CN101914567A - Method for expressing and identifying foreign genes in fungus - Google Patents
Method for expressing and identifying foreign genes in fungus Download PDFInfo
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- CN101914567A CN101914567A CN 201010232721 CN201010232721A CN101914567A CN 101914567 A CN101914567 A CN 101914567A CN 201010232721 CN201010232721 CN 201010232721 CN 201010232721 A CN201010232721 A CN 201010232721A CN 101914567 A CN101914567 A CN 101914567A
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Abstract
The invention relates to a plant transgenic breeding method. The foreign genes are expressed in fungus by using a fusarium oxysporum-identified CaMV 35S promoter so as to rapidly identify the gene function. The plant transgenic breeding method has the advantages of simpleness, convenience, rapidness, and capacities of shortening the plant transgenic breeding progress and finishing gene expression and preliminary functional identification within 24 days.
Description
Technical field:
The present invention relates to a kind of plant transgene breeding method, be specifically related to a kind of CaMV of utilization 35S promoter expression alien gene in fungi, the method for Rapid identification gene function.
Background technology:
In the plant transgene breeding method of prior art, foreign gene to be changed over to needs at first to express in eukaryote and functional verification usually, imports in the target plant again.Method commonly used at present is: by the transformation mode plant, as tobacco or Arabidopis thaliana, detect its expression and preliminary function, this gene is forwarded in the target plant again.This process is longer qualification cycle, generally needs just can finish in 6-8 month, and time-consuming, effort.
Fungi is the simplest eukaryote.Compare with transforming plant, the genetic transformation of fungi has saved loaded down with trivial details operating process such as sowing, results, tissue culture, and the cycle is short, easy to operation.Therefore, if can in fungi, realize expression of exogenous gene and function preliminary evaluation, will shorten the transgenic breeding process greatly.The method that transforms as fungi is a lot, but the method for generally acknowledging at present is the conversion by agriculture bacillus mediated (infecting).Realize that conversion process has been a proven technique, basic identical by the principle of Agrobacterium-mediated Transformation fungi and conversion plant.
At present, be used for the expression vector of higher plant genetic transformation, the employed promotor of its marker gene and functional gene is the CaMV35S promotor.CaMV35S is a kind of strong promoter from Caulimovirus, can be discerned by plant, is widely used in the plant transgene breeding.But regrettably, most fungies can't discern it, if change plant expression vector over to fungi, then can cause its resistant maker gene that has and functional gene to express in fungi.So, when scientist obtains a useful gene from prokaryotic organism (as bacterium) after, must be earlier in the such model plant of Arabidopis thaliana, do preliminary expression and Function Identification, and then change in the plant that needs, because the genetic transformation of Arabidopis thaliana is very easy to, convenient (Plant Transformation is difficult, main difficult become the seedling process in tissue culture), yet, this be considered at present most convenient efficiently process also need more than half a year.Therefore, it is necessary to set up quicker, a simple and easy to do exogenous gene expression authentication method in eukaryote.
Someone finds that Fusarium oxysporum can discern the CaMV35S promotor, and in the prior art, Shang Weijian has the people to use it in fungi expression of exogenous gene and Function Identification.
Summary of the invention:
Final purpose of the present invention be set up a kind of can be fast, save time, effortlessly in fungi to expressing from procaryotic new functional gene and the method for Function Identification.
Direct purpose of the present invention is to utilize agriculture bacillus mediated and CAMV35S promoter expression foreign gene and carry out the function preliminary evaluation.
In order to solve the problem of the used CAMV35S promotor of most fungi nonrecognition plant expression vectors, the inventor can discern the CaMV35S promotor to Fusarium oxysporum (Fusarium oxysporum) and study.Utilized this characteristics, the present invention has realized fast foreign gene being expressed the purpose of evaluation in fungi.
Fusarium oxysporum belongs to Deuteromycotina, Fusarium.Someone finds that Fusarium oxysporum can discern the CAMV35S promotor.The present invention experimental results show that, plant expression vector pCAMBIA1300 commonly used can directly transform the mutant that Fusarium oxysporum (Fusarium oxysporum) obtains to have hygromycin resistance, shows that sharp knife sickle spore bacterium can discern the CaMV35S promotor that the plant expression vector hygromycin phosphotransferase gene has really.This is a unique filamentous fungus that can discern the CaMV35S promotor of reporting up to now, and this discovery makes the binary vector that is applicable to Plant Transformation at present just can be used for transforming fungi without any need for revising.
