CN1446916A - Carrier for expressing specificity of bec gene cotton fiber - Google Patents
Carrier for expressing specificity of bec gene cotton fiber Download PDFInfo
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- CN1446916A CN1446916A CN 02103787 CN02103787A CN1446916A CN 1446916 A CN1446916 A CN 1446916A CN 02103787 CN02103787 CN 02103787 CN 02103787 A CN02103787 A CN 02103787A CN 1446916 A CN1446916 A CN 1446916A
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- 229920000742 Cotton Polymers 0.000 title claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 34
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- 239000000049 pigment Substances 0.000 claims abstract description 12
- 101000578200 Lens culinaris Non-specific lipid-transfer protein 6 Proteins 0.000 claims abstract description 9
- 101000972854 Lens culinaris Non-specific lipid-transfer protein 3 Proteins 0.000 claims abstract description 8
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- 102000004190 Enzymes Human genes 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 7
- 235000000177 Indigofera tinctoria Nutrition 0.000 claims description 6
- 229940097275 indigo Drugs 0.000 claims description 6
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 claims description 5
- 102000016680 Dioxygenases Human genes 0.000 claims description 4
- 108010028143 Dioxygenases Proteins 0.000 claims description 4
- 239000013600 plasmid vector Substances 0.000 claims description 4
- XAWPKHNOFIWWNZ-UHFFFAOYSA-N 1h-indol-6-ol Chemical compound OC1=CC=C2C=CNC2=C1 XAWPKHNOFIWWNZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000001590 oxidative effect Effects 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 claims description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 claims description 2
- 150000002475 indoles Chemical group 0.000 claims description 2
- JOUDBUYBGJYFFP-FOCLMDBBSA-N thioindigo Chemical compound S\1C2=CC=CC=C2C(=O)C/1=C1/C(=O)C2=CC=CC=C2S1 JOUDBUYBGJYFFP-FOCLMDBBSA-N 0.000 claims description 2
- 230000009261 transgenic effect Effects 0.000 claims 2
- JHFAEUICJHBVHB-UHFFFAOYSA-N 1h-indol-2-ol Chemical class C1=CC=C2NC(O)=CC2=C1 JHFAEUICJHBVHB-UHFFFAOYSA-N 0.000 claims 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims 1
- 108091000080 Phosphotransferase Proteins 0.000 claims 1
- 244000005700 microbiome Species 0.000 claims 1
- 102000020233 phosphotransferase Human genes 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 8
- 241000589155 Agrobacterium tumefaciens Species 0.000 abstract description 2
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
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- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
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Abstract
A genetic engineering process for improving the color of cotton fibres includes serially linking the promoters LTP12, LTP6 and LTP3 specific to cotton fibres with chromoenzyme gene to configure prokaryotic and eucaryotic expression carrier, transforming them to Agrobacterium tumefaciens, introducing the associative LTP12 (or LTP6 or LTP3)-bec gene to cotton of specific expression of chromenzyme gene in cotton fibres, and depositing the pigment on the cell wall of cotton fibres to generate specific color.
Description
The present invention relates to the genetically engineered field, be about to from rhodococcus
[3]The bec gene place under the control of the cotton fibre specific promoter LTP12 (or LTP6 or LTP3) that amplifies from the cotton gene group, make it at cotton fiber extension phase specifically expressing.Utilize can the encode characteristic of indoles dioxygenase of bec gene, make the indoles generation oxidizing reaction that produces in the tryptophan metabolism approach, the indolol of generation is spontaneous in conjunction with generating the indigo and thioindigo of pigment.This invention is having a wide range of applications aspect the cotton fiber modification of colour genetically engineered.
A kind of pigment enzyme of bec genes encoding-draw the diindyl dioxygenase, it is that the tryptophan metabolism approach is the key enzyme in the indigo route of synthesis
[2], the oxidizing reaction of its energy catalyzing indole makes it to take off a part glucose and generates indolol, and it very easily is oxidized to indigo
[5] [6] [7]
LTP12 is the starting element of the special fat transfer protein of cotton
[8] [9], it and other two kinds of starting element LTP6 and GH3 vary in size
[10] [11], but existing certain similarity between sequence integral body, near 5 ' non-translational region homology with the ATG upstream TATA frame shows very obviously.The LTP12 promotor can start the fat transfer protein in its expression of elongate fiber, the tandem gene that constitutes by LTP12 (or LTP6 or LTP3) and bec can cause the elongating stage of cotton fiber specifically expressing go out pigment, when pigment deposition then can present color in fiber core, can obtain chromatic cotton fiber.
