CN109371060B - Method for rapid transgenosis of euonymus persicifera - Google Patents
Method for rapid transgenosis of euonymus persicifera Download PDFInfo
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- CN109371060B CN109371060B CN201811430467.4A CN201811430467A CN109371060B CN 109371060 B CN109371060 B CN 109371060B CN 201811430467 A CN201811430467 A CN 201811430467A CN 109371060 B CN109371060 B CN 109371060B
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a rapid transgenosis method for euonymus persicae. The invention also discloses an identification method of the obtained transgenic plant. Compared with the traditional leaf disc method for transgenosis, the method obviously shortens the time of transgenosis, improves the transformation efficiency of the euonymus persicae transgenosis, reduces the workload of scientific researchers, provides a new way for efficient transformation and gene function analysis of the euonymus persicae, and also provides a new idea for transgenosis of other plants.
Description
Technical Field
The invention belongs to the technical field of plant improvement and new variety development, relates to a method for quickly transgenosis of euonymus persicaria, and particularly relates to a method for quickly transgenosis of euonymus persicae.
Background
The small deciduous tree of Euonymus Bungensis Maxim of Euonymus of Celastraceae of Euonymus of Russian is pleasurable, cold resistant, and more drought resistant, salt and alkali resistant, and water and moisture resistant, and has luxuriant branches and leaves, reddish color after early autumn frost, and beautiful capsules. The Euonymus alatus is high in resistance to wind and smoke, can improve ecological environment, purify air and absorb harmful gases such as sulfur dioxide and chlorine, is an important protection forest or landscaping ornamental tree species, and has important economic and ecological values. However, the capability of resisting diseases and insect pests is poor, so that the establishment of a Russian euonymus persicaria transgenic technology system has the advantage of high efficiency for the improvement of other euonymus plants, and the breeding process can be greatly shortened. The subject group establishes a rapid propagation technical system of the leaves of the Euonymus alatus by using a tissue culture technology, and at present, no report is found about a transgenic method of the Euonymus alatus, and no report is about the transgenic method in Euonymus.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for cultivating transgenic euonymus persicae by utilizing an agrobacterium-mediated method, provide technical support for improving insect resistance of the euonymus persicae, improving ornamental quality of the euonymus persicae and relevant basic research depending on transgenic technology, and have important reference significance for establishing transgenic methods for other plants of euonymus of Celastraceae.
The technical scheme is as follows: in order to solve the technical problems, the invention discloses a rapid transgenic method of euonymus persicae. The invention provides a high-efficiency and rapid transformation method, and solves the problems of long time consumption, low transformation efficiency and the like of a leaf disc transformation method.
Specifically, the method for rapid transgenosis of the euonymus persicaria comprises the following steps:
1) activating engineering bacteria: activating an agrobacterium tumefaciens C58 strain to obtain activated agrobacterium tumefaciens liquid;
2) genetic transformation: firstly, infecting euonymus persica callus with agrobacterium liquid, then carrying out co-culture in a co-culture medium, after the co-culture is finished, transferring the callus to a screening culture medium to screen a green resistant explant, transferring the green resistant explant to a subculture multiplication culture medium after 2 weeks to culture to obtain a large number of cluster buds, transferring the cluster buds to a bud elongation screening culture medium to carry out bud elongation induction culture, transferring the well-grown green plumule to a bud elongation screening culture medium to continue screening to obtain a green and vigorous resistant adventitious bud, cutting the robust adventitious bud with a certain length, transferring the robust adventitious bud to a rooting culture medium to obtain a rooted plant, and transplanting the rooted plant to a greenhouse for conventional management to obtain a transgenic plant after the plant is normally developed.
