CN104628840B - Plant stress tolerance related protein VrDREB2A, coding gene and application thereof - Google Patents
Plant stress tolerance related protein VrDREB2A, coding gene and application thereof Download PDFInfo
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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Abstract
The invention relates to the field of genetic engineering, and provides a plant stress tolerance related protein VrDREB2A, a coding gene VrDREB2A and application thereof. The amino acid sequence of the plant stress tolerance related protein VrDREB2A is shown in SEQ ID No. 1. Experiments prove that the VrDREB2A gene is introduced into arabidopsis thaliana by an agrobacterium transformation method, and a transgenic arabidopsis thaliana strain with remarkably improved salt resistance and drought resistance is screened by means of hygromycin primary screening, molecular detection, germination salt stress, later drought stress and the like. The invention has important value for cultivating salt-tolerant and drought-tolerant transgenic leguminous crops.
Description
Technical field
The present invention relates to genetic engineering field, specifically, be related to a kind of plant stress tolerance correlative protein VrDREB2A and
Its encoding gene and application.
Background technology
The serious growth hairs that govern plant such as abiotic stress is for example saline and alkaline, arid, low temperature, ozone, radiation and heavy metal
Educate, be the restraining factors of crop yield and quality.During long-term evolution, plant by the tolerance of cellular and molecular level and
The collaboration response of plant integral level forms a series of perfect Stress response mechanism.
DREB class transcription factor genes belong to AP2/EREBP genoids family, can be with adversity gene promoter region
DRE cis-acting elements combines, and participates in abiotic stress responsing reaction, strengthens the resistance of plant.There is scientist to demonstrate plan
Southern mustard DREB2A is dehydrated, high-salt stress induction, while DREB2A transcription factors can activate with DRE cis-acting elements one
The expression of serial target gene, such as rd29A, cor15a, rd17 and kin1, the product of these gene expressions are converse in Genes For Plant Tolerance
Different functions is played in answering, during this description of test DREB2A albumen directly participates in plant stress-resistance.Improving crop
To in the molecular breeding of environment-stress, DREB2A albumen is being imported into plant to the resistance that can directly improve plant.
Mung bean (Vigna radiata (Linn.) Wilczek.), belongs to pulse family, and in China, existing bimillennial cultivation is gone through
History.It is the important cereal crops in China.Because it has important nutritive value and medical value, as important functional form
Food is developed.In recent years, with the adjustment of crop mix and the change of people's diet structure, demand of the people to mung bean
Amount increasingly increases, and cultivated area soldier's year expands.Research show abiotic stress have a strong impact on Seeds Germination of Vigna radiata, growth of seedling,
Yield etc..In consideration of it, further investigation mung bean DREB2A participates in the mechanism of abiotic stress, it is stronger to filtering out drought resistance
The screening tool of kind and Drought-resistant Breeding material is of great significance, and solves the optimal way that mung bean produces under drought condition
Footpath it.
The content of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of plant stress tolerance correlative protein
VrDREB2A and its encoding gene and application.
In order to realize the object of the invention, present invention firstly provides a kind of plant stress tolerance correlative protein VrDREB2A, source
In mung bean (Vigna radiata (Linn.) Wilczek.), its amino acid sequence is as shown in SEQ ID No.1, or sequence warp
Replace and/or lack and/or add one or several amino acids formed amino acid sequences with equal function.
The replacement of one or several amino acid residues and/or missing and/or to be added to no more than 10 amino acid residual
The replacement of base and/or missing and/or addition.
Present invention also offers a kind of gene VrDREB2A related to plant stress tolerance, the foregoing plant of gene codified
Thing stress tolerance correlative protein VrDREB2A.
Further, the gene contains the nucleotide sequence as shown in 3-1160bp in SEQ ID No.2.
Further, when the gene is used to build recombinant vector, engineering bacteria, transgenic cell line or expression cassette,
Restriction enzyme site can be built in its end according to this area routine techniques.
In the specific embodiment of the present invention, the 1-6 positions nucleotides that the present invention provides a 5 ' ends is
NcoI restriction enzyme sites, 1161-1166 positions nucleotides are the nucleotide sequences of SpeI restriction enzyme site, and the nucleotide sequence is such as
Shown in SEQ ID No.2.
Present invention also offers the primer pair for expanding forementioned gene, the nucleotide sequence of the primer pair is respectively such as
Shown in SEQ ID No.3 and SEQ ID No.4.
Present invention also offers the carrier containing forementioned gene, the carrier is by forementioned gene insertion vector
The recombinant expression carrier obtained between pCAMBIA1302 NcoI and SpeI restriction enzyme sites.
