CN108841833A - A kind of DPBF1 recombinant fragment and its application - Google Patents
A kind of DPBF1 recombinant fragment and its application Download PDFInfo
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Abstract
The present invention provides a kind of DPBF1 recombinant fragment and its applications.DPBF1 gene conserved region II (AD1) and basic leucine zipper area (bZIP) are recombinated and are implemented on improved genetic transformation carrier pCAMBIA1301-GUSless, genetic transformation arabidopsis Columbia ecotype, transgenic arabidopsis rDPBF1 is obtained through screening and identification, and to be overexpressed the transgenic arabidopsis of the code area DPBF1 complete sequence (DPBF1) as control.Transcriptional level is analysis shows recombination can carry out transcriptional expression in transgenic arabidopsis.The transgenic line of acquisition shows advance flowering period, the form size of mature seed becomes larger, the superior phenotype that thousand grain weigth dramatically increases, drought resistance is remarkably reinforced compared with wild type.
Description
Technical field
The present invention relates to plant genetic engineering field, specifically a kind of arabidopsis DPBF1 recombinant fragment and its application.
Background technique
Abscisic Acid (ABA) is in the starting and maintenance of vegetable seeds and bud dormancy, plant to adverse circumstance especially water
It plays a major role in the response of point adverse circumstance.In addition, ABA is by affecting plant growth and development with the interaction of other plant hormones
All various aspects, such as at florescence and leaf senile.
Arabidopsis transcription factor DPBF1 is the basic leucine zipper class transcription factor of a response ABA signal, expression
By the induction of ABA and a variety of abiotic stress.DPBF1 can be combined with cis element ABRE, and then enhance downstream drought resisting, salt resistance
The expression of the correlation functions gene such as alkali, low-temperature resistance improves the ability of these abiotic stresses of plant resistant.Transcription factor DPBF1
Key regulatory effect is played during embryonic development advanced stage and ABA signal transduction, affects the maturation of vegetable seeds and is sprouted
The growth and development processes such as hair.Existing research is it is also shown that the arabidopsis for being overexpressed transcription factor DPBF1 gene changes at the florescence recently
Become, shows late blooming character.
However, having different opinions previously to whether DPBF1 gene enhances plant drought resistance, having been reported that, which can be enhanced, is also had been reported that
It cannot enhance.Since there is the regulatory regions of transcriptional activation in transcription factor DPBF1, and what these regulatory regions played a role
It is very not clear before type order, so that overall length DPBF1 gene constructed transgenic plant phenotype or be difficult to is presented or difficult
To repeat.Based on this, we construct the different mutants of transcription factor DPBF1 --- retain its transcriptional activation region and DNA
Transcriptional activation regulatory region is deleted in bond area, understands effect of the DPBF1 in plant growth and development process with this.We with
The transgenic arabidopsis of DPBF1 mutant gene building not only shows repeatable drought resistance, but also shows ahead of time
It blooms and the very significant new traits such as thousand grain weigth increase.
Problem of the existing technology：
(1) DPBF1 for drought resistance function and do not know, and have no DPBF1 recombinant fragment influence arabidopsis drought resisting
The report of property.
(2) have no that DPBF1 recombinant fragment influences the report in arabidopsis florescence.
(3) transcription regulatory region in view of transcription factor DPBF1 is related to the complicated regulatory mechanism of plant growth and development process, with
This attempts to illustrate that the process that DPBF1 is related in the intracorporal function of plant is more, mechanism is complicated, frequently results in the paradox of conclusion.
(4) prior art has no that DPBF1 increases the report of yield.
Summary of the invention
In view of the deficiencies in the prior art, the present invention provides a kind of DPBF1 recombinant fragment and its applications, utilize recombination
It is quasi- finally to realize overexpression transgenosis for DPBF1 gene conserved region II (AD1) and DPBF1 gene basic leucine zipper region
Advance flowering period, the drought resistance of southern mustard plant significantly increase, thousand grain weigth increases.
In order to achieve the above object, present invention employs the following technical solutions：
A kind of DPBF1 Protein reconstitution segment, sequence include：PMX-1, the conserved region II containing DPBF1 gene coding
(AD1, the 59th to the 79th amino acids residue) and basic leucine zipper area (bZIP, the 342nd to the 442nd amino acids residue)
2 segments, recombinant fragment are implemented on improved Genetic Transformation in Higher Plants carrier pCAMBIA1301-GUSless.PMX-1 is carried
Recombinant fragment AD1-bZIP the long 372bp of nucleotide sequence, nucleotide sequence such as sequence table SEQ ID NO：Shown in 1.
PMX-4, the full length sequence of the gene coding region containing DPBF1 are implemented in improved Genetic Transformation in Higher Plants carrier
On pCAMBIA1301-GUSless.The long 1323bp in the gene coding region overall length DPBF1 that pMX-4 is carried, nucleotide sequence such as sequence
Table SEQ ID NO：Shown in 2.
A kind of clone of DPBF1 Protein reconstitution segment and recombination method, include the following steps：
The transformation of 1.pCAMBIA1301 carrier
Multiple cloning sites (MCS) are located at the upstream plant promoter (35S promoter) in pCAMBIA1301 carrier, and
35S promoter downstream lacks the restriction enzyme site for target gene insertion.It is multiple cloning sites in elimination original vector to experimental implementation
Influence, the multiple cloning sites in pCAMBIA1301 carrier need to be removed, while the gus gene sequence in 35S promoter downstream being replaced
It is changed to the artificial synthesized Linker containing multiple cloning sites.
(1) in pCAMBIA1301 carrier multiple cloning sites removal
Multiple cloning sites in pCAMBIA1301 carrier are laggard with restriction enzyme EcoRI and HindIII double digestion
The connection of row flat end.HindIII digestion system is following (80 μ L):10 × M buffer, 8.0 12 μ L, Hind III inscribe of μ L, DNA
4.0 μ L of enzyme, sterile water complement to 80.0 μ L.Reaction solution is mixed, after being centrifuged about 30sec, the endonuclease reaction in 37 DEG C of water-baths
2h。
Digestion products bioengineering PCR product Purification Kit, purification process is referring to PCR product purification kit
Operating instruction.EcoRI digestion is used after purified product, EcoRI digestion system is following (100 μ L)：10 × quick cut buffer
10 40 μ L, Quickcut EcoRI of μ L, DNA, 2.0 μ L, sterile water complement to 100.0 μ L.
Reaction solution is mixed, after being centrifuged about 30sec, in 37 DEG C of reaction 15min.Digestion products PCR product purified reagent
Box purifying.To carry out flat end connection, need to carry out flat end processing to the DNA fragmentation of purifying, flat end reaction system is such as
Under (10 μ L)：PCAMBIA1301 after HindIII and EcoRI double digestion
>11 μ L, 10 × T4 DNA of μ l, 0.1%BSA of 0.1pmol, 1.7mmol/L dNTP Mixture
1 μ L, T4 DNA polymerase of polymerase buffer, 1 μ L, sterile water complement to 10 μ L.Above-mentioned reaction system is in 37 DEG C
1h is kept the temperature to complete the flat end of cohesive end.
Recirculation after linear carrier linked enzyme connection after flat end.Coupled reaction system is as follows：After flat end
3.5 μ L, 10 × Ligase buffer of linear carrier 1.5 μ L, T4 DNA Ligase (350U/ μ L) 1.5 μ L, sterile water complement to
15μL.16 DEG C of product after connection, converts bacillus coli DH 5 alpha competent cell overnight.Converted product is coated on containing Kan resistance
LB solid medium, 37 DEG C of overnight incubations.The single colonie that grows in picking resistant panel simultaneously extracts plasmid, with SacI and
SacII double digestion is identified, identifies that correct plasmid is named as pCAMBIA1301- Δ MCS.
Flat end will be carried out after restriction enzyme EcoRI and the HindIII digestion of the multiple cloning sites of pCAMBIA1301
Connection obtains plasmid pCAMBIA1301- Δ MCS, and carries out SacI and SacII double digestion mirror to pCAMBIA1301- Δ MCS
It is fixed.PCAMBIA1301- Δ MCS carrier forms the segment of 11837bp after SacI and SacII double digestion；pCAMBIA1301
Two segments are produced after SacI and SacII double digestion, a clip size is 9185bp, another segment is 2652bp
(Fig. 1).Thus speculate that the multiple cloning sites of pCAMBIA1301 carrier have been removed.
