CN100404681C - Specific expression starter and its use for cotton fibre - Google Patents
Specific expression starter and its use for cotton fibre Download PDFInfo
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- CN100404681C CN100404681C CNB2005100681884A CN200510068188A CN100404681C CN 100404681 C CN100404681 C CN 100404681C CN B2005100681884 A CNB2005100681884 A CN B2005100681884A CN 200510068188 A CN200510068188 A CN 200510068188A CN 100404681 C CN100404681 C CN 100404681C
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Abstract
The present invention discloses a specific expression promoter of a cotton fiber and an application thereof. The promoter is one of the following nucleotide sequences: 1) a DNA sequence of SEQ ID No. 1 in a sequence list, and 2) a nucleotide sequence which can be hybridized with the DNA sequence limited by SEQ ID No. 1 in the sequence list under the high precision condition. The promoter of the present invention can drive a target gene to specifically express in a cotton fiber, and has important meanings for breeding new fiber plant varieties, especially new cotton varieties.
Description
Technical field
The present invention relates to the specific expression promoter and the application thereof of vegetable fibre in the bioengineering field, particularly relate to a kind of specific expression promoter of cotton fiber and, especially cultivate the application in the new cotton variety cultivating the textile plant new variety.
Background technology
Cotton is extremely important textile industry raw material.Cotton fibre is to grow a kind of unicellular structure that forms by the epidermic cell on the ovule outer integument that is positioned at ovary.Its four periods of formation experience: the synthesis phase and the ripening stage of starting period, elongating stage (synthesizing of primary cell wall), secondary cell wall.Cotton ovule epidermic cell (except that pore epidermic cell and micropylar cell) all has the potential that is divided into cotton fibre, but has only 30% epidermic cell finally can form single celled fiber.
The Arabidopis thaliana epidermal hair is a kind of unicellular epidermal structure of specialization, be distributed widely in (Hulskamp M on blade, stem, lotus throne leaf, petal and the root, Misera S, Jurgens G.Genetic dissection of trichomecell devel opment in Arabidopsis.Cell, 76:555-566.1994).Because the growth course of this unicellular epidermal hair is simple, distribution at plant surface has certain pattern, and mutant does not change the others of development of plants, be convenient to do genetic analysis, therefore the growth course of Arabidopis thaliana epidermal hair has become a kind of modular system (Marks M D.Molecular genetic analysis oftrichome development in Arabidopsis.Annu.Rev.Plant Physiol of research vegetable cell destiny decision, Plant MolBiol, 48:137-163.1997).Studies show that, GL1 is the key gene that control Arabidopsis leaf trichome development starts, its encode MYB albumen of a kind of R2R3 type, form (OppenheimerD G by 228 amino acid, Herman P L, Sivakumaran S, Esch J, Marks M D.A myb gene required fortrichome differentiation in Arabidopsis is expressed in stipules.Cell, 67:483-493.1991); It is the gene of epidermal hair specific expression, only be expressed in (Larkin J C in children's epidermal hair that is in the etap in age, Oppenheimer D G, Pollock S, Marks M D.Arabidopsis GLABROUSIgene requires downstream sequences for function.Plant Cell, 5:1739-1748.1993).Arabidopis thaliana root hair is the another kind of unicellular epidermal hair that occurs in root.Determining of Arabidopis thaliana root hair by cell position, epidermic cell between 2 cells of cortex i.e. " H type " cell can develop into the root hair, and the epidermic cell that only links to each other with a tegumental cell is " N type " cell, often can only the non-root hair cell of bud into (Dolan L, Duckett C, Grierson C, LinseadP, Schneider K, Lawson E, Dean C, Poethig S, Roberts K.Clonal relations andpatterning in the root epidermis of Arabidopsis.Development, 120:2465-2474.1994).WER is the myb gene that control root hair is educated, and its a kind of R2R3 MYB albumen of encoding is made up of 203 amino-acid residues.The Northern analytical results shows that it only expresses in root and hypocotyl, and is only to express in " N " class cell in growing epidermis, and it is controlling " N " class cell development becomes non-root hair cell.WER in the Arabidopis thaliana and the sequence of GL1 have higher similarity.The consistence of WER and GL1 amino acid complete sequence is 57%, the consistence of DNA land is 91%, the amino acid consistence of coding region 3 ' end is 14/24, preceding 12 the amino acid whose consistence that relate to the R2 duplicon that combines with bHLH albumen are 100% (Lee M M, Schiefellbein J.Developmentally distinct MYB genes encode functionallyequivalent proteins in Arabidopsis.Development, 128:1539-1546.2001).WER shows the protein that WER is identical with the GL1 encoding function with a series of complementation tests of GL1, their different biological actions in development of plants are sequence difference (the Lee M M of promotor fully, Schiefellbein J.Developmentally distinct MYB genes encode functionally equivalent proteinsin Arabidopsis.Development, 128:1539-1546.2001).Verified at present, the complex body MYB-bHLH-WD40 that the startup of Arabidopis thaliana epidermal hair and Gen Mao is made up of three genes (GL1-GL3-TTG1) product at least controls, but each single-gene of also not getting rid of in them all has promoter action (Payne C T to the startup of epidermal hair, Zhang F, Lloyd A M, GL3 encodes a bHLH protein thatregulates trichome development in Arabidopsis through Interact ion with GL1and TTG1.Genetics, 156:1349-1362.2000).
The biological study result of development of plants shows that similar cell, tissue, allelotaxis's genetic mechanism has high conservative in the different plants.The epidermal hair of Arabidopis thaliana is a kind of unicellular epidermal structure of specialization, infers the genetic mechanism that is differentiated to form cotton fibre by the ovule epidermic cell, may be similar to the formation mechanism of Arabidopis thaliana epidermal hair.Known, GL1 (GLABROUS1) is one of oligogene of control Arabidopis thaliana epidermal hair differentiation with WER (WEREWOLF).Cotton fibre is as the unicellular epidermal hair on a kind of ovary ovule outer integument, and its growth Initiated Mechanism also is subjected to the control of MYB-bHLH-WD40 complex body regulatory mechanism probably.
Known at present, GhMYB109 (is called GhMYB9 again in disclosed another one related application " a kind of Myb transcription factor and encoding gene and the application of cotton ", number of patent application is 02145967.3,) 234 amino acid whose albumen of coding, characteristics with typical R 2R3 class myb transcription factor exist with single copy form in the cotton gene group.By finding relatively that with the full length sequence of AtMYBGL1 and AtWER their amino acid sequence homology reaches 51.2% and 59.1% respectively; With regard to the R2R3 conserved regions, homology is up to 82.1% and 84.0%.Northern analyze to find that GhMYB109 is the gene of a specifically expressing in cotton fibre, and finds in the fiber of initial phase this expression of gene is arranged promptly by Tissue in situ hybridization.Infer GhMYB109 (the Jingfeng Suo that may play an important role for the generation and the growth of cotton fibre thus, Xiaoe Liang, Li Pu, YangshengZhang, Yongbiao Xue.Identification of GhMYB109 encoding a R2R3 MYBtranscription factor that expressed specifically in fiber intials andelongating fibers of cotton (Gossypium Hirsutum L.) .Biochim Biophys Acta.1630 (1): 25-34.2003).
Summary of the invention
The purpose of this invention is to provide a kind of cotton fiber specifically-expressed promotor.
Cotton fiber specifically-expressed promotor provided by the present invention, name is called GhMYB109Pro, derives from Gossypium upland cotton (Gossypium hirsutum L.), is one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
The rigorous condition of described height can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized and washed film with 0.1 * SSPE under 65 ℃.
SEQ ID № in the sequence table: 1 by 2080 based compositions.
The expression vector that contains the cotton fibre specific promoter, transgenic cell line, host bacterium and the arbitrary segmental primer of the described promotor of amplification are to also belonging to protection scope of the present invention.The upstream primer of described primer centering and the distance between the downstream primer are between 50-2100 base, and the length of every primer of described primer centering is 15-30 base.