Present invention also offers the engineering bacteria containing forementioned gene.
Present invention also offers application of the forementioned gene in cultivating genetically modified plants and improving resistance of reverse.
Further, the application is to utilize foregoing recombinant expression carrier or engineering bacteria will be described related to plant stress tolerance
Gene VrDREB2A import purpose plant obtain resistance of reverse raising genetically modified plants.
Wherein, the resistance of reverse is drought tolerance and/or salt tolerance.
The plant is dicotyledon or monocotyledon, and the dicotyledon is preferably arabidopsis, such as
Columbia Arabidopsis thaliana ecotypes.
The beneficial effects of the present invention are：
The experiment proves that the present invention screens a transcription factor gene from legume mung bean
VrDREB2A, VrDREB2A is imported by arabidopsis by Agrobacterium-mediated Transformation method, by hygromycin primary dcreening operation, Molecular Detection, germination period
The means such as salt stress and Drought in later stage stress, screen salt resistance and transgenic arabidopsis strain that drought-resistant ability significantly improves.
The present invention is for cultivating salt tolerant, drought-resistant transgenosis legume has important value.
Brief description of the drawings
Fig. 1 is that carrier pCAMBIA1302-VrDREB2A of the present invention builds flow chart.
Fig. 2 is T1For the PCR testing results of transgenic arabidopsis；
Wherein：M：1kb DNA ladder, from down to up respectively 250bp, 500bp, 750bp, 1000bp, 1500bp,
2000bp；1：Negative control, H2O；3；Negative control, wildtype Arabidopsis thaliana；3：Positive control, pCAMBIA1302-VrDREB2A
Carrier；4-13 and 16：PCR identifies positive plant；14th, 15 and 17：PCR identifies negative plant.
Fig. 3 is T3For transgenic arabidopsis under normal growing conditions, the lower growth 7d's of 210mM and 220mM NaCl stress
Germination rate statistical chart.
Fig. 4 is T3For transgenic arabidopsis drought stress processing 14d survival rate statistical chart.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Experimental method used in following examples is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following examples.
Quantitative experiment in following examples, it is respectively provided with and repeats to test three times, results averaged.
In green No. 2 mung bean seeds：Cultivate that (seed is freely given to the public from Institute of Crop Science, Chinese Academy of Agricultural Science
Give).
Columbia Arabidopsis thaliana ecotypes (Columbia ecotype arabidopsis (columbia stain of
Arabidopsis thaliana as genetic background)：Purchased from salk companies.
Agrobacterium EHA105 is documented in Days to heading 7, a major quantitative locus
determining photoperiod sensitivity and regional adaptation in rice,He Gao et
Al, PNAS, 2014,111 (46), in 16337-16342, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science
.
The clone of embodiment 1VrDREB2A genes
1st, specific primer is designed to (VrDREB2A-5 ' and VrDREB2A-3 '), is synthesized by Invitrogen companies.
VrDREB2A-5’:5’-ATGGGTGCTTATGATCAAGTTTCTGTG-3’；
VrDREB2A-3’:5’-TCATTCCTTGCTTGCTAGCATTTCCTTTG-3’。
2nd, the RNA, reverse transcription cDNA of mung bean (Vigna radiata (Linn.) Wilczek.) whole plant are extracted；
3rd, using the cDNA of step 2 as template, with the specific primer of step 1 to VrDREB2A-5 ' and VrDREB2A-3 ' enter
Performing PCR expands, and reclaims pcr amplification product.
4th, the pcr amplification product of step 3 is sequenced.
Sequencing result is, the sequence of the gene of the PCR primer is the sequence 2 in sequence table, and code area is sequence 2 from 5 '
The 3-1166 positions nucleotides of end, it is NcoI restriction enzyme sites from the 1-6 positions nucleotides of 5 ' ends, 1161-1166 positions core
Thuja acid is SpeI restriction enzyme site.It is VrDREB2A by the unnamed gene, the albumen of the gene code is named as VrDREB2A, should
The amino acid sequence of albumen is that sequence 1 is made up of 385 amino acid residues shown in the sequence 1 in sequence table.
5th, above-mentioned PCR primer is inserted into pMD18T-simple carriers (being purchased from TaKaRa bio-engineering corporations), obtains carrier
PMD18T-simple-VrDREB2A, sequencing, is as a result by the sequence in sequence table for carrier pMD18T-simple-VrDREB2A
Row 2 insert the carrier that pMD18T-simple carriers obtain.
