CN102453718B - Plant bidirectional promoter BIGDB2 - Google Patents
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Abstract
The invention discloses a plant bidirectional promoter BIGDB2. The nucleotide sequence of the promoter is shown as a sequence 1 in a sequence table. Experiments prove that: the BIGDB2 can promote glucuronidase (GUS) expression after being inserted into the space between GUS and luciferase (LUC) in a vector pMOA34-G/L, and the BIGDB2 can also promote the GUS expression after being inserted into the space between the GUS and the LUC in the pMOA34-G/L reversely, so that the BIGDB2 has a bidirectional promoting function in arabidopsis thaliana.
Description
Technical field
The present invention relates to clone and the application of a kind of plant bidirectional promoter BIGDB2.
Background technology
Making plant obtain new proterties by the importing foreign gene can genetic stability be the final purpose of plant genetic engineering also.Caulimovirus CaMV 35S promoter is used by people as stable, exogenous promoter efficiently always widely.Yet because the appearance of tumor-necrosis factor glycoproteins can cause the gene silencing phenomenon, especially carry out a plurality of genes when importing in the transgenosis process, this phenomenon can be more serious, thereby had a strong impact on the acquisition of transfer-gen plant and offspring's stability.And carrying out two or more genes when importing, and need the identity of these expression of gene amounts, expression time and position usually, and then guarantee that transgene exercises normal function, use 35S promoter to be difficult to the requirement that reaches such.These all are problem demanding prompt solutions in the plant genetic engineering.
The gene silencing phenomenon people that cause for transgenosis have had more deep understanding, cause that transgene silencing has multiple factor, wherein topmost is exactly the generation of tumor-necrosis factor glycoproteins, when especially a plurality of genes import, the more artificial situation that has caused a plurality of 35S promoters to insert does not still have effective solution so far to this.Development along with genomics, multiple biological gene group order-checking is finished, it is found that the existence of bidirectional promoter through bioinformatic analysis, this carries out transgeneic procedure for we utilize the bidirectional promoter of plant self, provides possibility thereby avoid polygene to insert the gene silencing phenomenon that causes.
Summary of the invention
The object of the present invention is to provide a kind of dna fragmentation, derive from Arabidopis thaliana.
The nucleotide sequence of dna fragmentation provided by the invention is following 1) or 2) or 3):
1) nucleotide sequence shown in the sequence 1 in the sequence table;
2) under the rigorous condition of height with described 1) nucleotide sequence hybridization and have the nucleotide sequence of promoter activity;
3) with described 1) nucleotide sequence have the homology more than 65% and have the nucleotide sequence of promoter activity.
Sequence 1 in the sequence table is made up of 319 deoxyribonucleotides, is the intergenic sequence of arabidopsis gene AT4G12600 and AT4G12590.
The rigorous condition of above-mentioned height is meant, with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.2,7%SDS) in, 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7%SDS, isotope-labeled nucleotide fragments), 65 ℃ of hybridization 12hr; Abandon hybridization solution, (20mmol/L sodium phosphate buffer, pH7.2 5%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid I; (20mmol/L sodium phosphate buffer, pH7.2 1%SDS), wash film 30min for 65 ℃ to add film washing liquid II.
Dna fragmentation of the present invention (promoter active fragment) also comprise with from the segmental nucleotide sequence complementary nucleotide sequence of sequence in the sequence table 1.Term used herein " complementary " mean follow that basepairing rule produces meaning.
Those of ordinary skills can adopt known method at an easy rate, and the method for orthogenesis and point mutation is for example suddenlyd change to the nucleotide sequence of regulation and control fragment of the present invention.Those are through manually modified, has the nucleotide sequence 70% that separates the regulation and control fragment that obtains with the present invention or the Nucleotide of higher homology, as long as kept expressing the promoter activity of target gene, all be to be derived from nucleotide sequence of the present invention and to be equal to sequence of the present invention.
Term used herein " homology " refers to the sequence similarity with the natural acid sequence." homology " comprises that the nucleotide sequence with regulation and control fragment of the present invention has preferably 75% or higher, more preferably 85% or higher, even more preferably 90% or higher, and most preferably 95% or the nucleotide sequence of higher identity.Homology can be with the naked eye or computer software estimate.The software that uses a computer, the homology between two or more sequences can be used per-cent (%) expression, and it can be used for estimating the homology between the correlated series.
The recombinant vectors that contains above-mentioned dna fragmentation also belongs within protection scope of the present invention.
Above-mentioned recombinant vectors is that above-mentioned dna fragmentation inserts the recombinant vectors that the multiple clone site of carrier pMOA34-G/L is made;
Above-mentioned carrier pMOA34-G/L inserts the carrier that the multiple clone site of pMOA34 carrier obtains respectively with gus gene and LUC gene.
The transgenic cell line that contains above-mentioned dna fragmentation also belongs within protection scope of the present invention.
The reorganization bacterium that contains above-mentioned dna fragmentation also belongs within protection scope of the present invention.