One of purpose of this invention is that the characteristic with LTP12 (or LTP6 or LTP3) promotor and bec gene combines, make up a new carrier for expression of eukaryon, further to make the bigger effect of LTP12 (or LTP6 or LTP3) promotor performance, not only can be applied to the improvement of cotton fiber strength, also can be used for the improvement of cotton fiber color and luster.
Two of the purpose of this invention is that this carrier for expression of eukaryon can be used to make up the plasmid vector that can transform Agrobacterium, carries out the improvement of cotton fiber color and luster by Agrobacterium infestation method converting cotton.
The final purpose of this invention is by making up the cotton fiber specific expression vector of bec gene, utilize pollen tube passage method and Agrobacterium infestation method to make the bec gene in the efficient and special expression elongating stage of cotton fiber, thereby produce the pigment enzyme, synthetic colour in the plant amino acid pathways metabolism.Pigment deposition makes cotton fiber produce color in lumen, eliminated the harm that the traditional cotton dyeing keratin-fiber brings the mankind and environment
[1] [4], for the color and luster of genetically engineered improvement cotton fiber provides a brand-new approach.
The present invention mainly comprises following several respects technology:
The extraction of one cotton genomic dna
1. take by weighing the fresh blade of 4 grams as vegetable material, grinding powder in liquid nitrogen is used for DNA extraction.Powdered tissue is packed in the 50ml centrifuge tube, add the extraction damping fluid of 20ml ice bath, mix.2. in 2, centrifugal 20 minutes of 700g gets supernatant and adds the 8ml lysis buffer, and abundant vortex mixed is (this moment, genomic dna still was in the nuclear membrane protection down) evenly, 65 ℃ of warm baths 20-30 minute.3. add 10ml chloroform/primary isoamyl alcohol (24: 1), put upside down mixing, centrifugal 5 minutes of 12000rpm gets and changes over to mutually in the new centrifuge tube.4. add the ice-cold Virahol of 2/3 volume (about 5.6ml), room temperature left standstill 20-30 minute.Centrifugal 10 minutes of 6000rpm, the collecting precipitation thing.5. add the gentle vibration flushing of 1ml 70% ethanol 2～3 times, dry up.Add 65 ℃ of temperature of 500 μ l TE and bathed resuspended precipitation 10-30 minute.The polymerase chain reaction of two fiber-specific gene LTP12 promotors (Polymerase Chain Reaction PCR)
Sequence according to the LTP12 promotor of having delivered, we choose the one section sequence that is about 800bp, this sequence comprises the important regulating and controlling district of LTP12 promotor, required primer is synthetic by the learned company in Shanghai: upstream primer 5 '-GC GGT ACC AAA CAA TTA AGT ATT GAT ACC AGA-3 ', downstream primer 5 '-GG CTT AAG GAC ATT GAG CTA GCC ATA.Reaction system is 100ul, comprising: template (the total DNA of cotton) 1ul, dNTP Mixer 8ul, Mg
2+6ul, primer 1 2ul, primer 2 2ul, 10 * ExBuf (Mg
2+Free) 10ul, ddH
2O 70.5ul.Response procedures is: 94 ℃ of pre-sex change 3 minutes, and 94 ℃ of sex change 40 seconds, 58 ℃ of annealing 60 seconds, 72 ℃ were extended 60 seconds.30 circulations, 72 ℃ were extended termination reaction 10 minutes then.The structure of three bec gene fiber-specific expression vectors
We are carrier with pGEMT, and PTL12 promotor and bec gene are connected in series between the multiple clone site of this carrier.Connect the NOS terminator in the downstream of bec gene then, so just constitute the anther-specific expression carrier of bec gene.The required element of cotton fiber specific expression on this expression vector is forwarded on the pBI101.3, make between its LB and RB that places T-DNA.So just, be built into and be suitable for the binary vector that Agrobacterium is infected.
Marginal data
In legend (1), carrier pGEMTPTL12 is made up by carrier pGEMT, has inserted the LTP12 promotor in its multiple clone site; Carrier pUCPTL12 is made up by pUC19, has inserted the LTP12 promotor that is obtained through SphI and PstI double digestion by pGEMTPTL12 in its multiple clone site; Carrier pPTL221 is made up by pBI221, has inserted the LTP12 promotor that is obtained through SphI and XbaI double digestion by pUCPTL12 in its multiple clone site.
