CN102943075B - Gossypium hirsutum GhTCP2 gene promoter and application thereof - Google Patents
Gossypium hirsutum GhTCP2 gene promoter and application thereof Download PDFInfo
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Abstract
The invention relates to a gossypium hirsutum GhTCP2 gene promoter and application thereof. The sequence of the gossypium hirsutum GhTCP2 gene promoter is shown as SEQ ID No. 1. The invention discloses that the GhTCP2 gene promoter can make specific expression in axillary buds and flower buds of plants (arabidopsis, gossypium hirsutum, etc.), can provide a promoter for specific expression of an insect-resistant gene in gossypium hirsutum axillary buds and flower buds, helps to reduce the negative impact on gossypium hirsutum resulted from insect-resistant gene expression in other tissues, and improve the insect-resistant ability of gossypium hirsutum under the premise of without influence on cotton yield.
Description
Technical field
The invention belongs to genetically engineered field, specifically, relate to the application of cotton GhTCP2 gene promoter and tissue specific expression pattern thereof.
Background technology
Cotton (Gossypium Hirsutum) is as one of global most important cash crop, and its fiber is the main raw material(s) of mankind's textile industry.Meanwhile, the seed of cotton and oil plant are also indispensable good resources in the daily productive life of people, aspect livestock industry and diesel production, are playing an important role.But cotton diseases and insect pests is a lot, especially insect pest, directly affects output of cotton.Wherein, bollworm is important borer pest of cotton cotton buds and bolls phase, and main moth food flower bud, flower, bell, also take food tender leaf.This worm is the sociales of Chinese cotton region cotton buds and bolls phase insect, in recent years causes harm very rampant, and prediction and the integrated control of doing bollworm well seem very important.
TCP2 gene is under the jurisdiction of TCP transcription factor family, the TCP family member who finds the earliest comprises corn teosinte branched 1(TBI) cycloidea(CYC of gene, Common Snapdragon) gene and paddy rice PCF1, PCF2 gene, the ascend the throne origin of this family's title of these three members' english abbreviation initial group.TCP transcription factor protein member has unique secondary structure, the conservative DNA structure territory (TCPdomain) that approximately 60 amino acid forms is contained at its middle part, form an atypical alkaline helix-loop-helix structure [non-canonicalbasic-Helix-Loop-Helix (bHLH) structure], may participate in the dimerization of protein and the function of being combined with DNA.
Progress shows, TCP family member mainly expresses at the position of the Fast Growths such as the floral organ of plant, axil, apical meristem, axillalry bud, and this phenomenon shows, TCP family plays an important role in may and growing in cell fission.
Insect pest is one of important factor affecting output of cotton, and bollworm mainly takes food tender leaf and the square of cotton.The Bt gene that transgenic cotton against pests carries can effectively suppress bollworm infringement.The Bt gene of the transgenic cotton against pests of selling is in the market that the 35S promoter by Caulimovirus drives, plant, each has organized expression to this promotor, make its hetero-organization or the organ overexpression allos Bt gene that by bollworm, are not taken food, may be to the growing of cotton, the ability of resisting biological and abiotic stress impacts.
The GhTCP2 promotor the present invention relates to can replace the 35S promoter of the Caulimovirus in the transgene cotton of widespread use in the market, allow the main position that anti insect gene Bt takes food bollworm express, reduced the impact of anti insect gene ability of other except insect resistance capacity on transgene cotton.
Summary of the invention
The object of this invention is to provide cotton GhTCP2 gene promoter sequence, its tissue specific expression pattern and application.
The invention provides cotton GhTCP2 gene promoter, it has:
1) nucleotide sequence shown in SEQ ID No.1; Or
2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks and/or increases one or several Nucleotide; Or
3) under stringent condition with 1) nucleotide sequence of the DNA sequence dna hybridization that limits.
The invention provides the carrier that contains cotton GhTCP2 gene promoter.
In an embodiment of the present invention, the carrier that contains cotton GhTCP2 gene promoter is pBI121-GhTCP2.
The present invention also provides the host cell that contains above-mentioned carrier.