Above-mentioned dna fragmentation is also belonging within protection scope of the present invention as the application in the promotor.
Further, above-mentioned promotor is a bidirectional promoter.
Above-mentioned application specifically can be that above-mentioned dna fragmentation is in the application of driving purposes gene in plant interior expression.
Above-mentioned plant is dicotyledons or monocotyledons, as Arabidopis thaliana or tobacco.
Experiment showed, insert BIGDB2 of the present invention between GUS among the carrier pMOA34-G/L and the LUC after, BIGDB2 can start GUS to express; Can start GUS equally after oppositely inserting BIGDB2 between GUS among the pMOA34-G/L and the LUC and express, illustrate that BIGDB2 has the two-way startup function in Arabidopis thaliana.
Description of drawings
Fig. 1 is a pMOA34-G/L-BIGDB2 plant binary carrier collection of illustrative plates.
Fig. 2 is a transgenic arabidopsis GUS coloration result, and A is the cotyledon period seedling, and B is the seedling of two true leaves, and C is an inflorescence.
Fig. 3 is a transgenic arabidopsis GUS analytical results.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be ordinary method.
The acquisition of embodiment 1, promotor and detection thereof
One, the acquisition of promotor
According to the result of bioinformatic analysis, there are some potential two-way startup subsequences in the arabidopsis gene group.With the arabidopsis thaliana genomic dna is template, utilize forward primer (BIGDB2-F:5 '-GAGCTCTAGA
GCGGC CGCTCTAAACCCTAATTATCTCTCTTTCTCGTC-3 ') and reverse primer (BIGDB2-R:5 '-ACGC
GTCGACCCCGGGCTTCTTCTTCGTCTACGAGAGTTCTTTG-3 ') amplification, amplified production carries out agarose electrophoresis to be separated, reclaim and be connected on pEASY-T1 (the full formula King Company) cloning vector, among the transformed into escherichia coli XL1-BLUE (Agilent Technologies-Stratagene Products), determine that after checking order cloning vector is correct, the nucleotide sequence of amplified production (called after BIGDB2) is shown in sequence in the sequence table 1.
Two, the structure of recombinant expression vector
1, the structure of the intermediate carrier of tape label gene (pMOA34-G/L)
With pCambia1300-221 (CAMBIA company) carrier is masterplate, utilizes primer GUS-F:5 '-ACGC
GTCG ACATGTTACG TCCTGTAGAAACCCCAAC-3 ' and GUS-R:5 '-CAGCCG
ACCGGTTCATTGTTTGCCTCC CTGCTGC-3 ' amplifies the gus gene of 1812bp, after order-checking, be connected to by SalI and AgeI double digestion that (document of putting down in writing this material is Barrell on the pMOA34 carrier, P.J.and Conner, A.J. (2006) Minimal T-DNA vectors suitable for agricultural deployment of transgenic plants, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity), be built into the pMOA34-G intermediate carrier.
The product LUC gene of pGL3-Enhancer Vector (Promaga company) behind XbalI (mend flat) and XhoI double digestion and intermediate carrier pMOA34-G are connected with carrier behind the XhoI double digestion through SpeI (mend and put down), and the expression vector pMOA34-G/L of two marker gene GUS and LUC is carried in acquisition.
2, the structure of expression vector pMOA34-G/L-BIGDB2
Verify the plant expression vector pMOA34-G/L that the step 1 of correct cloning vector and step 2 obtains with SalI and NotI double digestion step 1, reclaim after the T4DNA ligase enzyme connects and obtain pMOA34-G/L-BIGDB2 plant expression vector (Fig. 1).
3, the structure of expression vector pMOA34-G/L-BIGDB2 (oppositely)
With BIGDB2 sequence reverses its direction among the pMOA34-G/L-BIGDB2, be built into pMOA34-G/L-BIGDB2 (oppositely) carrier.
With the cloning vector that has the correct sequence of BIGDB2 gene in the step 1 is template, utilizes primer BIGDB2-F2:5 '-GAGC
GTCGACTCTAAACCCTAATTATCTCTCTTTCTCGTC-3 ' and BIGDB2-R2:5 '-ACGC
GCGGCCGCCTTCTTCTTCGTCTACGAGAGTTCTTTG-3 ', obtain the reverse fragment of BIGDB2, after checking order, be connected to the plant expression vector pMOA34-G/L that the step 1 of step 2 obtains, obtain pMOA34-G/L-BIGDB2 (oppositely) plant expression vector with SalI and NotI double digestion.