In legend (2), bec is a kind of pigment enzyme gene, and it can give expression to the enzyme of catalysis committed step in the pigment building-up process.BamHI, EcoRI, PstI etc. are the restriction enzyme enzyme recognition site.PGEMT-7Z is a kind of e. coli plasmid vector, Amp
rBe amicillin resistance, the polyclone restriction enzyme site comprises ApaI, SphI, XbaI, XhoI, EcoRI, KpnI, SmaI, ClaI, HindIII, BamHI, SacI etc.
In legend (3), carrier pGEMbec is made up by pGEMT-7Z, has inserted the bec gene that is obtained through EcoRI and BamHI double digestion by pBbec in its multiple clone site.
In legend (4), carrier pPTLbec is made up by pPTL221, and it has inserted the bec gene that is obtained through XbaI and SacI double digestion by pGEMbec.
In legend (5), carrier pPTLbec101 is made up by pBI101.3, has inserted the LTP12 promotor and bec gene and the NOS terminator that are obtained through HindIII and SacI double digestion by pPTLbec in its multiple clone site.
Embodiment 1
The extraction of cotton genomic dna
1. take by weighing the fresh blade of 4 grams as vegetable material, grinding powder in liquid nitrogen is used for DNA extraction.Powdered tissue is packed in the 50ml centrifuge tube, add the extraction damping fluid of 20ml ice bath, mix.2.2 centrifugal 20 minutes of 700g gets supernatant and adds the 8ml lysis buffer, abundant vortex mixed is (this moment, genomic dna still was in the nuclear membrane protection down) evenly, 65 ℃ of warm baths 20-30 minute.
3. add 10ml chloroform/primary isoamyl alcohol (24: 1), put upside down mixing, centrifugal 5 minutes of 12000rpm gets and changes over to mutually in the new centrifuge tube.4。Add the ice-cold Virahol of 2/3 volume (about 5.6ml), room temperature left standstill 20-30 minute.Centrifugal 10 minutes of 6000rpm, the collecting precipitation thing.5. add the gentle vibration flushing of 1ml 70% ethanol 2～3 times, dry up.Add TE65 ℃ of temperature of 500 μ l and bathed resuspended precipitation 10-30 minute.
Embodiment 2
The amplification of LTP12 promotor and clone
Sequence according to the LTP12 promotor of having delivered, we choose the one section sequence that is about 800bp, this sequence comprises the important regulating and controlling district of LTP12 promotor, required primer is synthetic by the learned company in Shanghai: upstream primer 5 '-GC GGT ACC AAA CAA TTA AGT ATT GAT ACC AGA-3 ', downstream primer 5 '-GG CTT AAG GAC ATT GAG CTA GCC ATA.Reaction system is 100ul, comprising: template (the total DNA of cotton) 1ul, dNTP Mixer 8ul, Mg
2+6ul, primer 1 2ul, primer 2 2ul, 10 * ExBuf (Mg
2+Free) 10ul, ddH
2O 70.5ul.Response procedures is: 94 ℃ of pre-sex change 3 minutes, and 94 ℃ of sex change 40 seconds, 58 ℃ of annealing 60 seconds, 72 ℃ were extended 60 seconds.30 circulations, 72 ℃ were extended termination reaction 10 minutes then.After reaction is finished, get 20 μ l samples, carefully downcut the specific fragment that is positioned at about 800bp with disposable blade under the ultraviolet lamp, with being dissolved in 20 μ l ddH behind dna fragmentation recovery test kit recovery and the purifying through 1.0% agarose gel electrophoresis
2Among the O ,-20 ℃ of preservations are standby.Get 7 μ l PCR recovery product and mix, add T4 ligase and spend the night transformed competence colibacillus bacillus coli DH 5 alpha, screening positive clone in 4 ℃ of connections with 1 μ l pGEMT carrier.
Embodiment 3
The structure of bec gene fiber-specific expression vector
1. pGEMTPTL12 being cut gained LTP12 fragment with SphI, PstI enzyme mixes with the pUC19 carrier segments of cutting through SphI, PstI enzyme, add the T4 dna ligase, 4 ℃ of connections are spent the night, the transformed competence colibacillus bacillus coli DH 5 alpha, containing 37 ℃ of inversion overnight incubation on the LB substratum of 100ug/ul penbritin, promptly get pUCPTL12 through blue hickie screening.