The acquisition and functional study of the transgenic arabidopsis of embodiment 2
First, the acquisition of transgenic arabidopsis
1st, the structure of recombinant vector (pCAMBIA1302-VrDREB2A)
Building process is as shown in Figure 1.
(1) restriction enzyme NcoI and SpeI double digestion carrier pMD18T-simple-VrDREB2A is used, reclaims small pieces
Section.
(2) (Center for the are purchased from restriction enzyme NcoI and SpeI double digestion pCAMBIA1302
Application of Molecular Biology to International Agriclture, www.cambia.org),
Reclaim carrier framework.
(3) small fragment of step (1) is connected with the carrier framework of step (2), obtains connection product, by the connection product
Bacillus coli DH 5 alpha is converted, transformant is obtained, extracts the plasmid of transformant, send to sequencing, be as a result by sequence table for the plasmid
In sequence 2 insert obtained recombinant vector between pCAMBIA1302 NcoI and SpeI restriction enzyme sites, the plasmid is named as
pCAMBIA1302-VrDREB2A。
2nd, the acquisition of transgenic arabidopsis
(1) preparation of Agrobacterium competent cell
Picking Agrobacterium EHA105 single bacterium colonies are inoculated in 100ml YEB fluid nutrient mediums, 220rpm, 28 DEG C of shaken cultivations
To OD600=0.5；Sterile centrifugation tube is transferred to, 5000rpm centrifugation 5min, supernatant is removed, adds the 0.15M of 10ml precoolings CaCl2
The aqueous solution, gently suspension cell, places 20min on ice；4 DEG C, 5000rpm centrifugation 5min, remove supernatant, add containing for 4ml precoolings
10% glycerine (volumn concentration) and 0.15M CaCl2The aqueous solution, gently suspend；Obtain agrobacterium suspension (EHA105
Competent cell), it is sub-packed in sterile eppendorf tubes, often the μ l of pipe 200, quick-frozen 1min, freezes in -70 DEG C in liquid nitrogen.
(2) pCAMBIA1302-VrDREB2A converts Agrobacterium EHA105
Take 1 μ g pCAMBIA1302-VrDREB2A obtained above to add in 200 μ l EHA105 competent cells, mix,
Static 5min；Quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 5min, add 1ml YEB fluid nutrient mediums, 28 DEG C, 150rpm vibration trainings
Support 4h；5000rpm centrifuges 3min, abandons supernatant, adds 0.1ml YEB fluid nutrient mediums, again suspension cell；It is coated on and contains 50 μ
On the YEB solid plates of g/ml kanamycins and 50 μ g/ml rifampins, 28 DEG C of culture about 48h, transformant is obtained.By transformant
Bacterium solution PCR identifications are carried out, identification the primer is as follows：
5 ' primers：5’-ATGGGTGCTTATGATCAAGTTTCTGTG-3’；
3 ' primers：5’-TCATTCCTTGCTTGCTAGCATTTCCTTTG-3’.
As a result display obtains about 1160bp fragment, it was demonstrated that pCAMBIA1302-VrDREB2A is successfully transferred to EHA105
In.PCR is identified that positive transformant extracts plasmid again, sequencing is sent to, is as a result pCAMBIA1302- for the plasmid
VrDREB2A, further prove, carrier pCAMBIA1302-VrDREB2A has successfully been transferred in EHA105, will have been contained
PCAMBIA1302-VrDREB2A transformant is named as recombinational agrobacterium EHA105/pCAMBIA1302-VrDREB2A.
(3) arabidopsis thaliana transformation
1. recombinational agrobacterium EHA105/pCAMBIA1302-VrDREB2A is inoculated in into 10ml contains 50 μ g/ml kanamycins
In the YEB fluid nutrient mediums of 50 μ g/ml rifampins, 28 DEG C, 200rpm shaken cultivations stay overnight；
2. the previous day is converted with 1:50 ratios (volume ratio) are inoculated in YEB culture mediums of the 200ml containing identical antibiotic, are expanded
Big culture is 1.2-1.6 to OD600,5000rpm, 15min centrifugation collection bacterium, is resuspended in and penetrates into buffer solution (by sucrose, surface-active
Agent L-77 and water composition；The weight/mass percentage composition of sucrose is 5% sucrose, and L-77 volumn concentration is 0.05%), to make
OD600 is 0.8, as recombinational agrobacterium EHA105/pCAMBIA1302-VrDREB2A bacterium solutions；
3. Foral dip methods are used by the arabidopsis of Agrobacterium-mediated Transformation bud stage：In columbia Arabidopsis thaliana ecotypes
Cut off during (columbia stain of Arabidopsis thaliana as genetic background) bolting 4-5cm
Terminal inflorescence, grow axillary inflorescence, wound should be located above highest stem leaf when cutting, and be converted after about 4-5d, convert
Before to make soil fully drenched and remove big bud, only retain unopened small bud；During conversion by the whole strain of arabidopsis with
Flowerpot tips upside down in the container for filling 200ml recombinational agrobacterium EHA105/pCAMBIA1302-VrDREB2A bacterium solutions together to be soaked
2-3min, after immersion, flowerpot is taken out, side is put in pallet, covers black plastic cloth, plastic cloth is opened after 24hr, uprightly
Flowerpot is placed, carries out normal illumination cultivation, harvests T1In generation, turns VrDREB2A arabidopsis seeds.
