CN113684224A - Method for efficient genetic transformation of arabidopsis thaliana - Google Patents
Method for efficient genetic transformation of arabidopsis thaliana Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 12
- 230000002068 genetic effect Effects 0.000 title claims abstract description 7
- 241000219195 Arabidopsis thaliana Species 0.000 title claims description 10
- 241000219194 Arabidopsis Species 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 241000589158 Agrobacterium Species 0.000 claims abstract description 17
- 241000196324 Embryophyta Species 0.000 claims abstract description 14
- 241001052560 Thallis Species 0.000 claims abstract description 8
- 239000013612 plasmid Substances 0.000 claims abstract description 7
- 208000015181 infectious disease Diseases 0.000 claims abstract description 5
- 238000002791 soaking Methods 0.000 claims abstract description 5
- 230000009261 transgenic effect Effects 0.000 claims abstract description 5
- 230000001131 transforming effect Effects 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 230000001404 mediated effect Effects 0.000 claims description 5
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims description 4
- 235000017491 Bambusa tulda Nutrition 0.000 claims description 4
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- 235000015334 Phyllostachys viridis Nutrition 0.000 claims description 4
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- 239000011159 matrix material Substances 0.000 claims description 3
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- 238000012360 testing method Methods 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 3
- 238000012258 culturing Methods 0.000 abstract 1
- 238000003306 harvesting Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
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- 229920002684 Sepharose Polymers 0.000 description 1
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- 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)
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- 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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Abstract
The invention provides a method for high-efficiency genetic transformation of arabidopsis, which comprises the steps of constructing plasmids, transforming agrobacterium, collecting thalli, soaking arabidopsis inflorescences for infection, placing arabidopsis plants in a dark place for 16-24 hours, normally watering and culturing, harvesting seeds, directly screening positive seeds by screening markers, and obtaining positive transgenic plants after termination. The invention does not need to detect the single clone of the agrobacterium, thereby avoiding the step of agrobacterium PCR; the liquid culture of the agrobacterium liquid is not needed, and the liquid culture process and the time for centrifugally collecting the liquid are avoided. The invention improves the step of arabidopsis transformation, saves the time of experimental operation, improves the working efficiency of arabidopsis transformation operation, and enables the arabidopsis transformation operation to be carried out in large scale and batch.
Description
Technical Field
The invention relates to a method for high-efficiency genetic transformation of arabidopsis thaliana, belonging to the field of biology.
Background
Arabidopsis thaliana has become a widely used model plant in the field of plant research due to its small plant size, short life cycle, large number of seeds per plant, complete analysis of genome, and the like. In the research of arabidopsis thaliana, researchers and biotechnology service companies need to construct a large number of arabidopsis transgenic lines. The current common arabidopsis transformation method is an agrobacterium-mediated inflorescence infection method. The method comprises the following specific steps: (1) completing vector construction and verification in escherichia coli to obtain constructed plasmids; (2) transforming agrobacterium with plasmid, coating plate, selecting monoclone for verification and selecting positive monoclone; (3) selecting positive monoclonals to carry out small-amount liquid culture in 2mL of resistant LB culture medium; (4) 1:100 is inoculated into 500mL of resistant LB culture medium for mass culture to logarithmic growth phase (OD value is between 0.6 and 0.8); (5) e.coli thalli are obtained by centrifugation, 100mL of transformation liquid (1/2 MS, 5% sucrose) is used for resuspension; (6) before transformation, 0.02 percent of Silwet-77 is added into the bacterial liquid and the bacterial liquid is transferred into a beaker; (7) and (3) putting the arabidopsis inflorescence into the bacterial liquid and fully soaking for 3-5 minutes. (8) Wrapping the soaked plants with a preservative film, and placing the wrapped plants in a dark place for 16 to 24 hours to keep high humidity; (9) and (4) watering normally, fixing the plants by using bamboo sticks, and stopping watering when the seeds are mature. (10) oven-drying the harvested seeds at 37 ℃ for 3-5 days. (11) Positive shoots were screened by appropriate screening markers.
The conversion method has the following defects: (1) after agrobacterium transformation, monoclonals need to be selected, which is time-consuming; (2) the agrobacterium tumefaciens has a relatively thick cell wall and a low plasmid copy number, so that PCR of common bacterial liquid is difficult. (3) After the selection of the single clone, two steps of small amount of shake bacteria and large amount of shake bacteria are needed, and 3-4 days are consumed. (4) Finally, when a large amount of bacteria are shaken, the OD value condition of the bacteria liquid needs to be grasped to ensure that the transformed bacteria are in the logarithmic phase, which is tedious and time-consuming; (5) the collection of the thalli needs centrifugation, is time-consuming and labor-consuming, and is not suitable for the operation of one-time transformation and more construction.