Utilize any carrier that can guide foreign gene in plant, to express, promotor provided by the present invention is imported vegetable cell, obtain transgenic cell line and transfer-gen plant that goal gene is expressed in cotton fiber.
When utilizing promotor of the present invention to make up plant expression vector, can add encoding sequence or any enhanser of any gene thereafter.
For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, as the antibiotic marker thing that adds the alternative mark of plant or have resistance.
Carry promotor of the present invention expression vector can conventional biotechnological means imports vegetable cell by using that Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity are led etc., by the plant transformed host both can be monocotyledons, also can be dicotyledons.
Promotor of the present invention can driving purposes gene specifically expressing in cotton fibre, changes cotton fiber quantity, qualitative character, and to cultivating the textile plant new variety, it is significant particularly to cultivate new cotton variety.
The invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is the part physical map that contains the fusion vector of GhMYB109Pro promotor and gus gene
Fig. 2 drives the result of gus gene specifically expressing in cotton fibre for promotor GhMYB109Pro
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and used restriction enzyme reagent is all available from TaKaRa company.
Vegetable material:
At the cotton florescence, gather petal, bud, flower and young bell, to gather the preceding 3 days ovule of blooming, florescence ovule respectively and spend 1-3 days the ovule quick-frozen in liquid nitrogen in back with the utensil of sterilization ,-80 ℃ of preservations are standby.
The clone of embodiment 1, cotton fibre specific promoter GhMYB109Pro
Adopt Genomic Walker method clone cotton fibre specific promoter GhMYB109Pro, concrete grammar may further comprise the steps:
1, reference literature (Paterson A H, Brubaker C L, Wendel J F.A rapid methodfor extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLPor PCR analysis.Plant Mol Biol Rep, 11 (2): the method 122-127.1993) is extracted the genomic dna of cotton.
2, get the genomic dna of the cotton of four parts of (every part 2.5 μ g) steps 1 extractions, cut 12-24 hour with restriction enzyme EcoR V, Pvu II, Sca I and Stu I enzyme respectively.Enzyme is cut product carries out four kinds of enzymes of genomic dna being cut product carrying out purifying after 0.5% agarose gel electrophoresis detects, with purified product be dissolved in 20 μ LTE (10/0.1, pH7.5) in.
3, the sequence of the GenomicWalker Adaptor of design cloning promoter GhMYB109Pro nucleotide sequence, sequence is as follows:
5’-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3’；
3’-H
2N-CCCGACCA-PO
4-5’
Four kinds of enzymes of the purified genomic dna in the step 2 are cut product and are respectively got 4 μ L, add following substances respectively: 1.9 μ L Genomic Walker Adaptor (25 μ M), 1.6 μ L 10 * connection damping fluid and 0.5 μ L T
4Dna ligase (6units/ μ L).16 ℃ connect 12-24 hour earlier, again 70 ℃ of 5 minutes stopped reaction.Add 72 μ L TE (10/1, pH 7.5) then in every kind of reaction product, the enzyme that obtains EcoR V, Pvu II, ScaI and Stu I cotton genomic dna is cut the DNA library.
4, the 5 ' terminal sequence design primer according to GhMYB109Pro increases, and primer sequence is as follows:
AP1：5′-GTAATACGACTCACTATAGGGC-3’
GW1：5′-GAAGTGTGACTGTGTTGTTAAGAACCTG-3’
It is template that four kinds of enzymes getting the cotton genomic dna that step 3 obtains are cut each 1 μ L of DNA, each adds following substances: 40 μ L deionized waters, 5 μ L, 10 * PCR damping fluid, 1 μ L dNTP (10 μ M), 1 μ L AP1 (10 μ M), 1 μ L GW1 (10 μ M) and 1 μ L Ex Taq (5U/ μ L, Takara company) are as the reaction system of primary PCR.Carry out primary PCR reaction with MJ PCR instrument, reaction conditions is: 94 ℃ of 25s of elder generation, 72 ℃ of 3min, totally 7 circulations; 94 ℃ of 25s again, 67 ℃ of 3min, totally 32 circulations; Last 67 ℃ of 7min.After reaction finishes, the PCR product is carried out 1.5% agarose gel electrophoresis detect.