The technique means of the inventive method is:
Utilize the CaMV35S promotor to carry out the method for genetic expression and evaluation in fungi, its operation steps is as follows:
1, goal gene is cloned on the plant expression vector that has the CaMV35S promotor;
2, the carrier with step 1 gained transforms in the importing Agrobacterium;
3, infect Fusarium oxysporum with the resulting Agrobacterium of step 2, screening obtains positive transformant;
4, the goal gene that contains in the described positive transformant of step 3 is expressed and preliminary Function Identification.
In the above-mentioned steps 1, described plant expression vector can be this area plant expression vector commonly used, as pBI121 or pCAMBIA series etc.;
In the step 3, can select hygromycin resistance for use is selection markers.
In an example of utilization present method, the contriver has made up the epsp synthase gene plant expression vector of high-resistance glyphosate, utilize agriculture bacillus mediated genetic transforming method, transform the mutant of Fusarium oxysporum EPSPs gene knockout, can make the tolerance of its recovery glyphosate.The contriver has obtained EPSP synthase (the 5-enol form acetone shikimic acid-3-phosphate synthase) gene of a novel high-resistance glyphosate from prokaryotic organism, adopt method provided by the invention fast this gene to be carried out expression and function Rapid identification in eukaryote.Main operational steps is as follows:
1, the open reading frame sequence 5 ' end with epsp synthase gene connects the CAMV35S promotor, and 3 ' end connects the no terminator;
2, the complete epsp synthase gene that will connect is cloned into the multiple clone site (Fig. 1) of pCAMBIA1300;
3, the recombinant plasmid that obtains (expression vector) transforms and imports among the Agrobacterium AGL-1;
4, infect the mutants which had of Fusarium oxysporum glyphosate resistance disappearance with the Agrobacterium that contains recombinant plasmid,, and obtain single spore culture (Fig. 2) with hygromycin resistance label screening positive transformant;
5, extract the genomic dna of transformant,, adopt PCR method that positive transformant is carried out molecule checking (Fig. 3) at hygromycin phosphotransferase gene indoor design primer;
6, extract total RNA of transformant, reverse transcription is carried out genetic expression and is identified;
7, containing on the minimum medium flat board of glyphosate, carrying out the Function Identification of goal gene;
8, the expression and the function situation of analysis-by-synthesis assessment epsp synthase gene.
Specific operation process and result see embodiment for details.
The main innovative point of the present invention is to have selected Fusarium oxysporum for use.Because general fungi nonrecognition CAMV35S promotor transforms so the expression vector of commentaries on classics plant commonly used can not be used as fungi, can't be used as critical function expression of gene and Function Identification.
And the present invention utilizes Fusarium oxysporum can discern the characteristics of CAMV35S promotor, with the expression qualification process of plant critical function transgenosis, becomes by transforming fungi from revolving die formula plant Arabidopis thaliana commonly used and to identify.Because fungi and Arabidopis thaliana all are eukaryotes, thus can represent qualification result in Arabidopis thaliana at the qualification result in the fungi, this change or method innovation, easy, quick, shortened the process of plant transgene breeding.
Major advantage of the present invention:
1, technological operation is simple, laborsaving
Saved in greenhouse seedling, seed results, homozygote screening or tissue culture Cheng Miao trivial step such as (tobaccos), in the laboratory, just can finish.
2, the cycle short, fast, save time
Generally after plant expression vector construction is finished, can obtain positive transformant in 14 days, can obtain transformant pure culture bacterial strain in 18 days through molecule checking, comprise Agrobacterium-mediated Transformation 2 days, the sickle-like bacteria that needs to transform cultivate 2-3 days, altogether cultivate 2 days, transformant screening 4 days, obtain single bacterium colony 4 days and 4 days (seeing embodiment 1 for details) of molecule checking; Behind the single bacterium colony that obtains through the molecule checking, can finish expression of gene and function preliminary evaluation in 7 days, comprise and shake that bacterium is cultivated and RNA extraction, reverse transcription and express and identify (, can verify with molecule and carry out strain culturing simultaneously) for saving time.Gene function was identified and generally finished (for saving time, can identify with expression and carry out simultaneously) within 6 day.Concrete Time Calculation is referring to table 1.Therefore, after expression vector establishment was finished, employing the inventive method carries out genetic expression and Function Identification can be finished within 25 days, identified with arabidopsis thaliana transformation method commonly used at present and compare that shortened about half a year qualification cycle.