(2) in pCAMBIA1301- Δ MCS carrier gus gene sequence removal
Preliminary improved carrier pCAMBIA1301- Δ MCS is subjected to double digestion with NcoI and BstEII respectively to remove
Gus gene sequence.NcoI digestion system is following (100 μ L)：10 × K buffer
10.0 μ L, pCAMBIA1301- Δ MCS plasmid, 10 10 6 μ L of μ L, NcoI of μ L, BSA, sterile water complement to 100.0 μ
L.Reaction solution is mixed, after being centrifuged 30s, in 37 DEG C of endonuclease reaction 2h.
Endonuclease reaction liquid is recycled with PCR product Purification Kit, recovery product carries out digestion with BstEII later,
BstEII digestion system is following (20 μ L)：Returning after 10 × K buffer, 2.0 μ L, NcoI digestion pCAMBIA1301- Δ MCS plasmid
10 1 μ L of μ L, BstEII of product≤1 μ g, BSA is received, sterile water complements to 20.0 μ L.Reaction solution is mixed, it is anti-in 60 DEG C after centrifugation
Answer 2h, digestion products PCR product Purification Kit.
It is a pair of completely for following restriction enzyme site (NcoI EcoRI SalI KpnI BamHI SacI BstEII) synthesis
Complementary primer：
1301LinkerF：CATGGAATTCGTCGACGGTACCGGATCCGAGCTCG
1301LinkerR:GTCACCGAGCTCGGATCCGGTACCGTCGACGAATTC
By primer 1301LinkerF1 and 1301LinkerR after 60 DEG C of annealing 10min with above-mentioned linearisation after purification
Carrier carries out 16 DEG C of connections overnight, converts bacillus coli DH 5 alpha.Converted product is coated on kanamycins (50 μ g/ml of Kan) resistance
LB plate on, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium and stays overnight, extracts plasmid.Plasmid is sent
It is sequenced to Shanghai Sheng Gong Co., Ltd.Correctly improved pCAMBIA1301 vector plasmid will be sequenced to be named as
pCAMBIA1301-GUSless。
PCAMBIA1301- Δ MCS vector plasmid carries out digestion with NcoI and BstEII to remove GUS sequence, and to removal
The plasmid of GUS sequence carries out double digestion identification.Control vector pCAMBIA1301- Δ MCS is after NcoI and BstEII digestion, enzyme
Cut the small fragment of large fragment and a 2050bp that product is 9736bp；PCAMBIA1301-GUSless carrier through SacI and
The large fragment of 6700bp and the small fragment (Fig. 2) of about 3018bp are produced after SacII digestion.Digestion qualification result shows
GUS sequence in pCAMBIA1301-GUSless carrier has been removed.PCAMBIA1301-GUSless Vector map such as Fig. 3 institute
Show.
Conserved region II (AD1), basic leucine zipper area (bZIP) and the DPBF1 gene that 2.DPBF1 gene encodes are complete
The clone of sequence
(1) PCR amplification of DPBF1 gene conserved region II (AD1)
According to the DPBF1 gene order included in the sequence of pCAMBIA1301-GUSless plasmid and ncbi database, if
Count specific primer AD1F：CGGAATTCGGCAAGAACTTTGGGTCCAT and AD1R：GGGGTACC
CTCCTCTGCGTTCCAAATAG, primer both ends add restriction enzyme site EcoRI, KpnI and protection base, by bioengineering (on
Sea) Co., Ltd's synthesis.
Using pET32a-DPBF1 as the conserved region II of template primer amplified DPBF1 gene.The PCR of 50 μ L reacts
System is as follows：10 × PCR buffer (contain Mg2+), 5.0 4.0 μ L, TaKaRa rTaq DNA of μ L, dNTP (2.5mM)
Polymerase (5U/ μ L) 0.3 μ L, AD1-F (10 μM) 1.0 μ L, AD1-R (10 μM) 1.0 μ L, 1.0 μ L of template, sterile water are supplied
To 50.0 μ L.
Reaction condition：94 DEG C of initial denaturation 2min are recycled subsequently into 3step method：94 DEG C of denaturation 30sec, 40 DEG C of annealing
30sec, 72 DEG C of extension 5sec, after reacting 30 circulations, 72 DEG C of extension 10min；4℃99min.After the completion of PCR amplification, AD1 is taken
Pcr amplification product and DNA DL2000Marker electrophoresis is carried out in 1% Ago-Gel.100V electrophoresis 30min, it is ultraviolet
It is observed under lamp, Preliminary detection amplified production size.
(2) PCR amplification of DPBF1 basic leucine zipper area (bZIP)
According to the DPBF1 gene order included in the sequence of pCAMBIA1301-GUSless plasmid and ncbi database, if
Count specific primer DBDF：GGGGTACCAGGAAAAGAGTAGTGGATGGT and DBDR：
TCGAGCTCTTAGAGTGGACAACTCGGGT, restriction enzyme site KpnI, SacI and protection base are added in primer both ends, by biological work
The synthesis of journey (Shanghai) Co., Ltd..
The PCR amplification in the basic leucine zipper area (bZIP) of DPBF1 gene is carried out using pET32a-DPBF1 as template.50
The PCR reaction system of μ L is as follows：10 × PCR buffer (containing Mg2+) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, 0.5 μ L of template,
TaKaRa rTaq DNA polymerase (5U/ μ L) 0.3 μ L, DBD-F (10 μM) 1.0 μ L, DBD-R (10 μM) 1.0 μ L, it is sterile
Water complements to 50.0 μ L.
Reaction condition：94 DEG C of initial denaturation 2min are recycled subsequently into 3step method：94 DEG C of denaturation 30sec, 56 DEG C of annealing
30sec, 72 DEG C of extension 16sec, after reacting 30 circulations, 72 DEG C of extension 10min；4℃99min.After the completion of PCR amplification, PCR is taken
Amplified production and DNA DL2000 Marker carry out electrophoresis in 1% Ago-Gel.100V electrophoresis 30min, under ultraviolet lamp
Observation, Preliminary detection amplified production size.
(3) amplification of DPBF1 gene complete coding region
According to the DPBF1 gene order included in the sequence of pCAMBIA1301-GUSless plasmid and ncbi database, if
Count specific primer DPBF1F：5 '-CCGGAATTCACTAGAGAAACGAAGTTGACGT-3 and DPBF1R：
TCGAGCTCTTAGAGTGGACAACTCGGGT, restriction enzyme site EcoRI/SacI and protection base are added in primer both ends, by biology
The synthesis of engineering (Shanghai) Co., Ltd..
The PCR amplification of DPBF1 gene complete coding region is carried out using pET32a-DPBF1 as template.The PCR reactant of 50 μ L
It is as follows：10 × PCR buffer (containing Mg2+) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, 0.5 μ L, rTaq DNA of template
0.3 1.0 1.0 μ L of μ L, DPBF1-R (10 μM) of μ L, DPBF1-F (10 μM) of polymerase (5U/ μ L), sterile water complement to 50.0
μL。
Reaction condition：94 DEG C of initial denaturation 2min are recycled subsequently into 3step method：94 DEG C of denaturation 30sec, 56 DEG C of annealing
30sec, 72 DEG C of extension 1min18sec, after reacting 30 circulations, 72 DEG C of extension 10min；4℃99min.After the completion of PCR amplification,
Pcr amplification product and DNA DL2000 Marker is taken to carry out electrophoresis in 1% Ago-Gel.100V electrophoresis 30min is purple
It is observed under outer lamp, Preliminary detection amplified production size.
The pcr amplification product and DNA DL2000 Marker for taking each sequence carry out electrophoresis in 1% Ago-Gel,
100V electrophoresis 30min is observed under ultraviolet lamp.The fragment length of AD1 is 63bp, and the fragment length of bZIP is 303bp, and DPBF1 is compiled
The fragment length of code area overall length is 1323bp (Fig. 4).The molecular size range of pcr amplification product is consistent with expected theoretical value, tentatively
Determine that the PCR product amplified is required target fragment.