The present invention also provides the transformed plant cells that contains cotton GhTCP2 gene promoter.
The invention provides the application of cotton GhTCP2 gene promoter in preparation transgenic plant.
The invention provides the application of cotton GhTCP2 gene promoter expression pattern in plant tissue.
Wherein, described plant tissue is axillalry bud or bud.
Wherein, described expression pattern is for expressing anti insect gene.
Wherein, described plant is Arabidopis thaliana, paddy rice or cotton.
The present invention has following advantages and beneficial effect:
(1) the invention provides cotton GhTCP2 gene promoter (nucleotide sequence is as shown in SEQID No.1) and tissue specific expression pattern in plant axillalry bud and bud.
(2) by arabidopsis thaliana transformation wild-type Col-0, and carry out GUS dyeing, find GhTCP2 specifically expressing (Arabidopis thaliana, paddy rice, cotton etc.) in plant axillalry bud and bud, be expected to providing special promotor for express anti insect gene in the tender cotton boll of Young Cotton, to improve the insect resistance capacity of cotton.
Accompanying drawing explanation
Fig. 1 is GhTCP2 gene cotton different sites sxemiquantitative PCR result.
Fig. 2 is the carrier collection of illustrative plates that contains cotton GhTCP2 gene promoter.
Fig. 3 is that the carrier enzyme that contains cotton GhTCP2 gene promoter is cut the result; Wherein 1 with the enzyme of EcoRV and BamHI double digestion, cut result for empty carrier pBI121; 2 cut result with EcoRV and BamHI double digestion enzyme for pBI121-GhTCP2 carrier; M represents the 1kb DNA ladder of Bo Maide Bioisystech Co., Ltd.
Fig. 4 is GUS coloration result after cotton GhTCP2 gene promoter driving gus gene arabidopsis thaliana transformation, wherein 1. in Arabidopsis thaliana Seedlings, expresses; 2. at the tender root tip of children, divide expression; 3. in whole floral organ, there is expression; 4. at axil place, express; 5., after floral organ is amplified, find that expression amount is stronger in the tender bud of children.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art; If do not specialize, in embodiment, agents useful for same is commercially available.
The acquisition of embodiment 1 cotton GhTCP2 gene promoter
GhTCP2 gene promoter of the present invention is to adopt the new land of upland cotton (Gossypium hirsutum) kind that Genome Walking method buys from the market early to clone in No. 13 and obtain.Conserved sequence (its sequence is as shown in SEQ ID No.2), 5 ' RACE and 3 ' RACE flanking sequence in the middle of the GhTCP2 gene that before this laboratory, amplification obtains are spliced to primers GhTCP2ProSP1, the GhTCP2ProSP2 and the GhTCP2ProSP3 that according to splicing, obtain with SeqMan software.The total DNA of cotton gene group of take is template, with Genome Walking Kit AP primer of the same race, carries out nest-type PRC reaction amplification gene promoter region 3 times.By after 1000 times of 1stPCR reaction solution dilutions, get 1 μ L as the template of 2nd PCR reaction, preparation 2ndPCR reaction solution.Get after 1000 times of 2nd reaction solution dilutions, get 1 μ L as the template of 3rdPCR reaction, preparation 3rdPCR reaction solution.With four kinds of AP primers that provide in test kit, carry out three nest-type PRC reactions respectively, the sepharose with 1% carries out electrophoresis detection.Operation steps is as follows:
(1) preparation 1st PCR reaction solution:
Reaction conditions: 94 ℃ of 1min; 98 ℃ of 1min; 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 5 circulations; 94 ℃ of 30s; 25 ℃ of 3min; 72 ℃ of 2min; 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, 15 circulations; 72 ℃ of 10min.By after 1000 times of 1st PCR reaction solution dilutions, get 1 μ L as the template of 2nd PCR reaction.(2) preparation 2nd PCR reaction solution
Reaction conditions: 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 65 ℃ of 1min, 72 ℃ of 2min, 94 ℃ of 30s, 44 ℃ of 1min, 72 ℃ of 2min, 15 circulations; 72 ℃ of 10min.