Description of drawings:
Fig. 1 is with containing the Agrobacterium of plant expression vector pCAMBIA1300, directly infect Fusarium oxysporum, with hygromycin resistance as selection markers, resultant Fusarium oxysporum transformant.Illustrate that Fusarium oxysporum can discern the CAMV35S promotor that plant expression vector has.
Fig. 2 is cloned into plant expression vector with having CAMV35S promotor goal gene
Use the expression of plant transgene promotor CAMV35S control goal gene commonly used, after structure is finished, directly transform Fusarium oxysporum and carry out preliminary evaluation,, determine whether to transform plant according to qualification result.
Fig. 3 is the electrophoretogram of positive transformant Molecular Identification
At the indoor design primer of hygromycin phosphotransferase gene, the product size is 800bp, is that template is carried out pcr amplification with the genomic dna of transformant, the agarose gel electrophoresis with 1.0% (1-7 is different transformant).
Fig. 4 is that the Fusarium oxysporum that changes the IrrE gene is cultivated 4 days data of spore concentration later in the MM of different salt concn substratum.Original inoculating spores concentration is 0.1 * 10
6About/ml.
Embodiment
In following examples, carried out concrete experiment, proved that the inventive method is effective with EPSP synthase (the 5-enol form acetone shikimic acid-3-phosphate synthase) gene of high-resistance glyphosate.But need to prove that those skilled in the art take present method according to spirit of the present invention, also can be used for the detection of expression and the Function Identification of other goal gene.Therefore, the scope of this patent is not limited to the content of following embodiment.
Part material source used in following examples is as follows:
Fusarium oxysporum bacterial strain: from China Agriculture Academe Fertilizer Institute " Chinese agriculture microbial strains preservation administrative center (Agricultural Culture Collection of China, ACCC) ", bacterial strain deposit number: 31352.
Need to prove,, all can reach purpose of the present invention with any Fusarium oxysporum bacterial strain.
The CaMV35S promotor: from a kind of strong promoter of Caulimovirus, can be discerned by plant, the startup that has been widely used in foreign gene in the plant transgene breeding is expressed, and its sequence is known.
PCAMBIA series vegetable expression vector: buy from Australian Cambia institute.
PBI121: be plant transgene expression vector commonly used, can buy from plasmid vector service provider.
Hygromycin B: U.S. Amresco company product.
Epsp synthase gene: be the applicant by gathering pedotheque in the extreme contaminate environment of glyphosate, the total DNA of sample separation makes up cosmid library, screening glyphosate resistance transformant, sequential analysis, synthetic and the dna fragmentation of the high-resistance glyphosate that obtains.
Nos terminator: the expression termination element that is international eukaryotic gene.
PCAMBIA1300 plant expression vector: available from Australian Cambia institute.
Agrobacterium AGL-1: one of agrobacterium strains that fungi or plant transgene are commonly used, can buy from professional bacterial strain service provider.
The used plant expression vector pCAMBIA1300 of the present invention is from Australian Cambia institute, on it with the CAMV35S promotor be a kind of strong promoter from Caulimovirus, be widely used in the plant transgene breeding.
The present invention has obtained EPSP synthase (the 5-enol form acetone shikimic acid-3-phosphate synthase) gene of a novel high-resistance glyphosate from prokaryotic organism, its open reading frame nucleotide sequence total length is 1335 bases, and the coding total length is 445 amino acid.Adopt method provided by the invention to carry out Rapid identification to the expression and the function of this gene in eukaryote fast.Operation steps is as follows:
1, the structure of plant expression vector
1.1 the plasmid with the epsp synthase gene that contains above-mentioned high-resistance glyphosate is a template, open reading frame to this epsp synthase gene is carried out pcr amplification, employed 5 ' end primer adds 25 bases and CAMV35S promotor 3 ' end is complementary, and 3 ' terminal primer is introduced the HindIII restriction enzyme site;
1.2 design primer amplification CAMV35S promotor, 5 ' end primer is introduced the BamHI restriction enzyme site, and it is complementary that 25 bases of 3 ' end primer interpolation and epsp synthase gene are read 5 of frame ' end;
Link to each other 1.3 utilize the fusion round pcr that the CAMV35S promoter sequence is read frame with the exploitation of epsp synthase gene, obtain having the epsp synthase gene fragment of CAMV35S promotor;
1.4 the epsp synthase gene fragment of the band CAMV35S promotor that step 3 is obtained after cutting with BamHI and HindIII enzyme, is connected into that carrier pET28a that same enzyme cuts obtains recombinant plasmid pETGR-79 and with its transformed into escherichia coli BL21 (Promega company);
1.5 pETGR-79 and plant expression vector pCAMBIA1300 are cut with BamHI and HindIII enzyme respectively, reclaim the purpose fragment and connect, transformed into escherichia coli obtains having the plant expression vector (Fig. 1) of the epsp synthase gene of CAMV35S promotor.