5, the 5 ' terminal sequence design primer according to GhMYB109Pro increases, and primer sequence is as follows:
AP2：5’-ACTATAGGGCACGCGTGGT-3’
GW2：5’-GAGTAACTTGTCTTCCTCCATTGCCCATAAT-3’
Four kinds of primary PCR products of step 4 are diluted 50 times, respectively getting 1 μ L is template, each adds following substances: 40 μ L deionized waters, 5 μ L, 10 * PCR damping fluid, 1 μ L dNTP (10 μ M), 1 μ L AP2 (10 μ M), 1 μ L GW2 (10 μ M), 1 μ L Ex Taq (5U/ μ L) is as the reaction system of secondary PCR.Carry out secondary PCR reaction with MJ PCR instrument, reaction conditions is: 94 ℃ of 25s of elder generation, 72 ℃ of 3min, totally 5 circulations; 94 ℃ of 25s again, 67 ℃ of 3min, totally 20 circulations; Last 67 ℃ of 7min.After reaction finishes, the PCR product is carried out 1.5% agarose gel electrophoresis to be detected, recovery is the purpose fragment that the length of template amplification is about 3kb with the enzyme Qie Wenku of Sca I cotton genomic dna, with Wizard PCR Preps DNA Purification System (Promega company) purifying, with purified purpose fragment cloning in carrier pGEM-T Easy (Promega company), to contain the segmental recombinant vectors Transformed E of purpose .coli DH5 α competent cell, after the white screening of indigo plant, primer VT7 and Sp6 with carrier self carry out pcr amplification, select positive colony upgrading grain, check order with BeckmanCEQ2000, sequencing result shows that the purpose fragment has SEQ ID №: 1 nucleotide sequence, will contain the recombinant vectors called after pGEM-T-GhMYB109Pro of promotor GhMYB109Pro.
The functional verification of embodiment 2, promotor GhMYB109Pro
According to the dna sequence dna design primer of promotor GhMYB109Pro, primer sequence is as follows:
The GW2+XbaI:(upstream primer) 5 '-
TCTAGAGAGTAACTTGTCTTCCTCCATTGCCCATAAT-3 '
(band underscore part base is a restriction enzyme Xba I recognition site)
The GW10+SalI:(downstream primer) 5 '-ATA
GTCGACTGTGTCAAAGACGACTACTTGAG-3 ' (band underscore part base is a restriction enzyme Sal I recognition site)
With the recombinant vectors pGEM-T-GhMYB109Pro that contains promotor GhMYB109Pro is template, under the guiding of primer GW2+XbaI and primer GW10+SalI, pcr amplification promotor GhMYB109Pro, and make the sequence two ends add the recognition site of restriction enzyme Xba I and Sal I respectively.With the promotor GhMYB109Pro of pcr amplification with restriction enzyme Xba I be connected with the carrier pBI101.2 that has gus gene (Clontech company) that cuts through same enzyme enzyme after Sal I carries out double digestion, to connect product Transformed E .coli DH5 α competent cell, with the resistant panel screening positive clone that contains kantlex, obtain containing the fusion vector of promotor GhMYB109Pro and gus gene, called after pBI101.2-GhMYB109-GUS, the part physical map of this carrier as shown in Figure 1.Again the plasmid vector pBI101.2-GhMYB109-GUS that makes up is transformed Agrobacterium AGL1,, after identifying, obtain transgene cotton with agrobacterium tumefaciens dip method converting cotton.Getting transgene cotton spends back three days ovule to spend preceding 3 days with the florescence, spend 1-3 days the ovule in back to carry out the active detection of GUS, detection method reference literature (Weigel, D.andGalzebrook, J.Arabidopsis:A Laboratory Manual (Cold Spring Harbor, NY:ColdSpring Harbor Laboratory Press), p.243-248.2002).With resin vegetable material is carried out embedding then, the section (slice thickness is 10 μ m) under dark-field, observe, the result as shown in Figure 2, A is that the GhMYB109Pro promotor only drives gus gene specifically expressing in cotton fibre among Fig. 2; B is the wild-type contrast.Show that promotor GhMYB109Pro is the special promotor of fiber, it only drives gene specifically expressing in cotton fibre.