Wherein, the engineering bacteria activation of the step 1) comprises the following specific steps: stock solution of Agrobacterium C58 strain stored at-20 deg.C, taken out of refrigerator and thawed on ice. The YEB solid culture medium is placed on a super clean workbench, and the bacterial liquid is dipped by an inoculating loop and is scribed on a plate. Carrying out dark culture at 28 ℃ for 2-3 d to obtain a monoclonal antibody. Picking single clone with toothpick, inoculating into 250ml YEB liquid culture medium containing clarithromycin, placing in shaker at 28 deg.C and 200rpm, and shake culturing to OD600＝1～2。
Wherein, the step of infecting euonymus persicaria callus with agrobacterium liquid in the step 2) to obtain the explant comprises the following steps: cutting to induce 12-16 days loose pale yellow Euonymus persica callus with size of 1-1.5 cm, timely inoculating into a 9cm culture dish containing 10ml MS liquid culture medium, and adding OD600And (3) mixing 50 mu l of 0.4-0.6 of agrobacterium liquid, after 20-25 min of infection, continuously and gently oscillating in the infection process to ensure that the callus is fully contacted with the agrobacterium liquid, and filtering the liquid to obtain the agrobacterium liquid.
Wherein the co-culture medium is MS +0.5mg/L6-BA +0.5mg/L NAA +6.5g/L agar +30g/L sucrose, and the pH is 5.8.
Wherein the screening culture medium is MS +30g/L sucrose +6.5g/L agar +0.5mg/L6-BA +0.5mg/L LNAA + 250-300 mg/L timentin, and the pH is 5.8.
Wherein, the subculture multiplication culture medium: MS, 30g/L of sucrose, 6.5g/L of agar, 0.5mg/L of 6-BA, 0.3mg/L of LNAA, 200-250 mg/L of timentin, pH5.8, 121 ℃ and 20min of autoclaving.
Wherein the bud elongation screening culture medium is WPM +30g/L sucrose +6.5g/L agar +0.5mg/L6-BA +0.3mg/L LNAA +0.1mg/LGA3+150 mg/L-200 mg/L timentin, and the pH is 5.8.
Wherein the rooting medium is 1/2WPM, 20g/L sucrose, 6.0g/L agar, 2mg/LIBA, 0.05mg/LNAA + +0.2mg/LGA3 and 50-100 mg/L timentin, and the pH value is 5.8.
The euonymus persicae is a russian euonymus persicae.
The invention also comprises the identification method of the obtained transgenic plant, PCR amplification verification is carried out on 2 insect-resistant genes cry1Ac and SCK of the obtained transgenic plant, and amplified fragments of 1146bp and 384bp can be simultaneously obtained, namely the successful obtaining of the transgenic plant is indicated.
Wherein, the PCR amplification of the insect-resistant genes cry1Ac and SCK respectively corresponds to the following primer sequences:
Cry1Ac-F:5’-GCATTCCATACAACTGCTTG-3’
Cry1Ac-R:5’-TTCCATAGGCGAACTCTGTT-3’
SCK-F:5’-GCACCATCTTCTTTGCTCTC-3’
SCK-R:5’-CATCTTCATCCCTGGACTTG-3’。
has the advantages that: compared with the traditional leaf disc method for transgenosis, the method obviously shortens the time of transgenosis and improves the transformation efficiency of the euonymus persicae transgenosis. The infection ability of agrobacterium has a direct relation with transformation rate, and is also a main factor of transformation failure. The impregnation time of the agrobacterium has a great influence on the transformation efficiency, the strict control of the impregnation time is particularly important, and the long impregnation time of the agrobacterium by the explant can cause allergic reaction to result in browning, thereby seriously influencing the gene transformation efficiency; the time of infection is too short, and there is a possibility that the foreign DNA has not yet integrated into the chromosomal DNA of the explant. Therefore, the method controls the time for infecting the callus at 20-25 minutes, selects the callus for inducing for 12-16 days, induces and cultures the callus on a certain culture medium to ensure that the transformation efficiency reaches more than 40 percent, which is far more than that of the traditional leaf disc transformation method, reduces the workload of researchers, provides a new way for the efficient transformation and gene function analysis of euonymus persica, and also provides a new idea for the transgenosis of other plants.
