CN103421840A - Method for improving resistance of rape to Lepidoptera pests by transgenic technology - Google Patents
Method for improving resistance of rape to Lepidoptera pests by transgenic technology Download PDFInfo
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- CN103421840A CN103421840A CN2013103329711A CN201310332971A CN103421840A CN 103421840 A CN103421840 A CN 103421840A CN 2013103329711 A CN2013103329711 A CN 2013103329711A CN 201310332971 A CN201310332971 A CN 201310332971A CN 103421840 A CN103421840 A CN 103421840A
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Abstract
The invention belongs to the technical field of plant transgene, and particularly relates to a method for improving resistance of rape to Lepidoptera pests by a transgenic technology. The method is characterized in that a synthetic Bt pesticidal gene cry1C * is transgeneticized into the rape by an agrobacterium-mediated method; furthermore, through PCR (polymerase chain reaction) and Southern hybridization, whether an exogenous gene is integrated into a rape genome is detected; and expression conditions of the cry1C * gene in transcription and protein level are detected through real-time quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) respectively. Pest resistance identification results show that the resistance of the transgenic rape to the Lepidoptera pests is significantly improved. The method is simple in operation; through the method, the resistance of the rape to the Lepidoptera pests can be significantly improved, and the normal growth of a plant is not affected, so that the method is applicable to pest-resistant breeding of the rape.
Description
Technical field
The invention belongs to the rape field of transgenic technology, be specifically related to a kind of method of rape to the lepidoptera pest resistance that improve, the method can be used for the breeding of rape transgenic pest-resistant.
Background technology
Lepidopteran (Lepidoptera Linnaeus) insect is important crop pests, comprise the insects such as striped rice borer (Chilo supperssalis), yellow rice borer (Tryporyza incertulas), bollworm (Heliothis armigera), small cabbage moth (Plutella xylostella linnaeus) and cabbage caterpillar (Pierisrapae Linne), these lepidoptera pests cause serious harm to farm crop such as paddy rice, cotton and rapes.Have data to show, the Bt gene pairs lepidoptera pest that derives from bacillus thuringiensis (Bacillus thuringiensis) has significant resistance.The proteins encoded of this gene is commonly referred to delta-endotoxin (δ-endotoxins) or desinsection crystalline protein (insecticidal crystal protein, ICP), and molecular weight is generally 130-140kD.Cloned first Bt gene from the people such as Schnepf in 1981, after this constantly had dissimilar Bt gene to be found and to clone, the Bt gene of having found at present has kind more than 300 (Yang Daxing etc., 2008).Utilize the Bt gene to obtain the various crop such as transgenic pest-resistant cotton, paddy rice and rape, the plantation of insect-resistant transgenic crops, not only reduced spraying of sterilant, reduced the pollution to environment, and saved the production cost of farm crop.The cultivated area of Bt crop sustainable growth in worldwide in recent years, the cultivated area of global Bt crop in 2011 has reached 6,500 ten thousand hectares (James, 2011).
Rape is the important oil crops of worldwide extensively planting, and the Main Cultivation type comprises swede type rape, mustard type rape and turnip type rape.Lepidoptera pest is the main Field Pests of rape, badly influences the yield and quality of rape.Stewart etc. (1996) utilize agrobacterium-mediated transformation that the Bt killing gene cry 1Ac of synthetic is imported to rape the earliest, show that transfer-gen plant plays restraining effect to the growth of small cabbage moth, cabbage looper and Heliothis zea.Lin Liangbin etc. (2002) utilize Bt gene cry1 to transform rape, have obtained the transgenic line of high resistance cabbage caterpillar.
Compare the crops such as paddy rice, cotton, at present Bt transgene rape type is very few, big area for the production of the time easily cause insect to produce resistance.