3. the building of Genetic Transformation in Higher Plants carrier pMX-1
PCR purified product and pCAMBIA1301-GUSless carrier to AD1 carry out EcoRI/KpnI double digestion, and root
Glue recycling is carried out to corresponding electrophoretic band according to the raw work gel reclaims kit in Shanghai.Amplification is produced with T4 DNA ligase later
Object is connected on carrier pCAMBIA1301-GUSless.Coupled reaction system is following (15 μ L)：Glue recycles the AD1 piece after digestion
4.5 μ L of section, 3.5 μ L, 10 × Ligase buffer of linearized vector pCAMBIA1301-GUSless after the digestion of glue recycling
1.5 μ L, T4 DNA Ligase (350U/ μ L) 1.5 μ L, sterile water complement to 15 μ L.
Reaction solution is mixed, overnight, connection product converts bacillus coli DH 5 alpha for 16 DEG C of connections.Converted product is coated on card, and that is mould
On the LB plate of plain (50 μ g/ml of Kan) resistance, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium mistake
Night, extracting plasmid obtain First Contact Connections product.
PCR purified product and above-mentioned First Contact Connections product to bZIP carry out KpnI/SacI double digestion, and according to upper
Hai Shenggong gel reclaims kit carries out glue recycling to corresponding electrophoretic band.The glue of bZIP and First Contact Connections product is recycled
Product converts bacillus coli DH 5 alpha in 16 DEG C of connections overnight, connection product.Converted product is coated on kanamycins (50 μ g/ of Kan
Ml) on the LB plate of resistance, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium and stays overnight, extracts plasmid.
The recombinant plasmid of extracting is named as pMX-1.PMX-1 plasmid is sent and is sequenced in Shanghai bioengineering Co., Ltd, sequencing knot
Fruit is analyzed with DNAMAN software, is compared.Save the correct plasmid of sequencing result.
4. the building of Genetic Transformation in Higher Plants carrier pMX-4
PCR purified product and pCAMBIA1301-GUSless plasmid to the gene coding region DPBF1 complete sequence carry out
EcoRI/SacI double digestion, and glue recycling is carried out to corresponding electrophoretic band according to the raw work gel reclaims kit in Shanghai.It will
The glue recovery product of DPBF1 complete sequence and pCAMBIA1301-GUSless plasmid is in 16 DEG C of connections overnight.Coupled reaction system is such as
Shown in following table：The 4.5 μ L of DPBF1 genetic fragment of gel recycling, the linearized vector after digestion of gel recycling
3.5 μ L, 10 × Ligase buffer of pCAMBIA1301-GUSless 1.5 μ L, T4 DNA Ligase (350U/ μ L) 1.5 μ L,
Sterile water complements to 15 μ L.
Connection product converts bacillus coli DH 5 alpha.Converted product is coated on the LB of kanamycins (50 μ g/ml of Kan) resistance
On plate, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium and stays overnight, extracts plasmid.By the recombination of extracting
Plasmid is named as pMX-4.PMX-4 plasmid is sent and is sequenced in Shanghai bioengineering Co., Ltd, sequencing result DNAMAN
Software is analyzed, is compared.Save the correct plasmid of sequencing result.
5.pMX-1, pMX-4 plasmid convert Agrobacterium
PMX-1, pMX-4 plasmid convert Agrobacterium GV3101 respectively, and converted product is coated on containing gentamicin (40 μ g/
Ml), on the LB solid medium of rifampin (25 μ g/ml) and kanamycins (50 μ g/ml), 30 DEG C are cultivated two days.It picks them separately
The single bacterium grown in resistant panel drops down onto 4ml and contains gentamicin (40 μ g/ml), rifampin (25 μ g/ml) and kanamycins
In the LB liquid medium of (50 μ g/ml), after 30 DEG C are shaken incubation 12 hours slowly, takes 1 μ L as pcr template, carry out bacterium colony PCR mirror
Determine positive colony.Bacterium colony PCR identifies that positive colony method is as follows：The sterile water of 10 μ L is added in the 1.5mL EP pipe of sterilizing,
Taking monoclonal colonies with white lancet choicest, gently piping and druming is uniformly mixed into the sterile water of 10 μ L, and sterile water, which can be observed, to be become
Light milky, the template for taking 1 μ L to react as PCR carry out PCR identification using Taq archaeal dna polymerase.
Using AD1 F and DBD R as specific primer, the agrobacterium liquid containing pMX-1 plasmid is template；With DPBF1 F and
DBD R is specific primer, and the agrobacterium liquid containing pMX-4 plasmid is that template carries out PCR amplification respectively, verifies insetion sequence.
PCR product carries out electrophoresis detection in Ago-Gel.PCR of the pMX-1 plasmid in Agrobacterium identifies that product sheet segment length is
PCR of 372bp, the pMX-4 plasmid in Agrobacterium identifies that product sheet segment length is 1323bp (Fig. 5).Recombinant plasmid pMX-1,
PMX-4 has been transferred in Agrobacterium.
6. During Agrobacterium arabidopsis Columbia ecotype (Agrobacterium flower-dipping method arabidopsis thaliana transformation)
(1) arabidopsis is grown to after bolting, and the top of arabidopsis main inflorescence is cut while paying attention to avoiding injuring leaf life
Inflorescence prepares dip dyeing when side shoot stretches out 2-10cm to induce the generation of side inflorescence.It is subject to the more bud that do not open, and
Watering on the day before to needing the plant disseminated to mention；
(2) by the Agrobacterium inoculation containing genetic transformation carrier in 5ml LB liquid medium (gentamicin (40 μ g/
Ml), rifampin (25 μ g/ml) and kanamycins (50 μ g/ml)) in, 28 DEG C, 15 hours left sides of 200r/min shaking table shake culture
It is right；
(3) by the culture in upper step with 1：50 ratio is transferred to the LB Liquid Culture that 200ml contains corresponding antibiotic
In base, 28 DEG C, 200r/min shaking table culture 15 hours or so to OD600=0.8-1.0；
(4) 4000r/min is centrifuged 15min and collects thallus, is resuspended with permeabilization buffer (sucrose of 1/2MS culture medium+5%)
Thallus, be added Silwett L-77 make its final concentration of 0.03%；
(5) agrobacterium suspension for containing plasmid to be transformed respectively is drawn with 5ml syringe, and ready arabidopsis is planted
Each flower of strain is disseminated.The plant disseminated (T0 is for plant) cover is got up to keep humidity with freshness protection package；
(6) after the Arabidopsis plant after dip dyeing being cultivated 12h under dark condition, remove freshness protection package, normal culture is arrived
Fruit pod is mature, repeats dip dyeing 2-3 times during which to improve transformation efficiency；
(7) after fruit pod turns yellow, before cracking, several fruit pods are collected into 1.5ml centrifuge tube according to label, collect arabidopsis kind
Sub (T1 generation) stores after sufficiently drying, to screen transformant.
(8) transgenic plant that the screening of conversion pMX-1 plasmid obtains is named as Arabidopsis thaliana
RDPBF1, the transgenic plant that conversion pMX-4 plasmid screening obtains are named as Arabidopsis thaliana DPBF1.
7. the screening and identification of transgenic line seed
After the T1 after converting will be harvested for seed disinfection, it is layered on the 1/2MS solid medium containing 25 μ g/ml hygromycin.4
DEG C vernalization 2-3 moves back into plant illumination incubator normal light-exposed culture.Green plant (the bottle-green son of growth after two weeks
Leaf, root are longer) it may be transgenic positive plant, and non-transgenic plant cotyledon turns to be yellow, root is short, is unable to long-term surviving.It will be green
For color plantlet of transplant in soil, culture divides single plant to harvest seed to seed maturation (T2 is for seed).T2 is being contained into tide for seed
Continue screening on the MS culture medium of chloramphenicol resistance to T3 generation.
According to the sequence design specific primer of pMX-1 and pMX-4 plasmid to identify recombination in transgenic arabidopsis
In expression.It is extracted from and grows 7 days wild types and the RNA of transgenic arabidopsis seedling whole plant on 1/2MS culture medium, invert
For cDNA, identify recombination in transcriptional level by Reverse-Transcript PCR (RT-PCR) with specific primer
Expression.