2.pUCPTL12 be connected with SphI, XbaI double digestion pBI221 carrier segments through SphI, XbaI double digestion gained LTP12 fragment, the transformed competence colibacillus bacillus coli DH 5 alpha, the positive bacterium colony of picking, extract plasmid, cut evaluation through enzyme, to confirm that each element all is in correct connection, with this plasmid called after pPTL221.
3. the bec fragment that will downcut with EcoRI and BamHI enzyme from the plasmid vector that carries the bec gene is connected screening positive clone, called after pGEMbec with pGEMT-7Z carrier segments with EcoRI and BamHI double digestion.
4. will be connected screening positive clone, called after pPTLbec with 3800bp fragment with the bec fragment of the pGEMbec gained of XbaI and SacI double digestion with the pPTL221 gained of XbaI and SacI double digestion.
With pPTLbec with HindIII and SacI
-Double digestion gained fragment is connected to through HindIII, the pBI101.3 carrier segment of SacI double digestion, and screening positive clone is built into carrier pPTLbec101, and LTP12-bec-NOS is inserted between the LB and RB of T-DNA.This carrier can transform plant with the method that agrobacterium tumefaciens is infected.
The reference relevant with the present invention
1.A，Giri?AK，1989，Mutat?Res.223，313-319
2.Robin?J.Gouka?et?al.，2001，App.Env.Microbiol.June，2610-2616
3.Roychoudhury?R.H.et?al.，1989，J.Gen.Microbiol.135，1507-1513
4.Rannug?U.et?al.，1992，Mutat?Res.282，219-225
5.Tephen?H.&?David?R.W.，1992，J.Gen.Microbiol.138，205-209
6.Tephen?H.et?al.，1992，J.Gen.Microbiol.138，211-216
7.Tephen?H.et?al.，1990，J.Gen.Microbiol.136，1357-1363
8.Ma，D.P.et?al.，1997，Biochem.Biophys.Acta.1344，111-114
9.Ma，D.P.et?al.，1995，Biochem.Biophys.Acta.1257，81-84
10.Hsu，c.y.et?al.，1999，Plant?Science，143，63-70
11.John，M.E.et?al.，1992，Proc?Natl?Acad?Sci?USA，89，5769-5773
Claims (6)
1. a plasmid vector contains cotton fiber specific promoter, indoles dioxygenase (bec) gene, terminator and selection markers gene, it is characterized in that this promotor can regulate and control the bec gene in the special expression of cotton fiber extension phase.Bec expression of gene product is indoles dioxygenase (indol deoxydase), and this enzyme can the catalyzing indole oxidizing reaction generate indolol, and two molecule indolols are indigo in conjunction with generating pigment.
2. vector plasmid according to claim 1 is characterized in that 5 ' end of bec gene has been assembled cotton fiber specific promoter LTP12 (or LTP6 or LTP3), and this promotor can be regulated the bec gene at the specifically expressing elongating stage of cotton fiber.
3. vector plasmid according to claim 1 is characterized in that the NOS terminator that strengthens ability to express has been assembled in 3 ' end series connection of bec gene.
4. vector plasmid according to claim 1, it is characterized in that on this carrier, having assembled hygromix phosphotransferase (NTP-II) gene,, can use hygromycin selection as the selection markers of transgenic plant, as the selection markers of transgenic microorganism, can screen with kantlex.
5. vector plasmid according to claim 1 can produce another kind of pigment thioindigo if it is characterized in that the indigo replacement that an atom takes place of bec expression of gene product, presents pink.
6. vector plasmid according to claim 1, it is characterized in that with this carrier converting cotton, make the bec gene under the adjusting of LTP12 (or LTP6 or LTP3) promotor cotton fiber specifically-expressed, thereby produce indigo or pink or two kinds of simultaneous cotton fibers of pigment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 02103787 CN1446916A (en) | 2002-03-25 | 2002-03-25 | Carrier for expressing specificity of bec gene cotton fiber |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02103787 CN1446916A (en) | 2002-03-25 | 2002-03-25 | Carrier for expressing specificity of bec gene cotton fiber |
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| CN1446916A true CN1446916A (en) | 2003-10-08 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100404681C (en) * | 2005-04-29 | 2008-07-23 | 中国科学院遗传与发育生物学研究所 | Specific expression starter and its use for cotton fibre |
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2002
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100404681C (en) * | 2005-04-29 | 2008-07-23 | 中国科学院遗传与发育生物学研究所 | Specific expression starter and its use for cotton fibre |
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