Cry1C
*Gene be by the woods of State Key Laboratory of Crop Genetic Improvent support the army, professor Zhang Qifa transforms synthetic (Chinese invention patent number: ZL 02139081.9), they take the cry1Ca5 sequence as basis, the preferences synthetic used according to vegetable codon cry1C
*The encoding sequence of gene, and added the homing sequence that improves gene expression efficiency at its 5 ' end, added the tailing recognition sequence at 3 ' end simultaneously.Studies show that cry1C
*The transgenic paddy rice strain is to striped rice borer and yellow rice borer apparent altitude resistance: after inoculation striped rice borer in one age or yellow rice borer larva, the larval mortality in its 5 days is 100% or approaches 100%(Tang Wei, 2006).
Have not yet to see the relevant cry1C that utilizes
*Gene is cultivated the report of pest-resistant rape.Utilize cry1C
*Gene is cultivated has the novel B t transgene rape that insect resistance capacity is strong, for enriching the pest-resistant resource of rape, promotes that the development of rape breeding for pest resistance is significant.
Summary of the invention
The object of the invention is to overcome the defect of prior art, for the serious present situations that cause harm such as lepidoptera pest small cabbage moth, cabbage caterpillar in current rape production, provide a kind of transgenic technology of utilizing to improve the method for rape to the lepidoptera pest resistance.
The present invention is achieved through the following technical solutions:
(1) utilize cry1C
*The binary expression vector of gene (Chinese invention patent: ZL 02139081.9), transform rape (comprising swede type rape, mustard type rape or turnip type rape) by agriculture bacillus mediated method, the rape aseptic seedling hypocotyl of take is explant, hypocotyl segment after infecting Agrobacterium and carrying out common cultivation is transferred to callus inducing medium and the shoot regeneration substratum that contains selective agent, screening by selective agent, the regrowth that will have resistance is transferred in root media, the seedling after taking root is transferred to greenhouse and cultivated;
(2) transformed plant is carried out to PCR and Southern hybridization check, determine the transgenic positive strain, PCR detects and the amplification of Southern hybridization probe, and the special primer of use is as follows:
Forward primer: cry1C-F 5 '-TCGACATCTTGAACAACCTT-3 ';
Reverse primer: cry1C-R 5 '-CTACTTTTGTGCTCTTTCAA-3 ';
(3) by real-time quantitative RT-PCR to cry1C in the transgenic positive strain
*The transcriptional level of gene is detected, cry1C
*Amplimer as follows:
Forward primer: cry1C-193F 5 '-GGAATCGTTGGACCTTCTCA-3 ';
Reverse primer: cry1C-391R 5 '-CGATCACTCTGGTCCTGGTT-3 ';
The amplimer of reference gene BnActin is as follows:
Forward primer: actin-F 5 '-ACAGTGTCTGGATCGGTGGTTC-3 ';
Reverse primer: actin-R 5 '-TGCCTCATCATACTCAGCCTTG-3 ';
(4) strain is detected to transgenic positive to utilize enzyme linked immunosorbent assay (ELISA), determines Cry1C in plant
*Protein content;
(5) further transgenic line is carried out the insect-resistance evaluation of lepidoptera pest, the resistance level of clear and definite transgenic line, obtain the pest-resistant transgene rape strain;
Wherein:
Cry1C described in step (1)
*The nucleotide sequence of gene is as shown in SEQ ID NO:1;
Selective agent described in step (1), select corresponding selective agent according to the selective marker of the binary vector used, and selective agent wherein is careless ammonium phosphine;
Pcr amplified fragment described in step (2) and Southern hybridization probe sequence are as shown in SEQ ID NO:3, and size is 0.99kb;
Cry1C described in step (4)
*The aminoacid sequence of albumen is as shown in SEQ ID NO:2;
Medium component in above-mentioned steps is as follows:
Aseptic seedling culture base M
0: 1/2MS, 7g/L agar powder (Agargel), pH=5.8;
Liquid nutrient medium DM:MS, 30g/L sucrose, 100 μ M Syringylethanones, pH=5.8;
Be total to culture medium M
1: MS, 30g/L sucrose, 18g/L N.F,USP MANNITOL, 1mg/ L 2,4 dichlorophenoxyacetic acid, 0.3mg/L kinetin, 100 μ m Syringylethanones, 2.5g/L plant gel (Phytagel), pH=5.8;
Callus inducing medium M
2: MS, 30g/L sucrose, 18g/L N.F,USP MANNITOL, 1mg/ L 2,4 dichlorophenoxyacetic acid, 0.3mg/L kinetin, 20mg/L Silver Nitrate, 270mg/L Timentin, 5mg/L grass ammonium phosphine, 2.5g/L plant gel, pH=5.8;
Shoot regeneration substratum M
3: MS, 10g/L glucose, 0.25g/L wood sugar, 0.6g/L 2-(N-morpholine) ethane sulfonic acid, 0.1mg/L indolylacetic acid, the trans zeatin of 2mg/L, 270mg/L Timentin, 5mg/L grass ammonium phosphine, 2.5g/L plant gel, pH=5.8;
Root media M
4: 1/2MS, 10g/L sucrose, 0.5mg/L indolebutyric acid, 150mg/L Timentin, 8g/L agar powder, pH=5.8.