RT F1：cgggggactcttgaccatgga
RT R1：ctgcgcattctcttctttcaactggt
Expression of the recombination in transgenic arabidopsis is analyzed by RT-PCR.The RT-PCR electrophoresis of transgenic arabidopsis
As a result the specificity band with corresponding molecular size range is showed, control group wild type does not have band (Fig. 6).Show to recombinate base
Because transcriptional expression can be carried out in transgenic arabidopsis.
A kind of DPBF1 Protein reconstitution segment is promoting the application in arabidopsis Blooming.
(1) select identical flowerpot, be put into each flowerpot certain Nutrition Soil (Denmark's Pin Shi matrix 5,
PH5.5), the flowerpot for filling Nutrition Soil is placed in flat pallet, pallet adds water that the soil in disk in flowerpot is made independently to absorb water.
(2) by above-mentioned transgenic arabidopsis strain and Colombia's wild type seeds in 4 DEG C vernalization two days, dibbling is in soil
Surface, at concurrent 5, (24 ± 2 DEG C of temperature, humidity 20%, illumination condition is 16h illumination/8h dark, intensity of illumination for hot-house culture
5100lux).The undesirable seedling of upgrowth situation is removed after a week, and each flowerpot is made to retain seedling similar in 5 plants of upgrowth situations.
(3) greenhouse continues culture to blooming, and counts flowering time and upgrowth situation.
Plant bolting initially enters florescence, and wildtype Arabidopsis thaliana grows 30 days beginning boltings, transgenic arabidopsis
RDPBF1 and DPBF1 enters peduncle-growing period for rapeseed (Fig. 7) after growth 25 days and 27 days respectively.Transgenic arabidopsis rDPBF1 and DPBF1
The plant bolting time to show the phenotype bloomed ahead of time earlier than wild type.
A kind of DPBF1 Protein reconstitution segment is improving the application in arabidopsis thousand grain weigth.
(1) select identical flowerpot, be put into each flowerpot certain Nutrition Soil (Denmark's Pin Shi matrix 5,
PH5.5), the flowerpot for filling Nutrition Soil is placed in flat pallet, pallet adds water that the soil in disk in flowerpot is made independently to absorb water.
(2) by the seed of above-mentioned transgenic arabidopsis strain and Colombia's wild type in 4 DEG C vernalization two days, dibbling is in soil
Earth surface, at concurrent 5, (24 ± 2 DEG C of temperature, humidity 20%, illumination condition is 16h illumination/8h dark, and illumination is strong for hot-house culture
Spend 5100lux).The undesirable seedling of upgrowth situation is removed after a week, and each flowerpot is made to retain seedling similar in 5 plants of upgrowth situations.
(3) continue culture to seed maturation, choose fruit pod similar in form size and collect seed.
(4) each 1,000, the seed of wild type and transgenic line are taken, weighing.It is averaged in triplicate.
Plant growth later period seed is mature, and form is obviously (Fig. 8) bigger than wild type after absorbing water for the seed of transgenic line.
1000 mature seeds of each strain are weighed to weigh：Wild type seeds mass of 1000 kernel 18mg, transgenic arabidopsis rDPBF1 seed
Mass of 1000 kernel 24mg, transgenic arabidopsis DPBF1 thousand grain weigth 20mg (Fig. 9).Statistical analysis shows transgenic line and open country
The thousand grain weigth of raw type has highly significant difference.
A kind of DPBF1 Protein reconstitution segment is improving the application in arabidopsis drought resistance.
(1) select identical flowerpot, be put into each flowerpot equiponderant Nutrition Soil (Denmark's Pin Shi matrix 5,
PH5.5), the flowerpot for filling equivalent weight Nutrition Soil is placed in flat pallet, pallet adds water to make the nutrition in disk in flowerpot
Native autonomous water suction.
(2) by above-mentioned transgenic arabidopsis strain and Colombia's wild type seeds in 4 DEG C vernalization two days, dibbling is in soil
Surface, at concurrent 8, (24 ± 2 DEG C of temperature, humidity 20%, illumination condition is 16h illumination/8h dark, intensity of illumination for hot-house culture
5100lux).The undesirable seedling of upgrowth situation is removed after a week, and each flowerpot is made to retain seedling similar in 8 plants of upgrowth situations.
(3) after continuing culture 2 weeks, stop watering, drought stress is handled 10 days.
(4) arid rehydration, observation, statistics seedling survival situation after 1 week after 10 days.
To growth three weeks and the Arabidopsis thaliana Seedlings of the consistent wild type of condition of culture and transgenic line stopped watering, arid
Rehydration 1 week after 10 days, tolerance of the observation transgenic arabidopsis to arid.Wildtype Arabidopsis thaliana totally eight plants of seedling, eight plants of seedling
The dehydration of blade whole, withered death, i.e. survival rate of the wildtype Arabidopsis thaliana under drought stress is 0；Transgenic line
The survival rate of seedling of rDPBF1 is 87.5%, and the survival rate of seedling of transgenic line DPBF1 is 100%.Transgenic line blade
The active green of health is reverted to after rehydration, and can enter reproductive stage (Figure 10).
Detailed description of the invention
Fig. 1 is that electrophoresis result is identified in pCAMBIA1301- Δ MCS digestion of the present invention；
Wherein, 1：PCAMBIA1301- Δ MCS is through SacI and SacII digestion products；2：PCAMBIA1301 through SacI and
SacII digestion products；M：DL15000Marker.
Fig. 2 is that electrophoresis result is identified in pCAMBIA1301-GUSless digestion of the present invention；
Wherein, 1：PCAMBIA1301- Δ MCS is through NcoI and BstEII digestion products；2：pCAMBIA1301-GUSless
Through SacI and SacII digestion products；M：DL15000Marker.
Fig. 3 is the physical map of the improved Genetic Transformation in Higher Plants carrier pCAMBIA1301-GUSless of the present invention.
Fig. 4 is the agarose gel electrophoresis figure of the PCR amplification of AD1, bZIP and DPBF1 overall length of the present invention；
Wherein, 1：The pcr amplification product of AD1, fragment length 63bp；2：The pcr amplification product of bZIP, fragment length
303bp；3：The pcr amplification product of the complete coding region DPBF1, fragment length 1323bp；M：DL2000 molecular weight Marker.
Fig. 5 is the PCR identification of recombinant vector pMX-1, pMX-4 of the present invention in Agrobacterium；
Wherein, 1：The PCR of pMX-1 identifies product, fragment length 372bp；2：The PCR of pMX-4 identifies product, fragment length
1323bp；3：It is the negative control of progress using unloaded pCAMBIA1301-GUSless as template.M：DL2000 molecular weight
Marker。
Fig. 6 is that the present invention is overexpressed transgenic arabidopsis RT-PCR identification；
Wherein, it extracts on 1/2MS culture medium and grows 7 days transgenic lines and the total serum IgE of WT lines, be reversed to
RT-PCR analysis is carried out with specific primer after cDNA.For wild type (WT) as control, Actin2 is internal reference.Condition of culture：Temperature
24 ± 2 DEG C of degree, humidity 20%, illumination condition are 16 hours illumination/8 hour dark, intensity of illumination 5100lux.
Fig. 7 is that present invention overexpression transgenic Arabidopsis plants are bloomed in advance；
Wherein, it is overexpressed the bolting situation of transgenic Arabidopsis plants and WT lines after growth 5 weeks.Cultivate item
Part：24 ± 2 DEG C of temperature, humidity 20%, illumination condition is 16 hours illumination/8 hour dark, intensity of illumination 5100lux.
Fig. 8 is the seed size that the present invention is overexpressed transgenic Arabidopsis plants；
Wherein, the size after the mature seed of wild type and transgenic line impregnates one day in water compares；
Fig. 9 is the mass of 1000 kernel that the present invention is overexpressed transgenic Arabidopsis plants；
Wherein, the weight of wild type and every 1000 seeds of transgenic line.Student's t test examines wild type
Otherness between transgenic line, * * indicate P < 0.01 (difference is extremely significant).
Figure 10 is the drought resistance enhancing that the present invention is overexpressed transgenic plant；
Wherein, the upgrowth situation of drought stress Arabidopsis thaliana Seedlings before and after the processing.Wild type and overexpression transgenic arabidopsis
Plant grows after three weeks under same culture conditions, and simultaneously the growth of plant is survived after Osmotic treatment 10 days, rehydration 1 week for stopping watering
Situation.Each flowerpot shares 8 plants of seedling.