Drawings
FIG. 1 shows the callus of Euonymus alatus;
FIG. 2 adventitious buds of the differentiated transgenic Euonymus Alata Sieb;
FIG. 3 transgenic Euonymus persica plants;
FIG. 4 is the PCR amplification electrophoresis verification picture of the insect-resistant gene cry1Ac of the transgenic Euonymus Alata Sieb;
FIG. 5 is a PCR amplification electrophoresis verification diagram of insect-resistant gene SCK of transgenic Euonymus Alata Sieb;
FIG. 6 is a map of transformation plasmid PcRPBSCK35 sBT;
FIG. 7 is PCR amplification electrophoresis verification picture of cry1Ac gene and SCK gene of transgenic Euonymus Alata Sieb; lane 1 is DNA marker 2000bp, lanes 2-23, lane 25 are samples of transgenic Euonymus Alata Sieb 1-23, lane 24 is DNA of untransformed regenerated plant as negative control, the upper part of the figure is PCR electrophoresis result (normal product size 1146bp) using Cry1Ac-F/R primer, and the lower part of the figure is PCR electrophoresis result (product size 384bp) using SCK-F/R primer.
Detailed Description
To further illustrate the details of the present invention, several examples are set forth below, but the present invention should not be limited thereto.
The formula of the culture medium adopted by the invention is as follows:
co-culture medium: MS +0.5mg/L6-BA +0.5mg/L NAA +6.5g/L agar +30g/L sucrose pH5.8, 121 ℃, 20min autoclaving.
Screening a culture medium: MS, 30g/L of sucrose, 6.5g/L of agar, 0.5mg/L of 6-BA, 0.5mg/L of LNAA, 250-300 mg/L of timentin, pH5.8, 121 ℃ and 20min of autoclaving.
Subculture multiplication medium: MS, 30g/L of sucrose, 6.5g/L of agar, 0.5mg/L of 6-BA, 0.3mg/L of LNAA, 200-250 mg/L of timentin, pH5.8, 121 ℃ and 20min of autoclaving.
Shoot elongation medium:
WPM +30g/L sucrose +6.5g/L agar +0.5mg/L6-BA +0.3mg/LNAA +0.1mg/LGA3+150 mg/L-200 mg/L timentin PH5.8, 121 ℃, 20min autoclave sterilization.
Rooting culture medium:
1/2WPM +20g/L sucrose +6.0g/L agar +2mg/LIBA +0.05mg/LNAA +0.2mg/LGA3+50～100mg/L timentin pH5.8, 121 ℃, 20min autoclave.
YEB liquid medium: 5g/L of sucrose, 5g/L of beef extract, 1g/L of yeast powder, 4g/L of peptone, 7H2O4g/L of MgSO4 & 7H 4, pH7.0, 121 ℃, and autoclaving for 20 min. When YEB solid medium was prepared, 15g/L agar was added at first and the pH was 7.4. YEB kanamycin was added for selection, both in solid and liquid media. Sterilizing the culture medium under high pressure, taking out, placing on a clean bench, and pressing V without scalding handsCaramycin：VCulture mediumKanamycin was added at 1: 1000.
The ingredients of MS and WPM culture medium are shown in Table 1
TABLE 1 media composition (mg/L)
Example 1 preparation of transgenic Euonymus persica plants
1. Activating engineering bacteria: 2. mu.L of an engineered bacterium (Agrobacterium C58 strain, plasmid PcRPBSCK35SBt carrying bivalent insect-resistant gene, provided by the institute of genetics and development, Chinesota institute of sciences) stored at-80 ℃ was inoculated into 10ml YEB liquid medium containing antibiotic (kanamycin 200mg/L) in the T-DNA region of the plasmid vector containing 2 insect-resistant genes cry1Ac and SCK, and 1 plant selection marker NPT II gene, and cultured at 28 ℃ and 200rpm until the OD of the bacterial liquid is reached600＝1.0～2.0。
2. Genetic transformation:
2.1 Agrobacterium infection callus explants:
induction of callus:
collecting tender leaves of strong and healthy Russian Euonymus Alata Sieb branches, washing with washing powder to remove surface dirt, washing with running water for 30 min, washing with distilled water, soaking in 75% alcohol for 30s, and soaking in 0.1% HgCl2Soaking explant, sterilizing for 2-7min, washing with sterile water for 3 times, cutting the sterilized leaf into 0.5 × 0.5cm on a clean bench2Square leaf ofInoculating the leaf with the leaf right side up into a culture medium (the same as a screening culture medium) for inducing callus, and culturing in a culture bottle in a culture chamber with the culture conditions of illumination intensity of 2000lx, temperature of 23-28 ℃ and illumination time of 14h/d for 12-16 days to obtain light yellow callus.