By after 1000 times of 2nd PCR reaction solution dilutions, get 1 μ L as the template of 3rd PCR reaction.
(3) preparation 3rd PCR reaction solution, with 2nd PCR reaction solution application of sample amount, template is the 2nd PCR reaction product after diluting, gene specific primer GhTCP2ProSP3, reaction conditions, with 2nd PCR reaction conditions.
Get 1st, 2nd, each 5 μ L of 3rd PCR reaction solution, the sepharose of use 1% carries out electrophoresis.
Test kit comprises that 4 kinds of AP primers should carry out respectively nest-type PRC, and the 3rdPCR that gets band maximum cuts glue and reclaims electrophoretic band.Then be connected with T carrier (pMD19-T vector), transform competent escherichia coli cell, propose plasmid enzyme restriction checking.
The sequence correct according to order-checking, design primer GhTCP2PFF and GhTCP2PFR, take genomic dna as template, with TaKaRa PrimeSTAR HS DNA Polymerase withGC Bufer high-fidelity Taq enzymatic amplification promoter region.
50 μ L reaction systems are as follows:
Reaction conditions is: 98 ℃ of sex change 10s, and 60 ℃ of annealing 5s, 72 ℃ are extended 2min, 35 circulations.PCR product detects with 1% agarose gel electrophoresis, reclaims object band.Reclaim product and be connected with pEASY-Blunt Simpl Cloning Kit, transform intestinal bacteria, upgrading grain carries out enzyme and cuts evaluation, will identify that correct result checks order.
The primer sequence that table 1 the present invention relates to
Sequencing result shows, sequence 3 ' end matches with GhTCP2 sequence, illustrates that this sequence is the promoter region of GhTCP2 gene really, this promotor total length 1681bp, and its sequence, as shown in SEQID No.1, is wherein forgiven 5 ' the UTR district of 156bp.In GenBank, compare, found that this sequence is cloned into first.
Embodiment 2 cotton GhTCP2 gene sxemiquantitative PCR
1, use hundred Tyke general plant total RNA extraction reagent boxes (centrifugal column type) to extract total RNA of new land No. 13 Radix Gossypiis morning of 85 days of growth, stem, leaf, terminal bud, lateral bud, bud, concrete operation step is as follows:
(1) get 100mg plant tissue and grind fast fully in liquid nitrogen, in process of lapping, will keep in mortar, there is all the time liquid nitrogen.
(2) sample that grinds to form homogenate is transferred in 1.5mL RNase free centrifuge tube, adds 1mL lysate to put upside down and mix, 65 ℃ of incubation 5min so that ribosome decompose completely.4 ℃, the centrifugal 10min of 12000rpm, gets supernatant and moves to RNase free Filter column.
(3) 4 ℃, the centrifugal 45s of 10000rpm, collects lower filtrate in collection tube.
(4) at collection tube, add 1 times of volume 70% ethanol to mix, the solution obtaining is proceeded in adsorption column together with precipitation.
(5) 4 ℃, the centrifugal 45s of 10000rpm, discards waste liquid, and adsorption column is recovered to collection tube again.
(6) to adsorption column central authorities, add the DNase I working fluid of 80 μ L, room temperature is placed 15min.
(7) add 500 μ L protein liquid removals, 4 ℃ of 12 centrifugal 45s of 000rpm, discards waste liquid.
(8) add 700 μ L rinsing liquids, 4 ℃ of 12 centrifugal 45s of 000rpm, discards waste liquid.
(9) add 500 μ L rinsing liquids, 4 ℃ of 12 centrifugal 45s of 000rpm, discards waste liquid.
(10) adsorption column is put back in sky collection tube, 4 ℃ of 12 centrifugal 2min of 000rpm removes rinsing liquid as far as possible.
(11) take out adsorption column and put into the RNase free water that drips appropriate 65 ℃ of incubations without RNase centrifuge tube to adsorption film mid-way, room temperature is placed after 2min, and the centrifugal 1min of 12 000rpm collects RNA.
2, take the cDNA at each position of cotton is template, is sxemiquantitative RT-PCR, detects gene at the expression at each position of plant.