Three, transform plant and cultivation
1, imports Agrobacterium
The method that transforms by electric shock imports the pMOA34-G/L-BIGDB2 positive colony in the step 2 among the Agrobacterium EHA105, picking reorganization Agrobacterium mono-clonal bacterium colony changes in the 3-4ml LB nutrient solution and (contains 100 μ g/ml spectinomycins and 50 μ g/ml Rifampins), and 28 ℃ are shaken bacterium and spend the night.The bacterium liquid that cultivation obtains is transferred in 500ml LB (containing 100 μ g/ml spectinomycins and 50 μ g/ml Rifampins), and 28 ℃ are shaken bacterium and spend the night.When bacterium liquid OD600 value is 1.0, collect bacterium liquid and go into centrifuge tube, 4000rpm, 20min collect thalline.With the resuspended thalline of freshly prepared following solution: solvent is a water, solute is polyoxyethylene nonylphenol ether (NP-40) and sucrose, wherein, the concentration of polyoxyethylene nonylphenol ether (NP-40) (Fluka 74385) be 0.05% and the mass percentage concentration of sucrose be 5%.
2, arabidopsis thaliana transformation
Choose the healthy and strong plant of the environmental Arabidopis thaliana (the Biological resources center (ABRC) of Ohio State Univ-Columbus USA) of wild-type col-0 of bolting phase, the Agrobacterium that step 1 is obtained by vacuum infiltration method imports during Arabidopis thaliana spends, covered 1 day with plastic film, keep moistening.The Arabidopis thaliana plant that to handle then is put under 22～25 ℃ the illumination condition and makes its normal growth.Results seed, drying conditions are preserved stand-by down.
Four, genetic expression detects
1, the expression analysis of GUS, LUC in the transgenic arabidopsis
Choose in the step 3 10 not transgenic arabidopsis seeds of homophyletic system, on the 1/2MS substratum, sprout and growth, seedling and the inflorescence of getting cotyledon period seedling, two true leaves respectively carry out the GUS staining analysis, the GUS dyeing process is referring to Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions:b-glucuronidase as a sensitive and veratile gene fusion maker in higher plants.EMBO J 6:3901-7, analytical results such as Fig. 2, gus gene obtains expressing in the transfer-gen plant.
Choosing gus gene has the transgenic arabidopsis of expression, sprays the method for substrate by blade and carries out the LUC luminescence analysis, and the result does not observe fluorescence as scheduled.Insensitive for the LUC of tissue expression beyond the detection blade in view of the LUC luminescence analysis method that we select, we with BIGDB2 sequence reverses its direction among the pMOA34-G/L-BIGDB2, are built into pMOA34-G/L-BIGDB2 (oppositely) carrier in step 2.Change the mediation of this plant expression vector by agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain EHA105 over to Arabidopis thaliana, after obtaining the transgenic arabidopsis plant, carry out the GUS staining analysis, analytical results such as Fig. 3, gus gene obtains expressing in the transfer-gen plant.
Claims (5)
1. dna fragmentation, its nucleotide sequence is the nucleotide sequence shown in the sequence 1 in the sequence table.
2. the recombinant vectors that contains the described dna fragmentation of claim 1.
3. the transgenic cell line that contains the described dna fragmentation of claim 1.
4. the reorganization bacterium that contains the described dna fragmentation of claim 1.
5. the described dna fragmentation of claim 1 is as the application in the promotor;
Described promotor is a bidirectional promoter; Described application is that the described dna fragmentation of claim 1 is in the application of driving purposes gene in plant interior expression;
Described plant is an Arabidopis thaliana.
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| CN 201010521550 CN102453718B (en) | 2010-10-27 | 2010-10-27 | Plant bidirectional promoter BIGDB2 |
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| CN106893723B (en) * | 2015-12-17 | 2020-01-17 | 中国科学院遗传与发育生物学研究所 | Plant bidirectional promoter and application thereof |
| CN105624163B (en) * | 2016-04-06 | 2018-07-06 | 中国农业科学院生物技术研究所 | A kind of bidirectional promoter and its application from cotton |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6388170B1 (en) * | 2000-04-07 | 2002-05-14 | University Of Kentucky Research Foundation | Bidirectional promoters and methods related thereto |
| CN1936002A (en) * | 2005-09-19 | 2007-03-28 | 中国农业大学 | Method for separating bidirectional promoter and its use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6388170B1 (en) * | 2000-04-07 | 2002-05-14 | University Of Kentucky Research Foundation | Bidirectional promoters and methods related thereto |
| CN1936002A (en) * | 2005-09-19 | 2007-03-28 | 中国农业大学 | Method for separating bidirectional promoter and its use |
Non-Patent Citations (4)
| Title |
|---|
| Quan Wang et al..Searchong for bidirectional promoters in Arabidopsis thaliana.《BMC Bioinformatics》.2009,第10卷1-10. |
| Searchong for bidirectional promoters in Arabidopsis thaliana;Quan Wang et al.;《BMC Bioinformatics》;20090130;第10卷;1-10 * |
| 双向启动子构建及在毛白杨中瞬时表达研究;张春晓等;《北京林业大学学报》;20070131;第29卷(第1期);119-122 * |
| 张春晓等.双向启动子构建及在毛白杨中瞬时表达研究.《北京林业大学学报》.2007,第29卷(第1期),119-122. |
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