(4) screening of transgenic arabidopsis and Molecular Identification
1. the screening of transgenic arabidopsis
T1In generation, turns VrDREB2A arabidopsis seeds and is seeded on the MS solid plates of the hygromycin containing 80mg/L, 4 DEG C of vernalization 3d,
Put and 7d is cultivated in 22 DEG C of illumination boxs, transformant shows as that true leaf is dark green, and root is profound into culture medium, and transformant is moved
Into the MS culture mediums without antibiotic, the seedling of green is gone into breeding in soil after 6d, the seed of harvest is T2In generation, turns
VrDREB2A arabidopsis seeds.
2. DNA level detects
By T2In generation, turns VrDREB2A arabidopsis cultivating seeds into plant, extracts T2In generation, turns the base of VrDREB2A arabidopsis strains
Because group DNA is template, using plasmid p1302-VrDREB2A as positive control, the base of columbia Arabidopsis thaliana ecotypes (wild type)
Because group DNA is negative control, enters performing PCR with following primer and expand：
5 ' primers：5’-ATGGGTGCTTATGATCAAGTTTCTGTG-3’；
3 ' primers：5’-TCATTCCTTGCTTGCTAGCATTTCCTTTG-3’.
PCR system (25.O μ l)：10 × Ex Taq PCR Buffer 2.5,2.0 μ l of μ l, dNTP (25mM), 5 ' primers
(5pmol/ μ l) 1.0 μ l, the μ l of 3 ' primers (5pmol/ μ l), 1.0 μ l, ExTaq enzyme (5U/ μ l) 1.0, template (1 μ g/ μ l) 1.0 μ l,
ddH2O 16.5μl。
PCR programs are：The first round：94 DEG C of denaturation 5min；Second wheel：94 DEG C of denaturation 50sec, 52 DEG C of renaturation 50sec, 72 DEG C
Extend 1min, 30 circulations；Third round：72 DEG C of extension 10min.
After reaction terminates, 1.0% agarose gel electrophoresis detection pcr amplification product, as a result PCR amplifications as shown in Figure 2
Product electrophoretogram, M are 1kb DNA ladder, from down to up respectively 250bp, 500bp, 750bp, 1000bp, 1500bp,
2000bp, 1 is negative control, and 2 be negative control, and 3-13 and 16 is PCR positive plants, and 14,15 and 17 be PCR feminine gender plant；
As can be seen that the plant that can amplify VrDREB2A gene specifics band (about 1158bp) identifies positive T for PCR2In generation, turns
VrDREB2A Arabidopsis plants, obtain 12 PCR and identify positive T2In generation, turns VrDREB2A Arabidopsis plants, wherein three strain lives
Entitled L2, L5 and L8 strain (T2In generation, turns VrDREB2A arabidopsis).
L2, L5 and L8 strain are selfed respectively, obtain T3For seed, it is T to be cultivated3In generation, turns VrDREB2A and intends south
Mustard plant L2, L5 and L8.
2nd, the resistance of reverse identification of transgenic arabidopsis
Respectively by columbia Arabidopsis thaliana ecotypes (WT) seed, L2, L5 and L8 strain arabidopsis (T3In generation, turns VrDREB2A
Arabidopsis seed) carry out salt stress and drought stress experiment, each 90 seeds of strain.