The specific steps of strain propagation and infection are as follows:
stock solution of Agrobacterium C58 strain stored at-20 deg.C, taken out of refrigerator and thawed on ice. The YEB solid culture medium is placed on a super clean workbench, and the bacterial liquid is dipped by an inoculating loop and is scribed on a plate. Carrying out dark culture at 28 ℃ for 2-3 d to obtain a monoclonal antibody. Picking out single clone with toothpick, inoculating in 250ml YEB liquid culture medium, placing in 28 deg.C, shaking table at 200rpm, and shake culturing to OD6001-2. Will OD600Diluting the bacterial solution 1-2, centrifuging 10ml of the diluted bacterial solution in a centrifugal tube for 10min at 4500g, collecting thalli, removing supernate, resuspending the precipitate in10 ml of MS liquid culture medium to obtain an infection culture solution, and enabling OD to be OD600The range of 0.4 to 0.6.
Cutting and inducing 12-16 days of loose light yellow callus, wherein the size of the callus is 1-1.5 cm, inoculating the callus into infection culture solution, mixing uniformly, and after 20-25 min of infection, continuously and gently oscillating in the infection process to make the callus fully contact with the bacterial solution. And (4) draining the bacterial liquid, and respectively transferring explants infected by the agrobacterium into co-culture media.
2.2 Co-cultivation
A layer of sterile filter paper + MS +0.5mg/L6-BA +0.5mg/L NAA +6.5g/L agar +30g/L sucrose, pH5.8 was used as co-medium and the Agrobacterium-infected explants were cultured for 3 days at 25 ℃ in the dark.
2.3 screening culture:
after the co-culture is finished, selecting a light yellow callus which normally expands for 15 days to transfer to a screening culture medium (300 mg/L) and culturing for 2 weeks under the conditions of 22-26 ℃, illumination for 10-14 hours/day, 13-15 ℃ and dark culture for 8-10 hours/day, and then carrying out subculture proliferation;
2.4 subculture proliferation:
after 2 weeks, the resistant explants are green and can continue to grow and expand, adventitious buds are formed at the edges of the cut, the green resistant explants are transferred into a subculture multiplication medium (250 mg/L) to be cultured for 2 weeks under the conditions of 22-26 ℃, 10-14 hours/day of illumination, 13-15 ℃ and 8-10 hours/day of dark culture, and screening is continued for 2 weeks.
2.5 shoot elongation screening:
screening and culturing for 2 weeks to grow a large number of cluster buds on green nodular callus, transferring the adventitious buds to a bud elongation culture medium containing timentin of 150mg/L, and performing bud elongation induction for 20-30 days under the conditions of 22-26 ℃, illumination of 10-14 hours/day, 13-15 ℃ and dark culture of 8-10 hours/day. Subcultured every 2 weeks for about 2 weeks. At this time, the residual explants and the nodular calluses at the adventitious bud base part are gradually cut off, so that the buds are fully contacted with the culture medium. Through bud elongation screening, the resistant bud is green and grows vigorously, and the adventitious bud of the chimera gradually becomes light green or yellow and finally dies.
2.6 rooting culture:
cutting well-growing complete adventitious buds, transferring the well-growing complete adventitious buds into a rooting culture medium containing 50mg/L timentin, and obtaining a rooting plant after about 15 days. The culture environment conditions are as follows: 2000Lux 12h/d, 23-27 ℃.
