CN1446917A - GUS Carrier for expressing specificity of GUS gene plant anther induced by cyclomycin - Google Patents
GUS Carrier for expressing specificity of GUS gene plant anther induced by cyclomycin Download PDFInfo
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- CN1446917A CN1446917A CN 02103788 CN02103788A CN1446917A CN 1446917 A CN1446917 A CN 1446917A CN 02103788 CN02103788 CN 02103788 CN 02103788 A CN02103788 A CN 02103788A CN 1446917 A CN1446917 A CN 1446917A
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Abstract
The promoter T29 specific to the anthera for tobacco is combined with the tetracycline induction system to configune the TA29-Tx/TetR system induced by tetracycline for specific expression of anther tapetum. Under the control of said system, the tetracycline is applied to barnase gene for specific expression of barnase gene in said anther tapetum to generate a RNAase, which can kill cells to destroy anther tapetum, resulting in male sterility of plant.
Description
The present invention relates to plant genetic engineering field, be about under the control that gus gene places the chimeric promoters TA29-Tx that expresses through the plants flower pesticide specificity of transforming, make it after being subjected to tsiklomitsin and inducing, specifically expressing in the plant anther tapetum.Utilize this chimeric promoters that goal gene is expressed in specific time and space, thereby help the effect that we study its gene product.
TA29 is the promotor from tobacco samsum-TA29 gene
1This expression of gene is an anther-specific, and its product concentrates in the flower pesticide tapetum, generally is just to occur soon at postmeiotic, and content reaches the highest before After microspore mitosis, descends rapidly in flowering period.According to Koltunow etc.
2Research, the transcriptional control sequence of TA29 promotor-279 of its transcription initiation site upstream～-150bp between.This section sequence is to guarantee that the TA29 gene efficiently expresses necessary in the flower pesticide tapetum.And the sequence between the sequence between-207～-191 and-165～-158 is two pass key sequences of decision TA29 specifically expressing.Test shows: the TA29 promotor can be controlled foreign gene specific expression in plant anther
3.4
Tx is formed by the 35S promoter TATA frame both sides that three tumor-necrosis factor glycoproteinss insert CaMV, and specifically sequence is as follows
Wherein
Sequence derives from the promoter region of intestinal bacteria (E.coli) tetracyclin resistance operon.TetR (tet repressor) albumen of transposon Tn10 coding can be combined closely with above-mentioned manipulation sequence, stops the formation of transcription initiation mixture, thereby reaches the expression that suppresses downstream gene from transcriptional level.
The characteristic that is intended that TA29 promotor and Tx/TetR system of this invention combines, thereby creates a new system, and goal gene can either only be confined to the plant anther tapetum again and express by the tsiklomitsin abduction delivering.Final purpose of the present invention is can be by tsiklomitsin inductive Barnase gene plant anther specific expression vector by making up, be TA29-Tx-Barnase, the way of utilizing particle gun or agricultural bar mattress to infect, make this vector integration in the proteic Plant Genome of energy composing type high level expression TetR, when with the people for after applying tsiklomitsin and inducing, the Barnase gene is efficient and specific expression in plant anther, destroy the flower pesticide tapetum, cause pollen abortion, obtain male sterility line of plants, thereby create and a kind ofly utilize chemical induction to obtain the plants male sterility method, for the acquisition of male sterile line in the bilinear method breeding provides a brand-new approach.
The present invention mainly comprises following several respects technology: one. and the extraction of the total DNA of tobacco (with reference to the method that Wang Gejiao etc. provides, plant genetic transformation technology handbook, Fu Rongzhao etc. 1994)
5
1. get the fresh tobacco spire of 0.5g, add liquid nitrogen and be ground to dry powder, change over to and put into 500 μ l urine in advance
(8M urea, 500mM Tris.Cl pH8.0,350mM NaCl, 20mM in the centrifuge tube of plain damping fluid
EDTA, the suction filtration sterilization).
2. shake centrifuge tube, make vegetable material, add the phenol of 500 μ l in the damping fluid thorough mixing: chloroform (1:
1)。Abundant mixing.
3.10000rpm centrifugal 5 minutes, collect supernatant liquor, transfer in the clean centrifuge tube, add 500
The chloroform of μ l, extracting is one time again.
4. shift in the clean centrifuge tube of supernatant to, add 2 times of dehydrated alcohols that volume is ice-cold, mixing, quiet
Put precipitation 10 minutes.
5.15000rpm centrifugal 5 minutes, collecting precipitation, the washing with alcohol with 75% one time, drying at room temperature 10
Minute.Add 200 μ l ddH
2The O dissolving.