The present invention is easy and simple to handle, can significantly improve the resistance of rape to lepidoptera pest, simultaneously on the normal growth of plant without impact, there is important breeding value and productive value.
The accompanying drawing explanation
SEQ ID NO:1 is the cry1C of report
*The nucleotide sequence of gene, sequence total length 1896bp.
SEQ ID NO:2 is the Cry1C of report
*Protein sequence, 630 amino acid of encoding.
SEQ ID NO:3 is fragment and the Southern hybridization probe of pcr amplification, and the sequence total length is 0.99kb.
SEQ ID NO:4 is the forward primer of the special primer of PCR detection and Southern hybridization probe amplification use.
SEQ ID NO:5 is the reverse primer of the special primer of PCR detection and Southern hybridization probe amplification use.
SEQ ID NO:6 is real-time quantitative RT-PCR amplification cry1C
*Forward primer.
SEQ ID NO:7 is real-time quantitative RT-PCR amplification cry1C
*Reverse primer.
SEQ ID NO:8 is the forward primer of real-time quantitative RT-PCR amplification reference gene BnActin.
SEQ ID NO:9 is the forward primer of real-time quantitative RT-PCR amplification reference gene BnActin.
Fig. 1: the cry1C that the present invention uses
*The binary expression vector schematic diagram.Cry1C
*Gene is to be controlled and expressed by corn ubiquitin (Ubiquitin) promotor, in figure: the selective marker that the Bar gene is genetic transformation.
Fig. 2: cry1C
*The PCR of transfer-gen plant detects.Swimming lane situation: swimming lane M, 1kb marker; P, the empty plasmid transformed plant; NT, non-transformed plant; 1-19, transfer-gen plant.Arrow points 0.99kb cry1C
*The specific amplified fragment of gene.
Fig. 3: in the present invention to cry1C
*The Southern hybridization check of transformed plant is analyzed.Utilize total DNA(20 μ g that restriction enzyme Dra I is right) carry out enzyme and cut, hybridization probe is the cry1C of 0.99kb
*Specific amplified fragment (isotropic substance
32The P mark).In figure: NT: non-transformed plant contrast; Swimming lane 1-12: transfer-gen plant.
Fig. 4: utilize real-time quantitative RT-PCR to detect cry1C
*The expression amount of gene in wild-type and transfer-gen plant.The height of cylindricality has represented cry1C
*The mRNA relative level (mean+SD) of gene.In figure: WT is wild-type; 1C-6,1C-8,1C-28,1C-31 and 1C-34 are transgenic line.
Cry1C in Fig. 5, transgenic line young leaflet tablet
*The analysis of protein content (mean+SD).In figure: 1C-6,1C-8,1C-28,1C-31 and 1C-34 are transgenic line.
Fig. 6: the insect-resistance of transfer-gen plant is identified.50-60 bar second instar larvae is fed and plant taken pictures in 5 days on plants.In figure, WT is the wild-type plant; Tr is transfer-gen plant.