Sequence table
<160>1
<210>1
<211>2080
<212>DNA
＜213〉Gossypium upland cotton (Gossypium hirsutum L.)
<400>1
acgtcgcatg?ctcccggccg?ccatggcggc?cgcgggaatt?cgatttgtgt?caaagacgac 60
tacttgagac?tgtcaaaacc?tatacaacaa?aaaaaaatta?aaatcattct?cttaatttac 120
ccatttgaaa?tggatgccac?acataccatt?tgaaatatct?taaagagaac?tttcatttga 180
caccaccaat?gttgtcggaa?cctcaccaga?atttagtaac?tatactaacc?caaatgaaat 240
acatgacttt?cacgaaaaat?agattaaaac?acaagaaatc?taaaccaaag?ttatccttaa 300
tgtctaaaat?ccactagtta?tttgcccttc?ctttttcttt?cttcctcttc?tctatggtta 360
gcttctacca?aattgtcaag?atcatcatct?aacataaaaa?taccgtaaat?gattcttttc 420
tctttttttc?tagtctattc?tttttattag?aaaaaactat?aacctacata?ataaagttga 480
gcttgaaaag?aaaattggct?aatggaccac?atatttaaat?aatacccatt?taaaaatcat 540
gaatattaca?aatgtaataa?cacctgttaa?aggaaaggag?gtcacaaaca?aaagtgattt 600
gtacagagtt?ttttatcaat?gcaaagttgg?cgatatgata?tagcattgaa?taaaaccaat 660
aaccatttct?ttctctctct?atatatatac?acgtctttag?gatcttttat?gatgtacaaa 720
tttttcataa?ttttaatttt?taaattatcg?tggaggtgtt?acatgaggag?aagatcccta 780
taacactcaa?gccaaagcca?gatgagtcat?gaaatgatgt?ttagtatgtt?gcataatcct 840
aaatacctag?taaatataca?aggaaatgtc?attatttgtt?tactatttta?ctggaaaaat 900
attcctgttg?tatctatgtg?gctatgccat?caacttgtta?tcatctttca?tacaatgttt 960
tcacaatgtg?aagcacaagg?ctatcaacct?ttgtaaaaaa?ttgtatctat?cttgcctttt 1020
cttcttaact?gattggggta?taagtgcaaa?ttctatgtta?catggactcg?tctatgagta 1080
ttgaatatgg?gtaacacgaa?ataagcaaac?cctagtaaat?aagggacctc?ctaatggtgt 1140
gttgtaagct?taggtggaag?tgtagaaaag?aggaaaggga?aattgattgt?cccaaaaaag 1200
taagtgatgg?atttccgaat?ttagataaat?gtgcatttgc?taaggcatta?cggcccctac 1260
gtgcacggat?gtgatatggc?agttagttgt?tggtcataaa?tatctttgca?ttttctagtt 1320
tgagttcaag?tagtggggtt?tggatgagtg?attgggtgcg?gtgcagtgta?gtgcaatgcg 1380
tttagtttac?tttttatctc?acactacagt?atcattacaa?tatataatct?caccgtcact 1440
gctattttta?cactaaccgc?aagaaaatgt?accatctcat?acaaactaag?ccttagactt 1500
ataaactttt?aaaactcgca?tctaattcat?ttaattttaa?aaaaattaat?agattatatt 1560
tgaattacaa?catttgtaat?gtattctctt?aatccggaag?cgatgaactc?ttttgtttat 1620
atatagaaaa?aagaacacaa?taatttgggt?tatggatttt?ttttaacttt?aacatttacc 1680
attcatttta?atcctggaat?taatttagca?tgaatctaat?taagagagct?cacaaaatat 1740
gtgaagatgt?gtgcaaaagc?tattaagagt?aatttatcca?aatctgccat?ccatcatcat 1800
ctcatcatta?cctccatata?tacaaccaga?atcctacact?tacagctaag?aaaggaatat 1860
atatatattt?aaaacagcat?ttgtgtttca?aaaaaatcta?atccatatac?tgcaaaatcc 1920
ttgtgcatct?tcttcagagc?aaacggcaac?aactccaaca?aatgtccatg?aaaaaagaag 1980
gtgaaattct?atacaaaaag?ggattatggg?caatggagga?agacaagtta?ctcaatcact 2040
agtgaattcg?cggccgcctg?caggtcgacc?atatgggagg 2080
Claims (6)
1. cotton fiber specifically-expressed promotor, its base sequence is shown in SEQ ID NO:1.