2.7 transplanting of regeneration plants:
when the plants grow normally, carefully taking out the regenerated seedlings, cleaning the root agar, and transplanting the regenerated seedlings to turf: in a vermiculite 3:1(V/V) matrix, a film is attached to keep moisture, and the vermiculite is transplanted to a greenhouse for conventional management after survival.
2.8 identification of regenerated plants:
PCR identification of exogenous genes: extracting genome DNA by a CTAB method, and carrying out PCR identification on the screened insect-resistant plants, wherein the sizes of PCR amplified fragments of the 2 insect-resistant genes cry1Ac and SCK amplified by the invention are 1146bp and 384bp respectively; the untransformed regenerated plant DNA was used as a negative control, and the transformation plasmid PcRPBSCK35sBT was used as a positive control. The actual length of cry1Ac gene is 1848bp, the actual length of SCK gene is 411bp, the inventor considers that designing primers from the beginning and the end of gene fragment can cause the quality of the primers not to be good and bands are not easy to be amplified during PCR amplification, so the primer design is not designed from the beginning and the end, as long as the target fragment is turned in, the size of the product after designing the primers according to the proper position of the target fragment theoretically is consistent with the size amplified by PCR in the actual experiment.
cry1Ac gene:
SCK gene:
therefore, the primer design is designed by taking 1146bp and 384bp fragments selected from the middle segments of genes cry1Ac and SCK as templates, the middle sequence of the gray part is the target amplification sequence selected by the user, and the designed primers are as follows:
cry1 Ac-F5'-GCATTCCATACAACTGCTTG-3' 56 degree 1146bp
Cry1Ac-R:5’-TTCCATAGGCGAACTCTGTT-3’
5'-GCACCATCTTCTTTGCTCTC-3' 57 degree 384bp SCK-F
SCK-R:5’-CATCTTCATCCCTGGACTTG-3’
PCR amplification System:
10 μ l system
Extracting total DNA of a regenerated seedling of the transgenic Euonymus alatus by adopting a CTAB method, taking the total DNA as a template, and selecting the PCR amplification system to perform PCR amplification on Cry1Ac gene in a transgenic plant under the guidance of a primer pair Cry1Ac (Cry1Ac-F, Cry1Ac-R), wherein the PCR reaction conditions are as follows: firstly, the temperature is 95 ℃ for 5min, then the temperature is 94 ℃ for 30s, the temperature is 56 ℃ for 30s, and the temperature is 72 ℃ for 1min and 10s, and 30 cycles are carried out; finally 7min at 72 ℃. After the reaction is finished, carrying out 0.8% agarose gel electrophoresis detection on the PCR product, wherein the detection result is shown in figure 4, and the size of the PCR amplified fragment of cry1Ac gene is 1146 bp;
extracting total DNA of the regenerated seedlings of the transgenic Euonymus alatus by adopting a CTAB method, selecting the PCR amplification system by taking the total DNA as a template, and carrying out PCR amplification on SCK genes in transgenic plants under the guidance of a primer pair SCK (SCK-F, SCK-R), wherein the PCR reaction conditions are as follows: firstly, the temperature is 95 ℃ for 5min, then the temperature is 94 ℃ for 30s, the temperature is 57 ℃ for 30s, and the temperature is 72 ℃ for 30s, and 30 cycles are carried out; finally 7min at 72 ℃. After the reaction is finished, carrying out 0.8% agarose gel electrophoresis detection on the PCR product, wherein the detection result is shown in figure 5, and the PCR amplification fragment of the SCK gene is 384 bp;
as can be seen from FIGS. 4 and 5, the Cry1Ac gene and SCK gene can be successfully amplified by the transgenic plant of the present example, which indicates that the transgenic method of the present invention successfully obtains the transgenic Euonymus Alata Sieb.