Embodiment
Embodiment 1: the acquisition of rape transfer-gen plant
(1) with swede type rape spring varieties rape variety Westar(Westar kind nineteen eighty-two in Canada get the Green Light low erucic acid, the low sulfatide spring rape kind of plantation, be public business rape variety; Klassen etc., Can.J.Plant Sci.67:491-493(Apr.1987)) be transgene receptor.The Westar Semen Brassicae campestris is used to 75% Ethanol Treatment 1min, and 50%84 thimerosals are processed 8min, with after sterilized water punching cleaning seed 4-5 time, are seeded in Aseptic seedling culture base M
0On.Under 24 ℃, dark the cultivation 4 days, obtain the rape aseptic seedling.
(2) will transform cry1C
*Gene (gene order, see shown in SEQ ID NO:1) the agrobacterium strains GV3101(of binary expression vector (Fig. 1) is purchased from Invitrogen company) be inoculated in the LB liquid nutrient medium containing 50mg/L kantlex, 50mg/L gentamicin and 50mg/L Rifampin, on 28 ℃, the shaking table of 180-220r/min, shaken overnight is cultivated.Treat that Agrobacterium is cultured to OD
600, with the centrifugal 10min of 2500rpm rotating speed, outwell supernatant, with DM liquid nutrient medium Eddy diffusion thalline and shake up at=0.3 o'clock.
(3) will sow the seedling hypocotyl segment after 4 days, and be dipped in 30min in the work bacterium liquid prepared, the explant that will infect is transferred to common culture medium M
1Upper, be placed under 24 ℃ of dark conditions and cultivate altogether 48h.
(4) explant after cultivating is altogether transferred to callus inducing medium M
2Upper cultivation, condition is 24 ℃ and 16h illumination/8h dark.
(5), after cultivating 2 weeks, will transfer to shoot regeneration substratum M with the explant of obvious callus
3Upper, every 2-3 week is changed a fresh culture.
(6) when the green bud of regeneration grows to 2-3cm, after being cut, green bud transfers to root media M
4In take root.Seedling replanting after taking root, in native alms bowl, and is placed in to hot-house culture.
Medium component is as follows:
Aseptic seedling culture base M
0: 1/2MS, 7g/L agar powder (Agargel), pH=5.8;
Liquid nutrient medium DM:MS, 30g/L sucrose, 100 μ M Syringylethanones, pH=5.8;
Be total to culture medium M
1: MS, 30g/L sucrose, 18g/L N.F,USP MANNITOL, 1mg/ L 2,4 dichlorophenoxyacetic acid, 0.3mg/L kinetin, 100 μ m Syringylethanones, 2.5g/L plant gel (Phytagel), pH=5.8;
Callus inducing medium M
2: MS, 30g/L sucrose, 18g/L N.F,USP MANNITOL, 1mg/ L 2,4 dichlorophenoxyacetic acid, 0.3mg/L kinetin, 20mg/L Silver Nitrate, 270mg/L Timentin, 5mg/L grass ammonium phosphine, 2.5g/L plant gel, pH=5.8;
Shoot regeneration substratum M
3: MS, 10g/L glucose, 0.25g/L wood sugar, 0.6g/L 2-(N-morpholine) ethane sulfonic acid, 0.1mg/L indolylacetic acid, the trans zeatin of 2mg/L, 270mg/L Timentin, 5mg/L grass ammonium phosphine, 2.5g/L plant gel, pH=5.8;
Root media M
4: 1/2MS, 10g/L sucrose, 0.5mg/L indolebutyric acid, 150mg/L Timentin, 8g/L agar powder, pH=5.8;
By aforesaid method and step, the applicant obtains altogether 272 plant with careless ammonium phosphine resistance in 1258 explants, to the transgenosis T obtained
0Plant carries out PCR and Southern hybridization check.
