucleotides sequence is classified the paddy rice slice pinta bacterium HpaG of SEQ ID NO.1 as XoocGene fragment hpaG 28-126Obtain application in the kind that pest-resistant evil coerces importing plant.
2. described gene hpaG of claim 1 28-126The paddy rice slice pinta bacterium HpaG of coding XoocProtein fragments HpaG 10-42Application in the plant anti-insect evil is coerced, its aminoacid sequence are SEQ ID NO.2.
3. recombinant expression vector is characterized in that this recombinant expression vector is united to form by nucleotide sequence shown in the SEQ ID NO.1 and 44P promotor to express the pCAMBIA1300 carrier that combination and hyg gene be replaced by the manA gene and form.
4. the described recombinant expression vector of claim 3 is improving the application in coercing of transgenic crop disease resistance and/or output and/or pest-resistant evil.
5. application according to claim 4 is characterized in that described farm crop are wheat.
6. the application of the described recombinant expression vector of claim 3 in improving the transgenic crop disease resistance.
7. the application of the described recombinant expression vector of claim 3 in improving transgenic crop disease resistance and output.
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CN111138518A (en) * 2019-12-30 2020-05-12 山东农业大学 Expression and application of bacterial transposon component protein and truncation thereof

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