(1) cDNA the first chain is synthetic
1. at RNase free centrifuge tube, prepare following mixed solution:
2. 70 ℃ insulation 5min after rapidly on ice more than chilling 2min; Centrifugal several seconds mixed solution is gathered in pipe bottom;
3. add other inverse transcription reaction liquid:
4. 42 ℃, 60min; After 1～100 times of the cDNA solution dilution that obtains for pcr amplification.
(2) sxemiquantitative RT-PCR
Using cotton polyubiquitin gene (GhUBI) (AY189972) as the interior mark of RT-PCR reaction, and primer GhTCP2RTF and the GhTCP2RTR of design increase.Design gene specific upstream and downstream primer is as follows:
GhUBIF：5’-AAGACCTACACCAAGCCCAA-3’
GhUBIR：5’-AAGTGAGCCCACACTTACCA-3’
GhTCP2RTF：5’-CAAACTCAGTTCATGGTGTTC-3’
GhTCP2RTR：5’-CAGAAACCCAGTGATCTGATG-3’
First amplification interior label, adjusts PCR reaction system template add-on, makes the amount of amplification interior label amplified production identical.Use again the transcriptional expression level of corresponding template amount augmentation detection goal gene.The reaction system following (20 μ L) of PCR:
Reaction conditions is as follows: 94 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 30s, 25～40 circulations; 72 ℃ of 10min.Wherein 25 circulations of polyubiquitin gene (GhUBI) amplification, GhTCP2 gene amplification 40 circulations.Agarose gel electrophoresis with 2% detects the amplification of PCR.
Sxemiquantitative PCR result shows (Fig. 1), and GhTCP2 gene is specifically expressing in the axillalry bud of cotton and bud mainly, also has a small amount of expression in terminal bud.
Embodiment 3 cotton GhTCP2 gene promoter arabidopsis thaliana transformations
GhTCP2 gene promoter is cloned into plant expression vector pBI121(to be so kind as to give by the ocean academician of Lee family of Inst. of Genetics and Development Biology, CAS) obtain expression vector pBI121-GhTCP2, and in bacillus coli DH 5 alpha, expand numerous.By agrobacterium mediation converted method, the GhTCP2 gene promoter that pBI121 is carried proceeds to Arabidopis thaliana, obtains altogether 20 strain transgenic arabidopsis.
Gene clone is as follows to the process that carrier, carrier expand numerous and agrobacterium mediation converted in bacillus coli DH 5 alpha:
1, the structure of carrier
According to GhTCP2 gene promoter sequence design upstream and downstream primer GhTCP2ProEBF and GhTCP2ProEBR, by PCR, obtain GhTCP2 promoter fragment, at 5 ' end, introduce the flat end of EcoRV(), 3 ' end is introduced BamHI, front 7 AA containing GhTCP2, merge with gus gene translation skill.
50 μ L reaction systems are as follows:
PCR product is reclaimed, be connected with pEASY-Blunt Simpl Cloning Kit, transform competent escherichia coli cell, extract plasmid enzyme restriction and identify, will identify that correct bacterial strain is sent to company's order-checking.The bacterial strain that order-checking is correct extracts plasmid, with EcoRV and BamHI double digestion, reclaims GhTCP2 promotor object fragment.First with HindIII is mono-, cut, after cutting completely, with Klenow DNApolymerase, mend into flush end, heated and inactivated Klenow, then cut with BamHI is mono-, reclaim large fragment as carrier.The GhTCP2 promoter fragment of acquisition is connected and is spent the night with T4 ligase enzyme with carrier segments.
Linked system:
2, in bacillus coli DH 5 alpha, to expand numerous process as follows for carrier:
The molar weight of plasmid vector and external source segment is 1:3.Connection proceeds in DH5 α after spending the night, and the flat board being coated onto containing kalamycin resistance carries out positive-selecting.
The positive colony screening is cut evaluation with enzyme.Owing to respectively containing an EcoRI restriction enzyme site after GhTCP2 promotor 5 ' end and Transcription Termination point, so adopt EcoRI to enter single endonuclease digestion, detect, should cut 3699bp size strip.Carrier collection of illustrative plates is as Fig. 2, and enzyme is cut the result as Fig. 3.Finally the vector plasmid building is transformed to Agrobacterium GV3101.