Embodiment 2:PCR detects transgene rape
(1) adopt the CTAB method to extract total DNA(Hatmes etc., 1996 from the conversion rape leaf of anti-careless ammonium phosphine).
(2) detect cry1C
*The PCR primer of gene is cry1C-F(5 '-TCGACATCTTGAACAACCTT-3 ') and cry1C-R(5 '-CTACTTTTGTGCTCTTTCAA-3 ').
(3) 20 μ l PCR reaction systems comprise: 20ng DNA profiling, 1 * PCR damping fluid, 2mM MgCl
2, 2mM dNTPs, 0.4 μ Μ PCR primer and 1U Taq archaeal dna polymerase (purchased from Fermentas company), supplement ddH
2O to 20 μ l.
(4) PCR response procedures: 94 ℃ of denaturation 3min, 1 circulation; 94 ℃ of sex change 1min, 58 ℃ of renaturation 45s, 72 ℃ are extended 1min, totally 35 circulations; 72 ℃ are extended 10min, preserve 10min for 20 ℃.Add 10 μ l sample-loading buffers (98% deionized formamide, 10mM EDTA, the blue or green FF of 0.005% dimethylbenzene and 0.005% tetrabromophenol sulfonphthalein) in amplified production, the PCR product by 1% sepharose (containing 0.1% ethidium bromide) electrophoresis after, in the upper also observations of taking a picture of gel imaging system (Bio-Rad, Gel DocTM XR+).
Detected result as shown in Figure 2, detects and shows that, in 272 strain resistant plants, 42 strains are cry1C by PCR
*Positive transfer-gen plant, foreign DNA has been inserted in acceptor plant genome.
Embodiment 3: the Southern hybridization check of transgene rape
(1) the total DNA(of 20 μ g is comprised to cry1C
*The total DNA of transformed plant and the total DNA of non-transgenic adjoining tree) carry out enzyme with restriction enzyme DraI under 37 ℃ of conditions and cut, on 0.8% sepharose, electrophoresis spends the night.
(2), after electrophoresis, adopt the alkali transfer method that DNA is transferred on nylon membrane (purchased from Amersham Biosciences company).
(3) nylon membrane is transferred in 65 ℃ of Church damping fluids (0.25M NaPO4, pH7.2/0.7%SDS/1% bovine serum albumin (BSA)/1mM EDTA/50mg/L salmon sperm dna) and carried out the 6h prehybridization
(4) by the cry1C of 0.99kb
*Gene fragment (sequence is as shown in SEQ ID NO:3) is hybridized as probe.Probe adopt random primer labelling test kit (purchased from precious biotechnology Dalian company limited) carry out α-
32P dCTP mark.In the Church damping fluid, 65 ℃ of hybridization are spent the night, and wash after film to shield (FUJI FLA-9000) by phosphorus and scan and record result.
As shown in Figure 3, in the positive transformed plant of 42 strains, single copy transfer-gen plant has 6 strains to Southern hybrid experiment result, and other plant is 2-5 copy, and this result has further confirmed cry1C
*Gene has been incorporated in the rape genome.
Embodiment 4: anti insect gene cry1C in transgene rape
*The mensuration of transcriptional level
(1) get the blade of 100mg transfer-gen plant, with Trizol reagent (purchased from Invitrogen company, operation to specifications), extract total RNA.
(2) take the total RNA of 3 μ g is template, utilizes the cDNA of synthetic the first chain of M-MLV ThermoScript II (purchased from Fermentas company, operation to specifications).
(3) utilize SsoFast EvaGreen supermix test kit (Bio-Rad, according to the operation of test kit specification sheets) at real-time quantitative PCR detection system CFX96TM(Bio-Rad) upper to cry1C
*Transcriptional level is detected, and forward primer used: cry1C-193F(5 '-GGAATCGTTGGACCTTCTCA-3 ' increases) and reverse primer: cry1C-391R (5 '-CGATCACTCTGGTCCTGGTT-3 ').The forward primer of reference gene BnActin amplification: actin-F(5 '-ACAGTGTCTGGATCGGTGGTTC-3 ') and reverse primer: actin-R(5 '-TGCCTCATCATACTCAGCCTTG-3 ').The calculating of relative expression quantity adopts 2
-Δ Δ CTMethod.