2, the conversion of Agrobacterium
Change the plant expression vector that builds over to Agrobacterium and need 2 day time.
2.1 make the competent cell of Agrobacterium AGL-1 of ordinary method;
2.2 in every pipe competent cell, add the expression vector that about 1 μ g builds, ice bath 30min., liquid nitrogen flash freezer 1min, 37 ℃ melt 3min.
2.3 add 800 μ l YEP liquid nutrient mediums, after 28 ℃ of 150rpm recovered to cultivate 3h, the centrifugal 1min. of 5000rpm collected thalline.
2.4 the Agrobacterium thalline of collecting is coated on the YEP flat board (containing kantlex), to cultivate 2-3 days for 28 ℃, single bacterium colony that picking grows is identified transformant.
3, infect the cultivation of the preceding Agrobacterium of Fusarium oxysporum
The culturing process of Agrobacterium needs 2-3 days time.
3.1 get one of 50ml triangular flask, add MM liquid nutrient medium 20ml, add Kan and each 20 μ l of Rif of 50mg/ml respectively.Inoculation contains fresh Agrobacterium one ring of plant expression vector, 28 ℃, 200rpm shaking culture 48h.
3.2 get the 1.5ml EP pipe of two sterilizations, draw the cultured bacterium liquid of 1.5ml, 10000rpm, centrifugal 30s, with the resuspended thalline of 1ml IM liquid nutrient medium, the repeated centrifugation operation, and then with the resuspended thalline of 1ml IM.
3.3 draw 200 μ L bacterium liquid,, measure OD with 10 times of IM dilutions
600Value is calculated 10ml culture bacterium liquid consumption according to the O.D value that records, and makes final bacterium liquid OD
600=0.15.
3.4 add AS in the above-mentioned bacterium liquid of 10ml, making final concentration is 200 μ M/L.28 ℃, the 200rpm shaking table is cultivated 12h, makes OD
600Value is between 0.5-0.7, and is standby.
4, infect the preparation of preceding Fusarium oxysporum
Fusarium oxysporum is cultivated the 2-3 days time that needs, and carries out simultaneously but can cultivate with Agrobacterium.
4.1 get one of 100ml triangular flask, add CM liquid nutrient medium 20ml, Fusarium oxysporum EPSPs mutant that inoculation flat board or test tube are preserved, that preparation is used to transform is an amount of.
4.2 25 ℃, the 150rpm shaking table was cultivated 2-3 days.The bacterium liquid that takes a morsel with 10 times of microscopies of sterilized water dilution, calculates spore quantity;
4.3 according to the microscopy result, being diluted to spore concentration with sterilized water is 1 * 10
6About cfu/ml, standby.
5, the genetic transformation of Fusarium oxysporum
From infecting beginning to obtaining the transformant pure culture, need 12 days altogether, comprise following step.
5.1 cultivate altogether (2 days)
(1) the dull and stereotyped several piece of preparation IM (inducing culture), wherein every flat board (25ml) contains 20mMAS 50 μ L, and carefully the millipore filtration with sterilization is placed on the flat board, makes not have bubble as far as possible.
(2) draw ready Agrobacterium and Fusarium oxysporum spore suspension 100 μ L mixings respectively.
(3) 200 μ L Agrobacteriums and Fusarium oxysporum mixed solution are evenly coated on the millipore filtration, super clean bench dries up, and 25 ℃ of constant incubators are cultivated 48h.
5.2 the screening of positive transformant (4 days)
(1) the dull and stereotyped several piece of preparation PDA, wherein every flat board (25ml) contains 100mM Cef 50 μ L, 50mg/ml Hyg15 μ L, and the filter membrane that will finish common cultivation goes on this PDA flat board.
(2) continue 25 ℃ and cultivate 4d, occur until the transformant bacterium colony.
(3) the transformant list bacterium colony that obtains is forwarded on the PDA flat board that contains Cef and Hyg, continues to observe its growing state (Fig. 2).
5.3 the acquisition of transformant pure culture (4 days)
The positive transformant thalline that obtains is an amount of, suspend with sterilized water, microscopy calculates spore quantity, and carry out gradient dilution with sterilized water according to spore concentration, the bacterium liquid that takes a morsel is coated with flat board (PDA+Hyg), the colony number that makes every flat board is cultivated after 4 days for 25 ℃ and is obtained single bacterium colony (pure culture) between 30-50.