Disclosure of Invention
In order to solve the problems, the invention provides a more efficient agrobacterium-mediated arabidopsis genetic transformation method.
In order to achieve the purpose, the invention adopts the following technical scheme:
(1) the Km-resistant empty vector pDsRed0 was used as a test, and the sequence of the Km-resistant empty vector pDsRed0 is shown in SEQ ID NO. 1;
(2) transforming 100 mu L of agrobacterium-mediated state by adopting 1-2 mu g of pDsRed0 plasmid, and coating all transformed bacterial liquid into a culture dish with the diameter of 15cm, wherein the culture dish contains LB culture medium with 50mg/L Km resistance and 25mg/L Rif resistance;
(3) placing the culture dish at 28 ℃ for 3-4 days, cloning to 2-3mm, and directly collecting thalli by using a coating rod;
(4) resuspending the collected thallus with 100mL of transformation liquid, which is 1/2 MS culture medium containing 5% sucrose, to obtain a suspension;
(5) adding Silwet-77 with the final concentration of 0.02% into the suspension before transformation and transferring the bacterial liquid into a beaker;
(6) planting arabidopsis thaliana in a soil matrix in an artificial climate chamber until full-bloom stage, and cutting off grown fruit pods before transformation;
(7) placing the inflorescence of the arabidopsis into the suspension liquid and fully soaking for 3-5 minutes;
(8) wrapping the soaked arabidopsis plants with a preservative film, and placing the arabidopsis plants in a dark place for 16-24 hours to keep high humidity;
(9) watering normally, fixing the plants by bamboo sticks, and stopping watering when the seeds are mature;
(10) drying the harvested seeds in an oven at 37 ℃ for 3-5 days;
(11) and (4) selecting positive seeds through Ds-Red fluorescence, and planting the positive seeds to obtain transgenic positive plants.
The invention has the advantages that:
(1) the detection of the single clone of the agrobacterium is not needed, the step of agrobacterium PCR is avoided, and the time of single clone detection is saved;
(2) the clone population transformed by the agrobacterium is directly transformed, the steps of shaking a small amount of bacteria and expanding propagation are not needed, and the time is saved by at least 3-4 days.
(3) The agrobacterium colony growing on the plate solid culture medium can be used within 1 week and can be stored for a short time at 4 ℃, and the step of controlling OD value during liquid culture of thalli is avoided.
(4) The preparation of a large-volume liquid culture medium is not needed, and the thalli are obtained without centrifugation. The experimental steps are simplified, and the possibility is provided for a biological company to carry out large-scale transformation operation.
Drawings
FIG. 1 is a plot of the growth of Agrobacterium plaques of the present invention directly using these monoclonal populations for Agrobacterium-mediated transformation of Arabidopsis.
FIG. 2 is a diagram showing the structure of the vector pDsRed 0.
FIG. 3 comparison of positive seeds and non-fluorescent seeds. The positive seeds have obvious visible red fluorescence after blue light irradiation and red filter light filtration; non-fluorescent seeds have no visible red fluorescence.
Detailed Description
Culture medium formula
LB medium (1L): 10 g NaCl, 10 g yeast extract, 5 g trypsin powder.
5% sucrose 1/2 MS medium (1L): 2.25 g MS powder, 10 g sucrose, 10 g sepharose (pH = 5.8).
Example 1:
(1) the Km-resistant empty vector pDsRed0 was used as a test, and the sequence of the Km-resistant empty vector pDsRed0 is shown in SEQ ID NO. 1;
(2) mu.L of Agrobacterium infection was transformed with 1-2. mu.g of pDsRed0 plasmid (using the electrotransformation method, see Weigel, D., and Glazebrook, J. (2006).CSH protocols2006) All the transformed bacterial liquid is spread in a culture dish with the diameter of 15cm, and the culture dish contains LB culture medium with 50mg/L Km resistance and 25mg/L Rif resistance;
(3) placing the culture dish at 28 ℃ for 3-4 days, cloning to 2-3mm, and directly collecting thalli by using a coating rod;
(4) resuspending the collected thallus with 100mL of transformation liquid, which is 1/2 MS culture medium containing 5% sucrose, to obtain a suspension;
(5) adding Silwet-77 with the final concentration of 0.02% into the suspension before transformation and transferring the bacterial liquid into a beaker;
(6) planting arabidopsis thaliana in a soil matrix in an artificial climate chamber until full-bloom stage, and cutting off grown fruit pods before transformation;
(7) placing the inflorescence of the arabidopsis into the suspension liquid, and fully soaking for 3-5 minutes for infection;
(8) wrapping the soaked arabidopsis plants with a preservative film, and placing the arabidopsis plants in a dark place for 16-24 hours to keep high humidity;
(9) watering normally, fixing the plants by bamboo sticks, and stopping watering when the seeds are mature;
(10) drying the harvested seeds in an oven at 37 ℃ for 3-5 days;
(11) and (4) selecting positive seeds through Ds-Red fluorescence, and planting the positive seeds to obtain transgenic positive plants. .