1st, salt stress is tested
The arabidopsis seed of each strain is planted respectively and cultivated in the MS without NaCl and containing 210mM and 220mM NaCl
On base, 4 DEG C of vernalization 72hr, illumination cultivation 7d, daily light application time are 16 hours, and intensity of illumination is 100 μm of ol m-2s-1, carry out
Germination rate is calculated (using root long 1mm as sprouting standard).Statistical result as shown in figure 3, on the MS culture mediums for not containing NaCl,
The germination rate of columbia Arabidopsis thaliana ecotype seeds and L2, L5 and L8 strain arabidopsis (T3In generation, turns VrDREB2A arabidopsis kinds
Son) germination rate of seed do not have difference；But under salt stress treatment conditions, the germination rate of columbia Arabidopsis thaliana ecotype seeds
With L2, L5 and L8 strain arabidopsis (T3In generation, turns VrDREB2A arabidopsis seed) germination rate of seed has notable difference.Such as
On 210mM NaCl MS culture mediums, the germination rate of columbia Arabidopsis thaliana ecotype seeds is the germination rate of 15%, L2 seeds
Germination rate for 76.76%, L5 seeds is that the germination rate of 51.76%, L8 seeds is 65.83%, L2, L5 and L8 strain arabidopsis
The germination rate of seed is significantly higher than columbia Arabidopsis thaliana ecotypes, shows that being overexpressed VrDREB2A genes adds arabidopsis
Saline-alkaline tolerance.
2nd, drought stress is tested
The arabidopsis seed of each strain is planted on MS culture mediums respectively, it is (every after 4 DEG C of vernalization 72hr, illumination cultivation 7d
Its light application time is 16 hours, and intensity of illumination is 100 μm of ol m-2s-1), it is transferred to compost (Nutrition Soil:Vermiculite (v/v)=1:
3) in, normal growth is after 2 weeks, rehydration after Osmotic treatment 14d, while sets the control group of normal watering management, calculates and deposits after 3d
Motility rate (the survival strain number of survival strain number/control group of Osmotic treatment group).Experiment is set to be repeated three times, repeats each strain 30 every time
Grain seed.Count its data.Statistical result is as shown in Figure 4, it can be seen that the survival rate of columbia Arabidopsis thaliana ecotype seeds
The survival rate for being 44.76%, L2 seeds is that the survival rate of 75.13%, L5 seeds is that the survival rates of 68.89%, L8 seeds is
73.36%.Turn the survival rate of empty vector control arabidopsis seed with columbia Arabidopsis thaliana ecotype results without significant difference.
The survival rate of L2, L5 and L8 arabidopsis seed is significantly higher than columbia Arabidopsis thaliana ecotypes and turns empty vector control arabidopsis,
Show to be overexpressed the drought-resistant ability that VrDREB2A genes add arabidopsis.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
- A kind of 1. plant stress tolerance correlative protein VrDREB2A, it is characterised in that its amino acid sequence such as SEQ ID No.1 institutes Show.
- A kind of 2. gene related to plant stress toleranceVrDREB2A, it is characterised in that gene codified claim 1 institute The albumen stated.
- 3. gene according to claim 2VrDREB2A, it is characterised in that the gene contains as in SEQ ID No.2 Nucleotide sequence shown in 3-1160bp.
- 4. gene according to claim 3VrDREB2A, it is characterised in that its nucleotide sequence such as SEQ ID No.2 institutes Show.
- 5. for expanding gene described in claim 2VrDREB2APrimer pair, it is characterised in that the nucleotides of the primer pair Sequence is respectively as shown in SEQ ID No.3 and SEQ ID No.4.
- 6. contain any one of the claim 2-4 geneVrDREB2ACarrier.
- 7. contain any one of the claim 2-4 geneVrDREB2AEngineering bacteria.
- 8. the gene described in claim any one of 2-4VrDREB2AApplication in cultivating genetically modified plants and improving resistance of reverse.
- 9. application according to claim 8, it is characterised in that utilize the carrier described in claim 6 or claim 7 institute Gene of the engineering bacteria stated described in by claim any one of 2-4VrDREB2AImport purpose plant and obtain turning for resistance of reverse raising Gene plant.
- 10. application according to claim 9, it is characterised in that the resistance of reverse is drought tolerance and/or salt tolerance.
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| Accession ID:AEO50757.2，dehydration responsive element binding protein 2A [Vigna unguiculata];Sadhukhan,A.等;《NCBI Genbank》;20140721;DEFINITION、SOURCE及ORIGIN部分 * |
| VuDREB2A, a novel DREB2-type transcription factor in the drought-tolerant legume cowpea, mediates DRE-dependent;Ayan Sadhukhan等;《Planta》;20140717;第240卷;第646页右栏第2段及第650页右栏第2段 * |
| 绿豆种质资源芽期抗旱性鉴定;王兰芬等;《植物遗传资源学报》;20140408;第15卷(第3期);第498-503页 * |
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