6, the molecule of transformant checking
Transformant list bacterium colony shakes bacterium and cultivated 4 days, and a part is used to extract genomic dna, and another part is used to extract total RNA.
Extracting test kit with OMEGA company fungal gene group, extract the genomic dna of transformant, at hygromycin phosphotransferase gene indoor design primer, is template with the transformant genomic dna, adopts PCR method that positive transformant is carried out molecule checking (Fig. 3).
7, genetic expression is identified
This process approximately needs 7 days.Carrying out total RNA with the cultured bacterial strain of step 6 extracts.
Extract test kit in a small amount with bright the fungal rna in Shanghai, extract the total RNA of positive transformant.RT-PCR reactive system (K1002S) with promega company carries out reverse transcription and RT-PCR, and unconverted Fusarium oxysporum EPSPs mutant is contrast, carries out the genetic expression sxemiquantitative and identifies.Carry out the pcr amplification result at the foreign gene indoor design primer that imports and show that the gene of the new high-resistance glyphosate of importing has been realized normal expression under the control of CAMV35S promotor, and contrast Fusarium oxysporum EPSPs mutant does not amplify any band.
8, gene function is identified
This process approximately needs 6 days.But can verify with molecule, genetic expression identifies and to carry out simultaneously.
Fusarium oxysporum EPSPs mutant can not be grown in minimum medium (MM), and the positive transformant that changes the high-resistance glyphosate gene over to then can be grown.To be seeded in respectively through the positive transformant of identifying and contain 0,20,50,80,100,120,150,200,250, in the minimum medium of 300mM glyphosate concentration, cultivate 3 days for 25 ℃,, carry out the Function Identification of goal gene by detecting spore concentration.The result shows, when glyphosate concentration was 200mM, positive transformant still can normal growth, poor growth during 250mM, and the level that its glyphosate tolerant is described is between 200-250mM.
9, genetic expression and functional assessment
The expression and the function of analysis-by-synthesis assessment epsp synthase gene are asked the condition explanation, the plant expression vector CAMV35S promotor that makes up can normally start expression of exogenous gene, new high-resistance glyphosate gene have a resistance function, can be used for the transgenic breeding of plant.
The present invention has obtained a gene IrrE of global regulation from extremely corrosion-resistant abnormal cocci, this gene can improve colibacillary salt resistance ability.Adopt method provided by the invention to carry out Rapid identification to the expression and the function of this gene in eukaryote fast.Operation steps is as follows:
1, the structure of plant expression vector
1.1 the genomic dna with D.radiodurans R1 is a template, open reading frame to the IrrE gene is carried out pcr amplification, employed 5 ' end primer adds 25 bases and CAMV35S promotor 3 ' end is complementary, and 3 ' terminal primer is introduced the HindIII restriction enzyme site;
1.2 design primer amplification CAMV35S promotor, 5 ' end primer is introduced the BamHI restriction enzyme site, and it is complementary that 25 bases of 3 ' end primer interpolation and epsp synthase gene are read 5 of frame ' end;
Link to each other 1.3 utilize the fusion round pcr that the CAMV35S promoter sequence is read frame with the exploitation of IrrE gene, obtain having the IrrE gene fragment of CAMV35S promotor;
1.4 the IrrE gene fragment of the band CAMV35S promotor that step 3 is obtained after cutting with BamHI and HindIII enzyme, is connected into that carrier pET28a that same enzyme cuts obtains recombinant plasmid pETIrrE and with its transformed into escherichia coli BL21 (Promega company);
1.5 pETIrrE and plant expression vector pCAMBIA1300 are cut with BamHI and HindIII enzyme respectively, reclaim the purpose fragment and connect, transformed into escherichia coli obtains having the plant expression vector (Fig. 1) of the IrrE gene of CAMV35S promotor.
2, the conversion of Agrobacterium
With embodiment 1.
3, infect the cultivation of the preceding Agrobacterium of Fusarium oxysporum
Working method is with embodiment 1, but the Fusarium oxysporum bacterial strain uses wild-type.
4, infect the preparation of preceding Fusarium oxysporum
5, the genetic transformation of Fusarium oxysporum
6, the molecule of transformant checking
4,5,6 three working method are with embodiment 1.