Experimental result shows as Fig. 4: at T
3In transgenic line 1C-6,1C-8,1C-28,1C-31 and 1C-34, cry1C
*The relative level of gene mRNA exists significant difference.Wherein, the cry1C of strain 1C-8
*Expression amount is the highest, and the expression amount of strain 1C-34 is minimum, has the difference of 10 times of left and right between them.Other 3 strains show the transcriptional level of moderate.
Embodiment 5: Cry1C in transgene rape
*The detection of protein level
(1) Cry1C in the transgenic line blade
*Protein content utilizes the special ELISA test kit AP007(of Cry1C purchased from EnviroLogix company, according to the operation of test kit specification sheets) detected.
(2) absorbancy (OD value) is measured and is used MK3 type microplate reader (Thermo Labsystems Co., Chinese Shanghai), and measured value reads under 450nm wavelength condition.
(3) Cry1C
*The typical curve that protein mass (ng) utilizes standard model to make is calculated, the Cry1C of protein content to comprise in the fresh blade of every g
*Protein mass means.
Experimental result shows as Fig. 5, all contains Cry1C in transgenic line 1C-6,1C-8,1C-28,1C-31 and 1C-34
*Albumen, and protein level exists significant difference.The Cry1C of strain 1C-34
*Protein level minimum (20.52 ± 3.14ng/g), and the Cry1C of strain 1C-8
*Protein level the highest (799.32 ± 80.48ng/g).
Embodiment 6: detect transgene rape to the small cabbage moth resistance
The experiment of small cabbage moth resistance is carried out on transgenic line 1C-8.50-60 second instar larvae is placed in to transgenosis and the upper feeding of wild-type plant (25 ℃, 16h illumination/8h dark), the mortality ratio of 5 days " Invest, Then Investigate " small cabbage moths.
As shown in Figure 6, feeding is after 5 days for experimental result, although transgenic line 1C-8 shows slight causing harm, the mortality ratio of small cabbage moth still reaches 100%.On the wild-type plant, not only cause harm serious, and the small cabbage moth growth is normal, can be transitioned into for four length of times from three length of times, and complete the process of pupating.
Claims (3)
1. utilize transgenic technology to improve the method for rape to the lepidoptera pest resistance, it is characterized in that the following step:
(1) utilize cry1C
*The binary expression vector of gene, transform rape by agriculture bacillus mediated method, the rape aseptic seedling hypocotyl of take is explant, hypocotyl segment after infecting Agrobacterium and carrying out common cultivation is transferred on the callus inducing medium and shoot regeneration substratum that contains selective agent, the regrowth that will have resistance is transferred in root media, the seedling after taking root is transferred to greenhouse and cultivated;
(2) transformed plant is carried out to PCR and Southern hybridization check, determine the transgenic positive strain, PCR detects and the amplification of Southern hybridization probe, and the special primer of use is as follows:
Forward primer: cry1C-F 5 '-TCGACATCTTGAACAACCTT-3 ';
Reverse primer: cry1C-R 5 '-CTACTTTTGTGCTCTTTCAA-3 ';
(3) by real-time quantitative RT-PCR to cry1C in the transgenic positive strain
*The transcriptional level of gene is detected, cry1C
*Amplimer as follows:
Forward primer: cry1C-193F 5 '-GGAATCGTTGGACCTTCTCA-3 ';
Reverse primer: cry1C-391R 5 '-CGATCACTCTGGTCCTGGTT-3 ';
The amplimer of reference gene BnActin is as follows:
Forward primer: actin-F 5 '-ACAGTGTCTGGATCGGTGGTTC-3 ';
Reverse primer: actin-R 5 '-TGCCTCATCATACTCAGCCTTG-3 ';