6. get 5 μ l, with 0.8% agarose gel electrophoresis detection, quantitative.The polymerase chain reaction of two anther-specific gene TA29 promotors (Polymerase Chain Reaction, PCR)
According to the complete sequence of the TA29 promotor of having delivered, we have chosen between this gene 5 ' district-826～-69 one section as target sequence, and that this sequence comprises the TA29 anther-specific expression is necessary-279～-the 150bp sequence.Design P1, the P2 sequence is the PCR primer, 5 ' end primer P1:5 '---AGGAATTCAGTAATACACTTTTTGGT---3 '; 3 ' end primer P2:5 '---TACCCGGGCTTTTGTTGGAGCATTTC---3 '.Reaction system is 50 μ l, comprising template DNA 2 μ l (0.1 μ g/ μ l), P1 1 μ l (25pM), P2 1 μ l (25pM), 10xBuffer 5 μ l, dNTPs 2 μ l (2.5mM), ddH
2O 38 μ l, Taq enzyme 1 μ l.Response procedures is 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ were extended 30 circulations 1 minute; 72 ℃ were extended termination reaction 10 minutes then.Three. the structure of chimeric promoters
Chimeric promoters is made up of two portions: the TA29 promotor, and it can make its controlling gene at flower pesticide tapetum specifically expressing; Tx can combine closely with TetR albumen.When Tx being inserted into TA29 promotor TATA frame appropriate location, make chimeric promoters both have the tsiklomitsin inducing properties, keep flower pesticide tapetum specifically expressing characteristic again.Four. tsiklomitsin inductive gus gene anther-specific expression is cut out the structure of body
We are carrier with BinHygTx, and TA29 promotor and Tx sequence are concatenated between the multiple clone site of this carrier, add thereafter then to connect the Nos terminator in the downstream of gus gene by gus gene.So just, finished the structure of tsiklomitsin inductive gus gene anther-specific expression sanction body.The moment detection of expression of gus gene shows that this carrier is induced by tsiklomitsin not only, and is flower pesticide tapetum specifically expressing.This carrier is a binary vector, contains the border, the left and right sides of agrobatcerium T-DNA, can infect method with Agrobacterium and import plant.
Marginal data:
In legend (1), TA29 is to be template with the pGT89BN plasmid, according to Chen Qian etc.
6Article on sequences Design primer P1, P2 obtains by PCR method amplification, adds EcoRI and SmaI restriction enzyme site respectively at its two ends.PGEM-T is a kind of e. coli plasmid vector, and Amp is an amicillin resistance, and the restriction enzyme site on it is indicated as figure.
In the legend (2), carrier pGEM-TA29 is made up by pGEM-T, has inserted the TA29 promotor in its multiple clone site.Plasmid pUCA7-TX is that German Gottingen college professor C.Gatz is so kind as to give.
In the legend (3), carrier pUCA7-TA29-TX is made up by pUCA7-TX, and the TX sequence is positioned at TA29 promotor TATA box frame place just.
In the legend (4), carrier pBI221 and pGEM7Zf are this laboratory and preserve carrier.
In the legend (5), carrier pGEM-GUS goes up gus gene with pBI221 to downcut, and inserts pGEM7Zf carrier multiple clone site structure and forms, and purpose is to make the gus gene two ends obtain new restriction enzyme site.
In the legend (6), carrier pUCA7-TA29-TX and pGEM-GUS are respectively above-mentioned legend (3) and (5), and Bin-Hyg-TX is that German Gottingen college professor C.Gatz is so kind as to give.TA29-TX segment and gus gene are downcut from pUCA7-TA29-TX and pGEM-GUS respectively, displacement is fallen Bin-Hyg-TX and is gone up the 35S-Tx segment, be built into carrier B in-Hyg-TA29-TX-GUS, i.e. tsiklomitsin inductive gus gene plants flower pesticide specificity expression vector.This carrier both had been suitable for Agrobacterium and had infected conversion, also was suitable for particle gun and transformed.
The extraction of the total DNA of the embodiment 1 tobacco (method that provides with reference to Wang Gejiao etc.Plant genetic transformation technology handbook, Fu Rongzhao etc. 1994)
1. get the fresh tobacco spire of 0.5g, add liquid nitrogen and be ground to dry powder, change over to and put into 500 μ l in advance
(8M urea, 500mM Tris.Cl pH8.0,350mM NaCl, 20mM in the centrifuge tube of urea buffer solution
EDTA, the suction filtration sterilization).
2. shake centrifuge tube, make vegetable material, add the phenol of 500 μ l in the damping fluid thorough mixing: chloroform (1:
1)。Abundant mixing.
3.10000rpm centrifugal 5 minutes, collect supernatant liquor, transfer in the clean centrifuge tube, add 500
The chloroform of μ l, extracting is one time again.
4. shift in the clean centrifuge tube of supernatant ripple to, add 2 times of dehydrated alcohols that volume is ice-cold, mixing,
Staticly settled 10 minutes.