Specific embodiment
To keep the purposes, technical schemes and advantages of invention clearer, with reference to the accompanying drawing to specific implementation of the invention
Mode is described in detail.The example of these preferred embodiments is illustrated in the accompanying drawings.Shown in attached drawing and according to attached
The embodiments of the present invention of figure description are only exemplary, and the present invention is not limited to these embodiments.
Here, it should also be noted that, in order to avoid having obscured technical solution of the present invention because of unnecessary details,
Illustrate only in attached drawing with closely related structure and/or processing step according to the solution of the present invention, and relationship is omitted not
Big other details.
Embodiment 1
A kind of DPBF1 Protein reconstitution segment is present embodiments provided, sequence includes：PMX-1 is compiled containing DPBF1 gene
Code conserved region II (AD1, the 59th to the 79th amino acids residue) and basic leucine zipper area (bZIP, the 342nd to the 442nd
Amino acids residue) 2 segments, recombinant fragment is implemented in improved Genetic Transformation in Higher Plants carrier pCAMBIA1301-
On GUSless.The long 372bp of nucleotide sequence for the recombinant fragment AD1-bZIP that pMX-1 is carried, nucleotide sequence such as sequence table
SEQ ID NO：Shown in 1.
PMX-4, the full length sequence of the gene coding region containing DPBF1 are implemented in improved Genetic Transformation in Higher Plants carrier
On pCAMBIA1301-GUSless.The long 1323bp in the gene coding region overall length DPBF1 that pMX-4 is carried, nucleotide sequence such as sequence
Table SEQ ID NO：Shown in 2.
Embodiment 2
Clone and the recombination method for present embodiments providing a kind of DPBF1 Protein reconstitution segment, include the following steps：
The transformation of 1.pCAMBIA1301 carrier
Multiple cloning sites (MCS) are located at the upstream plant promoter (35S promoter) in pCAMBIA1301 carrier, and
35S promoter downstream lacks the restriction enzyme site for target gene insertion.It is multiple cloning sites in elimination original vector to experimental implementation
Influence, the multiple cloning sites in pCAMBIA1301 carrier need to be removed, while the gus gene sequence in 35S promoter downstream being replaced
It is changed to the artificial synthesized Linker containing multiple cloning sites.
(1) in pCAMBIA1301 carrier multiple cloning sites removal
Multiple cloning sites in pCAMBIA1301 carrier are laggard with restriction enzyme EcoRI and HindIII double digestion
The connection of row flat end.HindIII digestion system is following (80 μ L):10 × M buffer, 8.0 12 μ L, Hind III inscribe of μ L, DNA
4.0 μ L of enzyme, sterile water complement to 80.0 μ L.Reaction solution is mixed, after being centrifuged about 30sec, the endonuclease reaction in 37 DEG C of water-baths
2h。
Digestion products bioengineering PCR product Purification Kit, purification process is referring to PCR product purification kit
Operating instruction.EcoRI digestion is used after purified product, EcoRI digestion system is following (100 μ L)：10 × quick cut buffer
10 40 μ L, Quickcut EcoRI of μ L, DNA, 2.0 μ L, sterile water complement to 100.0 μ L.
Reaction solution is mixed, after being centrifuged about 30sec, in 37 DEG C of reaction 15min.Digestion products PCR product purified reagent
Box purifying.To carry out flat end connection, need to carry out flat end processing to the DNA fragmentation of purifying, flat end reaction system is such as
Under (10 μ L)：PCAMBIA1301 after HindIII and EcoRI double digestion
>11 μ L, 10 × T4 DNA of μ l, 0.1%BSA of 0.1pmol, 1.7mmol/L dNTP Mixture
1 μ L, T4 DNA polymerase of polymerase buffer, 1 μ L, sterile water complement to 10 μ L.Above-mentioned reaction system is in 37 DEG C
1h is kept the temperature to complete the flat end of cohesive end.
Recirculation after linear carrier linked enzyme connection after flat end.Coupled reaction system is as follows：After flat end
3.5 μ L, 10 × Ligase buffer of linear carrier 1.5 μ L, T4 DNA Ligase (350U/ μ L) 1.5 μ L, sterile water complement to
15μL.16 DEG C of product after connection, converts bacillus coli DH 5 alpha competent cell overnight.Converted product is coated on containing Kan resistance
LB solid medium, 37 DEG C of overnight incubations.The single colonie that grows in picking resistant panel simultaneously extracts plasmid, with SacI and
SacII double digestion is identified, identifies that correct plasmid is named as pCAMBIA1301- Δ MCS.
Flat end will be carried out after restriction enzyme EcoRI and the HindIII digestion of the multiple cloning sites of pCAMBIA1301
Connection obtains plasmid pCAMBIA1301- Δ MCS, and carries out SacI and SacII double digestion mirror to pCAMBIA1301- Δ MCS
It is fixed.PCAMBIA1301- Δ MCS carrier forms the segment of 11837bp after SacI and SacII double digestion；pCAMBIA1301
Two segments are produced after SacI and SacII double digestion, a clip size is 9185bp, another segment is 2652bp
(Fig. 1).Thus speculate that the multiple cloning sites of pCAMBIA1301 carrier have been removed.
(2) in pCAMBIA1301- Δ MCS carrier gus gene sequence removal
Preliminary improved carrier pCAMBIA1301- Δ MCS is subjected to double digestion with NcoI and BstEII respectively to remove
Gus gene sequence.NcoI digestion system is following (100 μ L)：10 × K buffer
10.0 μ L, pCAMBIA1301- Δ MCS plasmid, 10 10 6 μ L of μ L, NcoI of μ L, BSA, sterile water complement to 100.0 μ
L.Reaction solution is mixed, after being centrifuged 30s, in 37 DEG C of endonuclease reaction 2h.
Endonuclease reaction liquid is recycled with PCR product Purification Kit, recovery product carries out digestion with BstEII later,
BstEII digestion system is following (20 μ L)：Returning after 10 × K buffer, 2.0 μ L, NcoI digestion pCAMBIA1301- Δ MCS plasmid
10 1 μ L of μ L, BstEII of product≤1 μ g, BSA is received, sterile water complements to 20.0 μ L.Reaction solution is mixed, it is anti-in 60 DEG C after centrifugation
Answer 2h, digestion products PCR product Purification Kit.
It is a pair of completely for following restriction enzyme site (NcoI EcoRI SalI KpnI BamHI SacI BstEII) synthesis
Complementary primer：
1301LinkerF：CATGGAATTCGTCGACGGTACCGGATCCGAGCTCG
1301LinkerR:GTCACCGAGCTCGGATCCGGTACCGTCGACGAATTC
By primer 1301LinkerF1 and 1301LinkerR after 60 DEG C of annealing 10min with above-mentioned linearisation after purification
Carrier carries out 16 DEG C of connections overnight, converts bacillus coli DH 5 alpha.Converted product is coated on kanamycins (50 μ g/ml of Kan) resistance
LB plate on, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium and stays overnight, extracts plasmid.Plasmid is sent
It is sequenced to Shanghai Sheng Gong Co., Ltd.Correctly improved pCAMBIA1301 vector plasmid will be sequenced to be named as
pCAMBIA1301-GUSless。
PCAMBIA1301- Δ MCS vector plasmid carries out digestion with NcoI and BstEII to remove GUS sequence, and to removal
The plasmid of GUS sequence carries out double digestion identification.Control vector pCAMBIA1301- Δ MCS is after NcoI and BstEII digestion, enzyme
Cut the small fragment of large fragment and a 2050bp that product is 9736bp；PCAMBIA1301-GUSless carrier through SacI and
The large fragment of 6700bp and the small fragment (Fig. 2) of about 3018bp are produced after SacII digestion.Digestion qualification result shows
GUS sequence in pCAMBIA1301-GUSless carrier has been removed.PCAMBIA1301-GUSless Vector map such as Fig. 3 institute
Show.