(4) strain is detected to transgenic positive to utilize the ELISA method, determines Cry1C in plant
*Protein content;
(5) further transgenic line is carried out the insect-resistance evaluation of lepidoptera pest, the resistance level of clear and definite transgenic line, obtain the pest-resistant transgene rape strain;
Wherein:
Cry1C described in step (1)
*The nucleotide sequence of gene is as shown in SEQ ID NO:1;
Selective agent described in step (1), select corresponding selective agent according to the selective marker of the binary vector used, and selective agent wherein is careless ammonium phosphine;
Pcr amplified fragment described in step (2) and Southern hybridization probe sequence are as shown in SEQ ID NO:3;
Cry1C described in step (4)
*The aminoacid sequence of albumen is as shown in SEQ ID NO:2;
Medium component in above-mentioned steps is as follows:
Aseptic seedling culture base M
0: 1/2MS, 7g/L agar powder (Agargel), pH=5.8;
Liquid nutrient medium DM:MS, 30g/L sucrose, 100 μ M Syringylethanones, pH=5.8;
Be total to culture medium M
1: MS, 30g/L sucrose, 18g/L N.F,USP MANNITOL, 1mg/L 2,4 dichlorophenoxyacetic acid, 0.3mg/L kinetin, 100 μ m Syringylethanones, 2.5g/L plant gel (Phytagel), pH=5.8;
Callus inducing medium M
2: MS, 30g/L sucrose, 18g/L N.F,USP MANNITOL, 1mg/L 2,4 dichlorophenoxyacetic acid, 0.3mg/L kinetin, 20mg/L Silver Nitrate, 270mg/L Timentin, 5mg/L grass ammonium phosphine, 2.5g/L plant gel, pH=5.8;
Shoot regeneration substratum M
3: MS, 10g/L glucose, 0.25g/L wood sugar, 0.6g/L 2-(N-morpholine) ethane sulfonic acid, 0.1mg/L indolylacetic acid, the trans zeatin of 2mg/L, 270mg/L Timentin, 5mg/L grass ammonium phosphine, 2.5g/L plant gel, pH=5.8;
Root media M
4: 1/2MS, 10g/L sucrose, 0.5mg/L indolebutyric acid, 150mg/L Timentin, 8g/L agar powder, pH=5.8.
2. the transgenic technology of utilizing according to claim 1 improves the method for rape to the lepidoptera pest resistance, and it is characterized in that: the described conversion rape of step (1) comprises swede type rape, mustard type rape or turnip type rape.
3. the transgenic technology of utilizing according to claim 1 improves the method for rape to the lepidoptera pest resistance, it is characterized in that, the target fragment of the pcr amplification described in step (2) and Southern hybridization probe size are 0.99kb.
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| CN106258976A (en) * | 2016-08-24 | 2017-01-04 | 华中农业大学 | A kind of tissue culturing fast seedling-cultivating method of mustard type rape |
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| WO2017125040A1 (en) * | 2016-01-21 | 2017-07-27 | Sinobioway Bio-Agriculture Group Co. Ltd. | Plants having enhanced tolerance to insect pests and related constructs and methods involving insect tolerance genes |
| CN107475174A (en) * | 2017-08-16 | 2017-12-15 | 北京大北农生物技术有限公司 | The method for converting rape |
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| WO2017125040A1 (en) * | 2016-01-21 | 2017-07-27 | Sinobioway Bio-Agriculture Group Co. Ltd. | Plants having enhanced tolerance to insect pests and related constructs and methods involving insect tolerance genes |
| CN106258976A (en) * | 2016-08-24 | 2017-01-04 | 华中农业大学 | A kind of tissue culturing fast seedling-cultivating method of mustard type rape |
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| CN107475174A (en) * | 2017-08-16 | 2017-12-15 | 北京大北农生物技术有限公司 | The method for converting rape |
| CN107475174B (en) * | 2017-08-16 | 2020-08-28 | 北京大北农生物技术有限公司 | Method for transforming rape |
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