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> method for high-efficiency genetic transformation of arabidopsis thaliana
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 10005
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<213> Artificial sequence
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caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg agctgcccat 5220
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aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc 5880
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gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat 6240
ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg 6300
ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg 6360
tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca 6420
agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg 6480
taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc 6540
gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga 6600
tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag 6660
aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga 6720
agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga 6780
aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg 6840
gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag 6900
gggaaaaagg tcgaaaaggt ctctttcctg tggatagcac gtacattggg aacccaaagc 6960
cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt 7020
cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt 7080
taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca 7140
cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg 7200
cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca 7260
atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag 7320
gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 7380
gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 7440
tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga 7500
gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc 7560
ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt 7620
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 7680
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 7740
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 7800
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 7860
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 7920
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 7980
tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 8040
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 8100
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 8160
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 8220
gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 8280
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 8340
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 8400
ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgcatt 8460
ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct 8520
tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga 8580
tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat 8640
caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt 8700
gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg 8760
gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat 8820
tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg 8880
atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag 8940
ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca 9000
gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt 9060
ccagccatag catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc 9120
tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag 9180
gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg 9240
gtgatattct cattttagcc atttattatt tccttcctct tttctacagt atttaaagat 9300
accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa 9360
ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt 9420
atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct gtcatcgtta 9480
caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc 9540
gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg 9600
ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc 9660
cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg 9720
cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaatta 9780
attcctaggc caccatgttg ggcccggcgc gccgatgtat gtgacaaccc tcgggattgt 9840
tgatttattt caaaactaag agtttttgtc ttattgttct cgtctatttt ggatatcaat 9900
cttagtttta tatcttttct agttctctac gtgttaaatg ttcaacacac tagcaatttg 9960
gctgcagcgt atggattatg gaactatcaa gtctgtgacg cgtaa 10005
Claims (1)
1. A method for high-efficiency genetic transformation of arabidopsis thaliana is characterized by comprising the following steps: the method comprises the following steps:
(1) the Km-resistant empty vector pDsRed0 was used as a test, and the sequence of the Km-resistant empty vector pDsRed0 is shown in SEQ ID NO. 1;
(2) transforming 100 mu L of agrobacterium-mediated state by adopting 1-2 mu g of pDsRed0 plasmid, and coating all transformed bacterial liquid into a culture dish with the diameter of 15cm, wherein the culture dish contains LB culture medium with 50mg/L Km resistance and 25mg/L Rif resistance;
(3) placing the culture dish at 28 ℃ for 3-4 days, cloning to 2-3mm, and directly collecting thalli by using a coating rod;
(4) resuspending the collected thallus with 100mL of transformation liquid, which is 1/2 MS culture medium containing 5% sucrose, to obtain a suspension;
(5) adding Silwet-77 with the final concentration of 0.02% into the suspension before transformation and transferring the bacterial liquid into a beaker;
(6) planting arabidopsis thaliana in a soil matrix in an artificial climate chamber until full-bloom stage, and cutting off grown fruit pods before transformation;
(7) placing the inflorescence of the arabidopsis into the suspension liquid, and fully soaking for 3-5 minutes for infection;
(8) wrapping the infected arabidopsis plants with a preservative film, and placing the arabidopsis plants in a dark place for 16-24 hours to keep high humidity;
(9) watering normally, fixing the plants by bamboo sticks, and stopping watering when the seeds are mature;
(10) drying the harvested seeds in an oven at 37 ℃ for 3-5 days;
(11) and (4) selecting positive seeds through Ds-Red fluorescence, and planting to obtain positive transgenic plants.
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