5.15000rpm centrifugal 5 minutes, collecting precipitation, the washing with alcohol with 75% one time, drying at room temperature 10
Minute.Add 200 μ l ddH
2The O dissolving.
6. get 5 μ l, with 0.8% agarose gel electrophoresis detection, quantitative.
The pcr amplification and the clone of embodiment 2TA29 promotor
Complete sequence according to the TA29 promotor of having delivered, we have chosen between this gene 5 ' district-826～-69 one section as target sequence, the sequence between the sequence and-165～-158 that this sequence comprises the TA29 anther-specific expression between necessary-207～-191.Design P1, the P2 sequence is the PCR primer, 5 ' end primer P1:5 '---AGGAATTCAGTAATACACTTTTTGGT---3 '; 3 ' end primer P2:5 '---TACCCGGGCTTTTGTTGGAGCATTTC---3 '.Reaction system is 50 μ l, comprising template DNA 2 μ l (0.1 μ g/ μ l), P1 1 μ l (25pM), P2 1 μ l (25pM), 10xBuffer 5 μ l, dNTPs 2 μ l (2.5mM), ddH
2O 38 μ l, Taq enzyme 1 μ l.Response procedures is 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ were extended 30 circulations 1 minute; 72 ℃ were extended termination reaction 10 minutes then.
Get the 40ulPCR amplified production, 1% agarose gel electrophoresis with the band cutting-out of suitable size, reclaims pcr amplification product with dna fragmentation fast purifying/recovery test kit (being produced by Beijing ancient cooking vessel state biotech development center).With PCR product and 1: 1 in molar ratio the mixed of carrier pGEM-7Z that reclaims, the T4 Ligase that adds the production of Gibico company spends the night in 4 ℃ of connections.To connect product transformed competence colibacillus bacillus coli DH 5 alpha, screening positive clone.
The structure of embodiment 3 tsiklomitsin inductive gus gene anther-specific expression carriers
1. downcut TA29 fragment, agarose gel electrophoresis, recovery with the two enzymes of EcoRI/SmaI from plasmid pGEMT-TA29.
2. handle plasmid pUCA7-Tx with the two enzymes of EcoRI/EcoRV, agarose gel electrophoresis also reclaims the pUCA7-Tx carrier.
3. with the TA29 segment of above-mentioned recovery and the pUCA7-Tx carrier mol ratio mixing with 3: 1, add the T4 dna ligase, 4 ℃ of connections are spent the night.
4. will connect product and transform competent bacillus coli DH 5 alpha, on the LB substratum that contains 70 μ g/ml penbritins, be inverted overnight incubation for 37 ℃.
5. the picking positive colony extracts plasmid, by analysis of Restriction Endonuclease Profile, confirms that each element all is in correct connection, with this plasmid called after pUCA7-TA29-Tx.
6. GUS fragment (plasmid pBI221 cuts through the SacI enzyme, and the T4 archaeal dna polymerase is mended flat, cuts acquisition with the BamHI enzyme again) is inserted the pGEM-7Z carrier of handling through the two enzymes of BamHI/SmaI, obtain recombinant plasmid pGEM-7ZGUS.
7. TA29-Tx fragment (the pUCA7-TA29-Tx plasmid is handled the back through the two enzymes of EcoRI/BamHI and obtained) and GUS fragment (the pGEM-7ZGUS plasmid is handled the back through the two enzymes of BamHI/XbaI and obtained) are inserted in the BinHygTx carrier of handling through the EcoRI/XbaI double digestion, so just make the TA29-Tx-GUS segment be between the LB and RB of T-DNA, can change this carrier over to plant with the method that agrobacterium tumefaciens is infected.With this plasmid name BinHyg-TA29-Tx-GUS.Sketch map is as follows:
Embodiment 4 changes the external source goal gene in the tobacco cell technology by particle bombardment
The preparation of acceptor material: tobacco TetR2/3 (Nicotiana tabacum CV.Wisconsin 38) can composing type high level expression TetR albumen.With 70% ethanol sterilization 10 seconds, aseptic washing twice was again through 1 ‰ arsenic mercury sterilization 3～5 minutes with its seed, twice of aseptic washing, be tiled in then on the MS12 substratum that contains 50mg//L Kan, 1% agar, place between cultivation (16h illumination/every day, 25 ℃) to cultivate.When growing to 10～15cm, it is transplanted in the flowerpot, place greenhouse (15～28 ℃), treat that tobacco has just grown bud, get the flower bud sterilization of not blooming (method is with the tobacco seed sterilization together).Pollen is stripped out, with sterile razor blade every pollen is laterally cut into pieces, the square section places to contain the culture dish central authorities that the MS height oozes substratum (MS+0.4mol/L N.F,USP MANNITOL) up, and scope is the circle of diameter 3cm, cultivates in advance 5 hours.Its control material ovary is also handled with quadrat method through above-mentioned.