Conserved region II (AD1), basic leucine zipper area (bZIP) and the DPBF1 gene that 2.DPBF1 gene encodes are complete
The clone of sequence
(1) PCR amplification of DPBF1 gene conserved region II (AD1)
According to the DPBF1 gene order included in the sequence of pCAMBIA1301-GUSless plasmid and ncbi database, if
Count specific primer AD1F：CGGAATTCGGCAAGAACTTTGGGTCCAT and AD1R：GGGGTACC
CTCCTCTGCGTTCCAAATAG, primer both ends add restriction enzyme site EcoRI, KpnI and protection base, by bioengineering (on
Sea) Co., Ltd's synthesis.
Using pET32a-DPBF1 as the conserved region II of template primer amplified DPBF1 gene.The PCR of 50 μ L reacts
System is as follows：10 × PCR buffer (contain Mg2+), 5.0 4.0 μ L, TaKaRa rTaq DNA of μ L, dNTP (2.5mM)
Polymerase (5U/ μ L) 0.3 μ L, AD1-F (10 μM) 1.0 μ L, AD1-R (10 μM) 1.0 μ L, 1.0 μ L of template, sterile water are supplied
To 50.0 μ L.
Reaction condition：94 DEG C of initial denaturation 2min are recycled subsequently into 3step method：94 DEG C of denaturation 30sec, 40 DEG C of annealing
30sec, 72 DEG C of extension 5sec, after reacting 30 circulations, 72 DEG C of extension 10min；4℃99min.After the completion of PCR amplification, AD1 is taken
Pcr amplification product and DNA DL2000Marker electrophoresis is carried out in 1% Ago-Gel.100V electrophoresis 30min, it is ultraviolet
It is observed under lamp, Preliminary detection amplified production size.
(2) PCR amplification of DPBF1 basic leucine zipper area (bZIP)
According to the DPBF1 gene order included in the sequence of pCAMBIA1301-GUSless plasmid and ncbi database, if
Count specific primer DBDF：GGGGTACCAGGAAAAGAGTAGTGGATGGT and DBDR：
TCGAGCTCTTAGAGTGGACAACTCGGGT, restriction enzyme site KpnI, SacI and protection base are added in primer both ends, by biological work
The synthesis of journey (Shanghai) Co., Ltd..
The PCR amplification in the basic leucine zipper area (bZIP) of DPBF1 gene is carried out using pET32a-DPBF1 as template.50
The PCR reaction system of μ L is as follows：10 × PCR buffer (containing Mg2+) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, 0.5 μ L of template,
TaKaRa rTaq DNA polymerase (5U/ μ L) 0.3 μ L, DBD-F (10 μM) 1.0 μ L, DBD-R (10 μM) 1.0 μ L, it is sterile
Water complements to 50.0 μ L.
Reaction condition：94 DEG C of initial denaturation 2min are recycled subsequently into 3step method：94 DEG C of denaturation 30sec, 56 DEG C of annealing
30sec, 72 DEG C of extension 16sec, after reacting 30 circulations, 72 DEG C of extension 10min；4℃99min.After the completion of PCR amplification, PCR is taken
Amplified production and DNA DL2000 Marker carry out electrophoresis in 1% Ago-Gel.100V electrophoresis 30min, under ultraviolet lamp
Observation, Preliminary detection amplified production size.
(3) amplification of DPBF1 gene complete coding region
According to the DPBF1 gene order included in the sequence of pCAMBIA1301-GUSless plasmid and ncbi database, if
Count specific primer DPBF1F：5 '-CCGGAATTCACTAGAGAAACGAAGTTGACGT-3 and DPBF1R：
TCGAGCTCTTAGAGTGGACAACTCGGGT, restriction enzyme site EcoRI/SacI and protection base are added in primer both ends, by biology
The synthesis of engineering (Shanghai) Co., Ltd..
The PCR amplification of DPBF1 gene complete coding region is carried out using pET32a-DPBF1 as template.The PCR reactant of 50 μ L
It is as follows：10 × PCR buffer (containing Mg2+) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, 0.5 μ L, rTaq DNA of template
0.3 1.0 1.0 μ L of μ L, DPBF1-R (10 μM) of μ L, DPBF1-F (10 μM) of polymerase (5U/ μ L), sterile water complement to 50.0
μL。
Reaction condition：94 DEG C of initial denaturation 2min are recycled subsequently into 3step method：94 DEG C of denaturation 30sec, 56 DEG C of annealing
30sec, 72 DEG C of extension 1min18sec, after reacting 30 circulations, 72 DEG C of extension 10min；4℃99min.After the completion of PCR amplification,
Pcr amplification product and DNA DL2000 Marker is taken to carry out electrophoresis in 1% Ago-Gel.100V electrophoresis 30min is purple
It is observed under outer lamp, Preliminary detection amplified production size.
The pcr amplification product and DNA DL2000 Marker for taking each sequence carry out electrophoresis in 1% Ago-Gel,
100V electrophoresis 30min is observed under ultraviolet lamp.The fragment length of AD1 is 63bp, and the fragment length of bZIP is 303bp, and DPBF1 is compiled
The fragment length of code area overall length is 1323bp (Fig. 4).The molecular size range of pcr amplification product is consistent with expected theoretical value, tentatively
Determine that the PCR product amplified is required target fragment.
3. the building of Genetic Transformation in Higher Plants carrier pMX-1
PCR purified product and pCAMBIA1301-GUSless carrier to AD1 carry out EcoRI/KpnI double digestion, and root
Glue recycling is carried out to corresponding electrophoretic band according to the raw work gel reclaims kit in Shanghai.Amplification is produced with T4 DNA ligase later
Object is connected on carrier pCAMBIA1301-GUSless.Coupled reaction system is following (15 μ L)：Glue recycles the AD1 piece after digestion
4.5 μ L of section, 3.5 μ L, 10 × Ligase buffer of linearized vector pCAMBIA1301-GUSless after the digestion of glue recycling
1.5 μ L, T4 DNA Ligase (350U/ μ L) 1.5 μ L, sterile water complement to 15 μ L.
Reaction solution is mixed, overnight, connection product converts bacillus coli DH 5 alpha for 16 DEG C of connections.Converted product is coated on card, and that is mould
On the LB plate of plain (50 μ g/ml of Kan) resistance, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium mistake
Night, extracting plasmid obtain First Contact Connections product.
PCR purified product and above-mentioned First Contact Connections product to bZIP carry out KpnI/SacI double digestion, and root simultaneously
Glue recycling is carried out to corresponding electrophoretic band according to the raw work gel reclaims kit in Shanghai.By the glue of bZIP and First Contact Connections product
Recovery product converts bacillus coli DH 5 alpha in 16 DEG C of connections overnight, connection product.Converted product is coated on kanamycins (Kan 50
μ g/ml) resistance LB plate on, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium and stays overnight, extracts matter
Grain.The recombinant plasmid of extracting is named as pMX-1.PMX-1 plasmid is sent and is sequenced in Shanghai bioengineering Co., Ltd, is sequenced
As a result it analyzed, compared with DNAMAN software.Save the correct plasmid of sequencing result.
4. the building of Genetic Transformation in Higher Plants carrier pMX-4
PCR purified product and pCAMBIA1301-GUSless plasmid to the gene coding region DPBF1 complete sequence carry out
EcoRI/SacI double digestion, and glue recycling is carried out to corresponding electrophoretic band according to the raw work gel reclaims kit in Shanghai.It will
The glue recovery product of DPBF1 complete sequence and pCAMBIA1301-GUSless plasmid is in 16 DEG C of connections overnight.Coupled reaction system is such as
Shown in following table：The 4.5 μ L of DPBF1 genetic fragment of gel recycling, the linearized vector after digestion of gel recycling
3.5 μ L, 10 × Ligase buffer of pCAMBIA1301-GUSless 1.5 μ L, T4 DNA Ligase (350U/ μ L) 1.5 μ L,
Sterile water complements to 15 μ L.
Connection product converts bacillus coli DH 5 alpha.Converted product is coated on the LB of kanamycins (50 μ g/ml of Kan) resistance
On plate, 37 DEG C of overnight incubations.Picking is grown in the single colonie of resistant panel, shakes bacterium and stays overnight, extracts plasmid.By the recombination of extracting
Plasmid is named as pMX-4.PMX-4 plasmid is sent and is sequenced in Shanghai bioengineering Co., Ltd, sequencing result DNAMAN
Software is analyzed, is compared.Save the correct plasmid of sequencing result.