7, genetic expression is identified
Change the total RNA of IrrE gene masculine transformant list bacterium colony and extract, use TRIzol reagent (GIBCO-BRL), RNase-free DNase (TaKaRa company) is used to remove DNA and pollutes.RT-PCR reactive system (K1002S) with promega company carries out reverse transcription and RT-PCR, and the wild-type Fusarium oxysporum is contrast, carries out the genetic expression sxemiquantitative and identifies.Carry out the pcr amplification result at the IrrE gene indoor design primer that imports and show that the new IrrE gene of importing has been realized normal expression under the control of CAMV35S promotor, and contrast Fusarium oxysporum wild-type does not amplify any band.
8, gene function is identified
Wild-type Fusarium oxysporum bacterial strain can normal growth in liquid-based basal culture medium (MM).To be seeded in respectively and contain 0,10,15,20 through the positive transformant of identifying (is contrast with the wild type strain), 25,30,40,50,75, in the liquid-based basal culture medium of 100mM NaCI concentration, cultivated 4 days for 25 ℃,, carry out the Function Identification of goal gene by detecting spore concentration.The result shows that when wild type strain was 15mM in NaCI concentration, the speed of growth was suppressed, and can not grow when 25mM is above.And that the transgenic positive transformant is grown when 25mM is normal substantially, and its complete repressed salt concn illustrates that the importing of its IrrE gene has improved the salt tolerant level (Fig. 4) of Fusarium oxysporum more than 30mM.
Table 1 the inventive method is carried out genetic expression and Function Identification required time
Claims (5)
1. the method for an expression alien gene in fungi it is characterized in that utilizing Fusarium oxysporum identification CaMV35S promotor, and promotor gene is expressed.
2. the described method of claim 1, its operation steps is as follows:
1) goal gene is cloned on the plant expression vector that has the CaMV35S promotor;
2) carrier with the step 1) gained transforms in the importing Agrobacterium;
3) with step 2) resulting Agrobacterium infects Fusarium oxysporum, and screening obtains positive transformant;
4) goal gene that contains in the described positive transformant of step 3) is expressed and Function Identification.
3. the described method of claim 2, the described plant expression vector of step 1) is pBI121 or pCAMBIA series;
4. claim 2 or 3 described methods, the step 3) hygromycin resistance is a selection markers.
5. express the epsp synthase gene of high-resistance glyphosate and the method for function Rapid identification for one kind in fungi, main operational steps is as follows:
1) the open reading frame sequence 5 ' end with epsp synthase gene connects the CAMV35S promotor, and 3 ' end connects the no terminator;
2) the complete epsp synthase gene that will connect is cloned into the multiple clone site of pCAMBIA1300;
3) recombinant plasmid that obtains (expression vector) transforms and imports among the Agrobacterium AGL-1;
4) infect the mutants which had that the Fusarium oxysporum glyphosate resistance lacks with the Agrobacterium that contains recombinant plasmid,, and obtain single spore culture with hygromycin resistance label screening positive transformant;
5), adopt PCR method that positive transformant is carried out the molecule checking at hygromycin phosphotransferase gene indoor design primer;
6) genomic dna and the total RNA that extracts transformant respectively carries out the genetic expression evaluation;
7) containing on the minimum medium flat board of glyphosate, carrying out the Function Identification of goal gene.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058275A (en) * | 2017-05-27 | 2017-08-18 | 山东大学 | The thio isorhamnose synthase gene Zmsqd1m of corn UDP and its application in plant tolerant to low-phosphorus is improved |
| CN109485704A (en) * | 2018-11-27 | 2019-03-19 | 温州大学 | A kind of expression system of meningococcus fHbp albumen |
-
2010
- 2010-07-16 CN CN 201010232721 patent/CN101914567A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 《PHYTOPATHOLOGY》 20010228 E. D. Mullins et al Agrobacterium-Mediated Transformation of Fusarium oxysporum: 第91卷, 第2期 2 * |
| 《分子植物育种》 20030430 朱玉等 草甘膦生物抗性和生物降解及其转基因研究 第435-441页 第1 卷, 第4 期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058275A (en) * | 2017-05-27 | 2017-08-18 | 山东大学 | The thio isorhamnose synthase gene Zmsqd1m of corn UDP and its application in plant tolerant to low-phosphorus is improved |
| CN109485704A (en) * | 2018-11-27 | 2019-03-19 | 温州大学 | A kind of expression system of meningococcus fHbp albumen |
| CN109485704B (en) * | 2018-11-27 | 2022-04-19 | 温州大学 | Expression system of meningococcal fHbp protein |
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