The preparation of particulate bullet: claim the 60mg bronze in 1ml 70% alcohol, fully vortex leaves standstill 15min, the centrifugal 5min of 1500rpm.Remove supernatant liquor, add 1ml distilled water, abundant vortex, the centrifugal 5min of 1500rpm removes supernatant liquor, triplicate.Bronze is changed in the 1ml50% glycerine, make the bronze suspension of 60mg/ml.Get 50 μ l suspension, add 1 μ g/ μ l plasmid DNA, 5 μ l, 2.5mol/L CaCl by turning round and look at preface
250 μ l and 0.1mol/L spermidine 20 μ l, vortex 10min, the centrifugal 5sec of 1500rpm removes supernatant liquor.250 μ l, 100% ethanol is washed once, vortex, and the centrifugal 5min of 1500rpm removes supernatant liquor, repeats once.At last, bronze is suspended in 60 μ l, 100% ethanol.
Get 6 μ l bag by the bronze suspension of DNA, equipment particle gun (PDS-1000 type, U.S. BioRod company makes), each culture dish bombards once, and the distance between bullet and the tobacco cell is 6cm.After the bombardment, culture dish is cultivated down in suitable condition.Whole bombardment process is carried out under sterile state.
Reference related to the present invention
1.Seurinck，J.et?al.，1990，Trends?Biochem.Sci.14，450-454
2.A.M.Koltunow，J.Truettner，R.B.Goldberg?et?al.(1990)Different?Temporaland?Spatial?Gene?Expression?Patterns?Occur?During?Anther?Development.ThePlant?Cell?2.1201-1224
3.Mariani，C.et?al.，1990，Nature?347，737-741
4.mariani，C.et?al.，1992，Nature?357，384-387
5. Fu Rong is clear etc., and 1994, plant genetic transformation technology handbook, China Science Tech Publishing House, Beijing
6. Chen Qian, Wang Yingchun, the structure of flower pesticide distinctive embedment promoters such as Zhang Liming and the acquisition Journal of Agricultural Biotechnology of male sterile transgenic arabidopsis, 2001,9 (1): 62-64
Claims (5)
1. plasmid vector contains and is subjected to that tsiklomitsin is induced, the chimeric promoters of anther-specific expression, beta-glucuronidase (β-glucuronidase, GUS) gene, terminator and selection markers gene.It is characterized in that being in the gus gene under this chimeric promoters control, only after induced by tsiklomitsin just at plant anther tapetum specifically expressing.
2. vector plasmid according to claim 1, it is characterized in that gus gene 5 ' end assembled be subjected to the chimeric promoters TA29-Tx that tsiklomitsin is induced, the flower pesticide tapetum is specific expressed, this chimeric promoters can regulate gus gene be subjected to tsiklomitsin induce the back in the plant anther tapetum specifically expressing.
3. vector plasmid according to claim 1 is characterized in that having assembled the NOS terminator that strengthens ability to express in 3 ' end series connection of gus gene.
4. vector plasmid according to claim 1 is characterized in that having assembled the HPT-II gene on this carrier, as the selection markers of transgenic plant, can use hygromycin selection; As the selection markers of transgenic microorganism, can screen with kantlex.
5. vector plasmid according to claim 1 is characterized in that on this carrier with ribonuclease gene (Barnase) displacement gus gene.After being subjected to tsiklomitsin and inducing, the Barnase gene efficiently expresses and obtains male sterility line of plants at the flower pesticide tapetum under above-mentioned chimeric promoters control.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02103788 CN1446917A (en) | 2002-03-25 | 2002-03-25 | GUS Carrier for expressing specificity of GUS gene plant anther induced by cyclomycin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 02103788 CN1446917A (en) | 2002-03-25 | 2002-03-25 | GUS Carrier for expressing specificity of GUS gene plant anther induced by cyclomycin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993878A (en) * | 2010-10-14 | 2011-03-30 | 南京农业大学 | RRNA mosaic promoter and expression vector containing same |
| CN102816793A (en) * | 2012-08-22 | 2012-12-12 | 陕西师范大学 | Single carrier bidirectionally started Tet-on inducible expression system, its construction method and application |
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2002
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993878A (en) * | 2010-10-14 | 2011-03-30 | 南京农业大学 | RRNA mosaic promoter and expression vector containing same |
| CN101993878B (en) * | 2010-10-14 | 2012-04-18 | 南京农业大学 | RRNA mosaic promoter and expression vector containing same |
| CN102816793A (en) * | 2012-08-22 | 2012-12-12 | 陕西师范大学 | Single carrier bidirectionally started Tet-on inducible expression system, its construction method and application |
| CN102816793B (en) * | 2012-08-22 | 2014-09-10 | 陕西师范大学 | Single carrier bidirectionally started Tet-on inducible expression system, its construction method and application |
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