5.pMX-1, pMX-4 plasmid convert Agrobacterium
PMX-1, pMX-4 plasmid convert Agrobacterium GV3101 respectively, and converted product is coated on containing gentamicin (40 μ g/
Ml), on the LB solid medium of rifampin (25 μ g/ml) and kanamycins (50 μ g/ml), 30 DEG C are cultivated two days.It picks them separately
The single bacterium grown in resistant panel drops down onto 4ml and contains gentamicin (40 μ g/ml), rifampin (25 μ g/ml) and kanamycins
In the LB liquid medium of (50 μ g/ml), after 30 DEG C are shaken incubation 12 hours slowly, takes 1 μ L as pcr template, carry out bacterium colony PCR mirror
Determine positive colony.Bacterium colony PCR identifies that positive colony method is as follows：The sterile water of 10 μ L is added in the 1.5mL EP pipe of sterilizing,
Taking monoclonal colonies with white lancet choicest, gently piping and druming is uniformly mixed into the sterile water of 10 μ L, and sterile water, which can be observed, to be become
Light milky, the template for taking 1 μ L to react as PCR carry out PCR identification using Taq archaeal dna polymerase.
Using AD1 F and DBD R as specific primer, the agrobacterium liquid containing pMX-1 plasmid is template；With DPBF1 F and
DBD R is specific primer, and the agrobacterium liquid containing pMX-4 plasmid is that template carries out PCR amplification respectively, verifies insetion sequence.
PCR product carries out electrophoresis detection in Ago-Gel.PCR of the pMX-1 plasmid in Agrobacterium identifies that product sheet segment length is
PCR of 372bp, the pMX-4 plasmid in Agrobacterium identifies that product sheet segment length is 1323bp (Fig. 5).Recombinant plasmid pMX-1,
PMX-4 has been transferred in Agrobacterium.
6. During Agrobacterium arabidopsis wild type (Agrobacterium flower-dipping method arabidopsis thaliana transformation), steps are as follows：
(1) arabidopsis is grown to after bolting, and the top of arabidopsis main inflorescence is cut while paying attention to avoiding injuring leaf life
Inflorescence prepares dip dyeing when side shoot stretches out 2-10cm to induce the generation of side inflorescence.It is subject to the more bud that do not open, and
Watering on the day before to needing the plant disseminated to mention；
(2) by the Agrobacterium inoculation containing genetic transformation carrier in 5ml LB liquid medium (gentamicin (40 μ g/
Ml), rifampin (25 μ g/ml) and kanamycins (50 μ g/ml)) in, 28 DEG C, 15 hours left sides of 200r/min shaking table shake culture
It is right；
(3) by the culture in upper step with 1：50 ratio is transferred to the LB Liquid Culture that 200ml contains corresponding antibiotic
In base, 28 DEG C, 200r/min shaking table culture 15 hours or so to OD600=0.8-1.0；
(4) 4000r/min is centrifuged 15min and collects thallus, is resuspended with permeabilization buffer (sucrose of 1/2MS culture medium+5%)
Thallus, be added Silwett L-77 make its final concentration of 0.03%；
(5) agrobacterium suspension for containing plasmid to be transformed respectively is drawn with 5ml syringe, and ready arabidopsis is planted
Each flower of strain is disseminated.The plant disseminated (T0 is for plant) cover is got up to keep humidity with freshness protection package；
(6) after the Arabidopsis plant after dip dyeing being cultivated 12h under dark condition, remove freshness protection package, normal culture is arrived
Fruit pod is mature, repeats dip dyeing 2-3 times during which to improve transformation efficiency；
(7) after fruit pod turns yellow, before cracking, several fruit pods are collected into 1.5ml centrifuge tube according to label, collect arabidopsis kind
Sub (T1 generation) stores after sufficiently drying, to screen transformant.
(8) transgenic plant that the screening of conversion pMX-1 plasmid obtains is named as Arabidopsis thaliana
RDPBF1, the transgenic plant that conversion pMX-4 plasmid screening obtains are named as Arabidopsis thaliana DPBF1.
7. the screening and identification of seed
After the T1 after converting will be harvested for seed disinfection, it is layered on the 1/2MS solid medium containing 25 μ g/ml hygromycin.4
DEG C vernalization 2-3 moves back into plant illumination incubator normal light-exposed culture.Green plant (the bottle-green son of growth after two weeks
Leaf, root are longer) it may be transgenic positive plant, and non-transgenic plant cotyledon turns to be yellow, root is short, is unable to long-term surviving.It will be green
For color plantlet of transplant in soil, culture divides single plant to harvest seed to seed maturation (T2 is for seed).T2 is being contained into tide for seed
Continue screening on the MS culture medium of chloramphenicol resistance to T3 generation.
According to the sequence design specific primer of pMX-1 and pMX-4 plasmid to identify recombination in transgenic arabidopsis
In expression.It is extracted from and grows 7 days wild types and the RNA of transgenic arabidopsis seedling whole plant on 1/2MS culture medium, invert
For cDNA, identify recombination in transcriptional level by Reverse-Transcript PCR (RT-PCR) with specific primer
Expression.
RT F1：cgggggactcttgaccatgga
RT R1：ctgcgcattctcttctttcaactggt
Expression of the recombination in transgenic arabidopsis is analyzed by RT-PCR.The RT-PCR electrophoresis of transgenic arabidopsis
As a result the specificity band with corresponding molecular size range is showed, control group wild type does not have band (Fig. 6).Show to recombinate base
Because transcriptional expression can be carried out in transgenic arabidopsis.
Embodiment 3
It present embodiments provides a kind of DPBF1 Protein reconstitution segment and is promoting the application in arabidopsis Blooming.
(1) select identical flowerpot, be put into each flowerpot certain Nutrition Soil (Denmark's Pin Shi matrix 5,
PH5.5), the flowerpot for filling Nutrition Soil is placed in flat pallet, pallet adds water that the soil in disk in flowerpot is made independently to absorb water.
(2) by above-mentioned transgenic arabidopsis strain and Colombia's wild type seeds in 4 DEG C vernalization two days, dibbling is in soil
Surface, at concurrent 5, (24 ± 2 DEG C of temperature, humidity 20%, illumination condition is 16h illumination/8h dark, intensity of illumination for hot-house culture
5100lux).The undesirable seedling of upgrowth situation is removed after a week, and each flowerpot is made to retain seedling similar in 5 plants of upgrowth situations.
(3) greenhouse continues culture to blooming, and counts flowering time and upgrowth situation.
Plant bolting initially enters florescence, and wildtype Arabidopsis thaliana grows 30 days beginning boltings, transgenic arabidopsis
RDPBF1 and DPBF1 enters peduncle-growing period for rapeseed (Fig. 7) after growth 25 days and 27 days respectively.Transgenic arabidopsis rDPBF1 and DPBF1
The plant bolting time to show the phenotype bloomed ahead of time earlier than wild type.
Embodiment 4
It present embodiments provides a kind of DPBF1 Protein reconstitution segment and is improving the application in arabidopsis thousand grain weigth.
(1) select identical flowerpot, be put into each flowerpot certain Nutrition Soil (Denmark's Pin Shi matrix 5,
PH5.5), the flowerpot for filling Nutrition Soil is placed in flat pallet, pallet adds water that the soil in disk in flowerpot is made independently to absorb water.
(2) by the seed of above-mentioned transgenic arabidopsis strain and Colombia's wild type in 4 DEG C vernalization two days, dibbling is in soil
Earth surface, at concurrent 5, (24 ± 2 DEG C of temperature, humidity 20%, illumination condition is 16h illumination/8h dark, and illumination is strong for hot-house culture
Spend 5100lux).The undesirable seedling of upgrowth situation is removed after a week, and each flowerpot is made to retain seedling similar in 5 plants of upgrowth situations.
(3) continue culture to seed maturation, choose fruit pod similar in form size and collect seed.
(4) each 1,000, the seed of wild type and transgenic line are taken, weighing.It is averaged in triplicate.
Plant growth later period seed is mature, and form is obviously (Fig. 8) bigger than wild type after absorbing water for the seed of transgenic line.