2. the expression vector that contains the described promotor of claim 1.
3. the transgenic cell line that contains the described promotor of claim 1.
4. the host bacterium that contains the described promotor of claim 1.
5. the described promotor of claim 1 is in the application in the specifically expressing in cotton fibre of driving purposes gene.
6. the application of the described promotor of claim 1 in cultivating new cotton variety.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100681884A CN100404681C (en) | 2005-04-29 | 2005-04-29 | Specific expression starter and its use for cotton fibre |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100681884A CN100404681C (en) | 2005-04-29 | 2005-04-29 | Specific expression starter and its use for cotton fibre |
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| CN1854297A CN1854297A (en) | 2006-11-01 |
| CN100404681C true CN100404681C (en) | 2008-07-23 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB2005100681884A Expired - Fee Related CN100404681C (en) | 2005-04-29 | 2005-04-29 | Specific expression starter and its use for cotton fibre |
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| CN (1) | CN100404681C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002501A (en) * | 2010-11-10 | 2011-04-06 | 北京大学 | Promoter of cotton KCS 12 gene and application thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503691B (en) * | 2008-02-04 | 2012-05-02 | 中国科学院上海生命科学研究院 | Plant epidermal hair specific expression promoter F1F1 and use thereof |
| CN101967483B (en) * | 2010-09-26 | 2013-03-27 | 北京大学 | Promoter of cotton FDH gene and application thereof |
| CN104611338B (en) * | 2015-02-28 | 2018-11-16 | 中国科学院遗传与发育生物学研究所 | GhGL3 gene promoter, carrier and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446916A (en) * | 2002-03-25 | 2003-10-08 | 中国科学院遗传与发育生物学研究所 | Carrier for expressing specificity of bec gene cotton fiber |
| WO2004053129A1 (en) * | 2002-12-12 | 2004-06-24 | The Australian National University | Methods and means for modulating cellulose biosynthesis in fiber producing plants |
-
2005
- 2005-04-29 CN CNB2005100681884A patent/CN100404681C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446916A (en) * | 2002-03-25 | 2003-10-08 | 中国科学院遗传与发育生物学研究所 | Carrier for expressing specificity of bec gene cotton fiber |
| WO2004053129A1 (en) * | 2002-12-12 | 2004-06-24 | The Australian National University | Methods and means for modulating cellulose biosynthesis in fiber producing plants |
Non-Patent Citations (1)
| Title |
|---|
| 外源纤维改良基因对棉花纤维品质的影响研究. 张震林等人.华中农业大学学报，第23卷第2期. 2004 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002501A (en) * | 2010-11-10 | 2011-04-06 | 北京大学 | Promoter of cotton KCS 12 gene and application thereof |
Also Published As
| Publication number | Publication date |
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| CN1854297A (en) | 2006-11-01 |
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