In order to further obtain the transformation rate of the transgenic plant of the invention, 23 transgenic euonymus ruscus and 1 untransformed euonymus ruscus are respectively extracted, PCR amplification is carried out on the Cry1Ac gene and the SCK gene according to the PCR amplification system and the PCR amplification conditions, the repeated results of 3 times of parallel experiments are the same, the electrophoresis results of the PCR products are shown in figure 7, lane 1 is DNA marker 2000bp, lanes 2-23 and lane 25 are transgenic euonymus ruscus 1-23 samples, lane 24 is untransformed regenerated plant DNA as a negative control, and in the figure, lanes 2, 3, 7-10, 12, 16, 20 and 23 can be amplified to obtain Cry1Ac gene fragments (product size 1146bp) and SCK gene fragments (product size 384bp) lanes 4, 5, 6, 11, 13, 14, 15, 17, 18, 19, 21, 22 and 25, and Cry1Ac gene fragments and SCK gene fragments can not be simultaneously amplified to obtain Cry1 gene fragments and SCK gene fragments, therefore, the transformation rate of the transgenic plant of the present invention was found to be 43.5% by calculation.
Sequence listing
<110> Jinling science and technology institute
<120> method for rapid transgenosis of euonymus persicaria
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1848
<212> DNA
<213> cry1Ac Gene (cryac)
<400> 1
atggacaaca acccaaacat caacgaatgc attccataca actgcttgag taacccagaa 60
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tccttgacac agtttctgct cagcgagttc gtgccaggtg ctgggttcgt tctcggacta 180
gttgacatca tctggggtat ctttggtcca tctcaatggg atgcattcct ggtgcaaatt 240
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gaaggattga gcaatctcta ccaaatctat gcagagagct tcagagagtg ggaagccgat 360
cctactaacc cagctctccg cgaggaaatg cgtattcaat tcaacgacat gaacagcgcc 420
ttgaccacag ctatcccatt gttcgcagtc cagaactacc aagttcctct cttgtccgtg 480
tacgttcaag cagctaatct tcacctcagc gtgcttcgag acgttagcgt gtttgggcaa 540
aggtggggat tcgatgctgc aaccatcaat agccgttaca acgaccttac taggctgatt 600
ggaaactaca ccgaccacgc tgttcgttgg tacaacactg gcttggagcg tgtctggggt 660
cctgattcta gagattggat tagatacaac cagttcagga gagaattgac cctcacagtt 720
ttggacattg tgtctctctt cccgaactat gactccagaa cctaccctat ccgtacagtg 780
tcccaactta ccagagaaat ctatactaac ccagttcttg agaacttcga cggtagcttc 840
cgtggttctg cccaaggtat cgaaggctcc atcaggagcc cacacttgat ggacatcttg 900
aacagcataa ctatctacac cgatgctcac agaggagagt attactggtc tggacaccag 960
atcatggcct ctccagttgg attcagcggg cccgagttta cctttcctct ctatggaact 1020
atgggaaacg ccgctccaca acaacgtatc gttgctcaac taggtcaggg tgtctacaga 1080
accttgtctt ccaccttgta cagaagaccc ttcaatatcg gtatcaacaa ccagcaactt 1140
tccgttcttg acggaacaga gttcgcctat ggaacctctt ctaacttgcc atccgctgtt 1200
tacagaaaga gcggaaccgt tgattccttg gacgaaatcc caccacagaa caacaatgtg 1260
ccacccaggc aaggattctc ccacaggttg agccacgtgt ccatgttccg ttccggattc 1320
agcaacagtt ccgtgagcat catcagagct cctatgttct cttggataca ccgtagtgct 1380
gagttcaaca acatcatcgc atccgatagt attactcaaa tccctgcagt gaagggaaac 1440
tttctcttca acggttctgt catttcagga ccaggattca ctggtggaga cctcgttaga 1500
ctcaacagca gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1560
ccatccacat ctaccagata tagagttcgt gtgaggtatg cttctgtgac ccctattcac 1620
ctcaacgtta attggggtaa ttcatccatc ttctccaata cagttccagc tacagctacc 1680
tccttggata atctccaatc cagcgatttc ggttactttg aaagtgccaa tgcttttaca 1740
tcttcactcg gtaacatcgt gggtgttaga aactttagtg ggactgcagg agtgattatc 1800
gacagattcg agttcattcc agttactgca acactcgagg ctgaatga 1848
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gttactactg cagccatgga tctgaaccac ctcggaagta atcatcatga tgactcaagc 180
gatgaacctt ctgagtcttc agaaccatgc tgcgattcat gcatctgcac taaatcaata 240