1000 mature seeds of each strain are weighed to weigh：Wild type seeds mass of 1000 kernel 18mg, transgenic arabidopsis rDPBF1 seed
Mass of 1000 kernel 24mg, transgenic arabidopsis DPBF1 thousand grain weigth 20mg (Fig. 9).Statistical analysis shows transgenic line and open country
The thousand grain weigth of raw type has highly significant difference.
Embodiment 5
It present embodiments provides a kind of DPBF1 Protein reconstitution segment and is improving the application in arabidopsis drought resistance.
(1) select identical flowerpot, be put into each flowerpot equiponderant Nutrition Soil (Denmark's Pin Shi matrix 5,
PH5.5), the flowerpot for filling equivalent weight Nutrition Soil is placed in flat pallet, pallet adds water to make the nutrition in disk in flowerpot
Native autonomous water suction.
(2) by above-mentioned transgenic arabidopsis strain and Colombia's wild type seeds in 4 DEG C vernalization two days, dibbling is in soil
Surface, at concurrent 8, (24 ± 2 DEG C of temperature, humidity 20%, illumination condition is 16h illumination/8h dark, intensity of illumination for hot-house culture
5100lux).The undesirable seedling of upgrowth situation is removed after a week, and each flowerpot is made to retain seedling similar in 8 plants of upgrowth situations.
(3) after continuing culture 2 weeks, stop watering, drought stress is handled 10 days.
(4) arid rehydration, observation, statistics seedling survival situation after 1 week after 10 days.
To growth three weeks and the Arabidopsis thaliana Seedlings of the consistent wild type of condition of culture and transgenic line stopped watering, arid
Rehydration 1 week after 10 days, tolerance of the observation transgenic arabidopsis to arid.Wildtype Arabidopsis thaliana totally eight plants of seedling, eight plants of seedling
The dehydration of blade whole, withered death, i.e. survival rate of the wildtype Arabidopsis thaliana under drought stress is 0；Transgenic line
The survival rate of seedling of rDPBF1 is 87.5%, and the survival rate of seedling of transgenic line DPBF1 is 100%.Transgenic line blade
The active green of health is reverted to after rehydration, and can enter reproductive stage (Figure 10).
Beneficial effect：
(1) demonstrating the transgenic plant for being overexpressed the code area DPBF1 complete sequence and recombinant fragment for the first time has drought resistance.
(2) it is overexpressed the transgenic plant advance flowering period of recombinant fragment, shortens plant growth period, to be used for agricultural production
Early-medium maturing variety is cultivated.
(3) code area DPBF1 complete sequence is compared, recombinant fragment sequence is shorter.
(4) find that the recombinant fragment of the code area DPBF1 can promote seed morphology to increase, increase by thousand of seed for the first time
Weight, side, which reflects the recombinant fragment, can be improved yield
(5) code area DPBF1 complete sequence is compared, the transgenic line phenotype for being overexpressed recombinant fragment outstanding becomes apparent
(thousand grain weigth, advance flowering period).
The above is only the specific embodiment of the application, it is noted that for the ordinary skill people of the art
For member, under the premise of not departing from the application principle, several improvements and modifications can also be made, these improvements and modifications are also answered
It is considered as the protection scope of the application.
<110>Lanzhou University of Science & Technology
<120>A kind of DPBF1 recombinant fragment and its application
<160> 2
<210> 1
<211> 372
<212> DNA
<213>Arabidopsis（Arabidopsis thaliana）
<400> 1
ggcaagaactttgggtccatgaacatggacgagtttcttgtctctatttggaacgcagaggagggtaccagga
aaagagtagtggatggtccagtggagaaagtagtggagagaagacagaggaggatgatcaagaaccgcgagtctgct
gctagatctagagcaagaaaacaagcatatacagtggaattggaagctgaacttaaccagttgaaagaagagaatgc
gcagctaaaacatgcattggcggagttggagaggaagaggaagcaacagtattttgagagtttgaagtcaagggcac
aaccgaaattgccgaaatcgaacgggagattgcggacattgatgaggaacccgagttgtccactctaa
<210> 1
<211> 1323
<212> DNA
<213>Arabidopsis（Arabidopsis thaliana）
<400> 2
atggtaactagagaaacgaagttgacgtcagagcgagaagtagagtcgtccatggcgcaagcgagacataatg
gaggaggtggtggtgagaatcatccgtttacttctttgggaagacaatcctctatctactcattgacccttgacgag
ttccaacatgctttatgtgagaacggcaagaactttgggtccatgaacatggacgagtttcttgtctctatttggaa
cgcagaggagaataataacaatcaacaacaagcagcagcagctgcaggttcacattctgttccggctaatcacaatg
gtttcaacaacaacaataacaatggaggcgagggtggtgttggtgtctttagtggtggttctagaggcaacgaagat
gctaacaataagagagggatagcgaacgagtctagtcttcctcgacaaggctctttgacacttccagctccgctttg
taggaagactgttgatgaggtttggtctgagatacatagaggtggtggtagcggtaatggaggagacagcaatggac
gtagtagtagtagtaatggacagaacaatgctcagaacggcggtgagactgcggctagacaaccgacttttggagag
atgacacttgaggatttcttggtgaaggctggtgtggttagagaacatcccactaatcctaaacctaatccaaaccc
gaaccaaaaccaaaacccgtctagtgtaatacccgcagctgcacagcaacagctttatggtgtgtttcaaggaaccg
gtgatccttcattcccgggtcaagctatgggtgtgggtgacccatcaggttatgctaaaaggacaggaggaggaggg
tatcagcaggcgccaccagttcaggcaggtgtttgctatggaggtggcgttgggtttggagcgggtggacagcaaat
gggaatggttggaccgttaagcccggtgtcttcagatggattaggacatggacaagtggataacataggaggtcagt
atggagtagatatgggagggctaaggggaaggaaaagagtagtggatggtccagtggagaaagtagtggagagaaga
cagaggaggatgatcaagaaccgcgagtctgctgctagatctagagcaagaaaacaagcatatacagtggaattgga
agctgaacttaaccagttgaaagaagagaatgcgcagctaaaacatgcattggcggagttggagaggaagaggaagc
aacagtattttgagagtttgaagtcaagggcacaaccgaaattgccgaaatcgaacgggagattgcggacattgatg
aggaacccgagttgtccactctaa
Claims (6)
1. a kind of DPBF1 recombinant fragment, which is characterized in that the DPBF1 recombinant fragment nucleotide sequence such as sequence table SEQ ID
NO：Shown in 1.
2. a kind of acquisition methods of DPBF1 recombinant fragment, which is characterized in that include the following steps：
(1) transformation of pCAMBIA1301 carrier；(2) conserved region II (AD1), the basic leucine zipper area of DPBF1 gene coding
(bZIP) and the clone of the gene coding region DPBF1 complete sequence；(3) building of Genetic Transformation in Higher Plants carrier pMX-1；(4) plant
The building of genetic transformation carrier pMX-4；(5) pMX-1, pMX-4 plasmid convert Agrobacterium；(6) During Agrobacterium arabidopsis brother human relations
It is more environmental than sub-；(7) screening and identification of transgenic line seed.
3. a kind of acquisition methods of DPBF1 recombinant fragment according to claim 2, it is characterised in that step (1) packet
It includes：(1) in pCAMBIA1301 carrier multiple cloning sites removal；(2) gus gene sequence in pCAMBIA1301- Δ MCS carrier
Removal.
4. such as sequence table SEQ ID NO：DPBF1 recombinant fragment described in 1 is promoting the application in arabidopsis Blooming.
5. such as sequence table SEQ ID NO：DPBF1 recombinant fragment described in 1 is improving the application in arabidopsis thousand grain weigth.
6. such as sequence table SEQ ID NO：DPBF1 recombinant fragment described in 1 is improving the application in arabidopsis drought resistance.
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| CN113234730A (en) * | 2021-05-11 | 2021-08-10 | 兰州理工大学 | Recombinant gene fragment 4IIA1bZIP and application thereof |
| CN113234730B (en) * | 2021-05-11 | 2023-11-10 | 兰州理工大学 | Recombinant gene fragment 4IIA1bZIP and application thereof |
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