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<400> 5
gcaccatctt ctttgctctc 20
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<213> downstream primer (SCK-R)
<400> 6
catcttcatc cctggacttg 20
Claims (3)
1. A rapid transgenic method of Euonymus persicae is characterized in that in the genetic transformation step, a transgenic plant is obtained by infecting callus of Euonymus persicae with Agrobacterium, and the method specifically comprises the following steps:
1) activating engineering bacteria: activating an agrobacterium tumefaciens C58 strain to obtain activated agrobacterium tumefaciens liquid;
2) genetic transformation: firstly, infecting euonymus persicinus callus with agrobacterium liquid, then carrying out co-culture in a co-culture medium, after the co-culture is finished, transferring the callus to a screening culture medium to screen a green resistant explant, transferring the green resistant explant to a subculture multiplication culture medium after 2 weeks to culture to obtain a large number of cluster buds, transferring the cluster buds to a bud elongation screening culture medium to carry out bud elongation induction culture, transferring the well-grown green plumule to a bud elongation screening culture medium to continue screening to obtain a green and vigorous resistant adventitious bud, cutting the robust adventitious bud with a certain length, transferring the robust adventitious bud to a rooting culture medium to obtain a rooted plant, transplanting the rooted plant to a greenhouse for conventional management to obtain a transgenic plant after the plant is normally developed, and infecting the euonymus persicinus callus with agrobacterium liquid to obtain the explant: cutting to induce 12-16 days loose pale yellow Euonymus persica callus with size of 1-1.5 cm, inoculating into a 9cm culture dish containing 10ml MS liquid culture medium, adding OD6000.4-0.6 mu L of agrobacterium liquid, uniformly mixing, after 20-25 min of infection, continuously and gently oscillating in the infection process to make callus fully contact with the agrobacterium liquid, and draining the liquid to obtain the agrobacterium liquid, wherein the co-culture medium is MS +0.5mg/L6-BA +0.5mg/L NAA +6.5g/L agar +30g/L sucrose, the pH =5.8, the screening culture medium is MS +30g/L sucrose +6.5g/L agar +0.5mg/L6-BA +0.5mg/L NAA + 250-300 mg/L timentin, the pH =5.8, the subculture multiplication culture medium is MS +30g/L sucrose +6.5g/L agar +0.5mg/L6-BA +0.3mg/L A + 200-250 mg/L timentin, the pH =5.8, and the bud elongation screening culture medium is WPM +30g/L sucrose +6.5g/L agar +0.5mg/L timentin BA +0.3mg/L NAA +0.1mg/L GA3+150 mg/L-200 mg/L timentin and PH =5.8, wherein the rooting medium is 1/2WPM +20g/L sucrose +6.0g/L agar +2mg/L IBA +0.05mg/L NAA + +0.2mg/L GA3+ 50-100 mg/L timentin and PH = 5.8.
2. The method for rapid transgenosis of euonymus persicae as claimed in claim 1, wherein PCR amplification verification is performed on 2 insect-resistant genes cry1Ac and SCK of the obtained transgenic plant, and 1146bp and 384bp of amplified fragments can be obtained simultaneously, which indicates that the transgenic plant is successfully obtained.
3. The method for rapidly transgenosis by euonymus persicae as claimed in claim 2, wherein the sequences of primers respectively corresponding to the PCR amplification of the insect-resistant genes cry1Ac and SCK are as follows:
cry1Ac-F:5’-GCATTCCATACAACTGCTTG-3’，
cry1Ac-R:5’-TTCCATAGGCGAACTCTGTT-3’，
SCK-F:5’-GCACCATCTTCTTTGCTCTC-3’，
SCK-R:5’-CATCTTCATCCCTGGACTTG-3’。
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