3, the step of conversion Agrobacterium competent cell is as follows:
(1) get the Agrobacterium of-70 ℃ of preservations in the flat lining out of YEP containing 50 μ g/mL Rifampins (Rifampicin), cultivate 2 ~ 3d for 28 ℃.
(2) picking list colony inoculation is in 5mL YEP liquid nutrient medium, 28 ℃ of shaking culture 12 ~ 16h of 220rpm.
(3) get 2mL bacterium liquid and transfer in 100mL YEP liquid nutrient medium, 28 ℃ of 220rpm shaking culture are to OD
600=0.4 ~ 0.6.
(4) proceed to aseptic centrifuge tube, the centrifugal 5min of 5000rpm, abandons supernatant.
(5) every 50mL bacterium liquid adds the 0.1M CaCl2 solution of 5mL precooling, and suspension cell, places 20min on ice gently.4 ℃ of centrifugal 5min of 5000rpm, abandon supernatant.
(6) every 50mL bacterium liquid adds the 0.1M CaCl2 solution containing 15% glycerine of 2mL precooling, gently suspension cell.
(7) agrobacterium suspension is sub-packed in the aseptic EP pipe of 1.5mL, every pipe 100 μ L(can be frozen in-70 ℃).
(8) plasmid DNA of getting about 1 μ g joins in 100 μ L Agrobacterium competent cells, mixes rear ice bath 30min.
(9) liquid nitrogen flash freezer 5min, then at 37 ℃ of water-bath 5min.
(10) horse back ice bath 2min, adds 800 μ L YEP liquid nutrient mediums, after 28 ℃ of 175rpm shaking culture 2 ~ 4h, is coated in containing on the YEP solid plate of 50 μ g/ml kantlex, cultivates 2 ~ 3d to forming single bacterium colony for 28 ℃.
(11) picking list bacterium colony, PCR checking.Positive colony is for conversion of plant.
4, agriculture bacillus mediated Arabidopis thaliana Col-0 conversion process is as follows:
Arabidopis thaliana acceptor material is prepared: Arabidopis thaliana (buying from Arabidopis thaliana biological study center (arabidopsis biological research centre)) is transplanted, its main tongue grows to 10 ~ 15cm, generates 1 ~ 2 angle and can be used for conversion test really time.For obtaining more inflorescence, terminal bud can be removed, impel more lateral bud growths, before conversion, cut off the bud of having bloomed and the angle having grown up to really.
Picking activated and the mono-bacterium colony of GV3101 that contains object carrier in 5mL YEB liquid nutrient medium (kantlex 50mg/L, Rifampin 50mg/L) in, 12 ~ 16h is cultivated in 28 ℃ of 220rpm concussions, get 20 ~ 60 μ L bacterium liquid and be coated with and contain the upper 28 ℃ of dark culturing 2 ~ 3d of corresponding antibiotic flat board, collection thalline is dissolved in 30mL liquid YEB makes OD
600value reaches 2.0, by bacterium liquid join containing in the plastics bag of 120mL conversion medium for transforming.
Inflorescence is immersed to conversion fluid 10s, shake off gently unnecessary bacterium liquid, be sidelong and 16 ~ 24h is processed in lucifuge moisturizing, the plant having transformed is put into and on culturing rack, continues illumination cultivation.After 1 week with same method again transfection once.Collect T
0for seed.
5, the screening process of transgenic positive seedling is as follows:
T
0for seed, after sterilization, be resuspended in 0.05% agar-agar soln (40 μ L seed/ml agar).Containing card, receiving the MS culture medium flat plate cover plant 1mL mixture of mycin 90x90x25mm, 4 ℃ of refrigerator vernalization 3d, transgenic positive seedling (T
0for plant) can grow up to true leaf, the seedling containing goal gene fragment can not survive, by positive transplantation of seedlings.
The GUS dyeing of embodiment 4 cotton GhTCP2 gene promoter arabidopsis thaliana transformation positive plants
Choose the transgenic plant in the embodiment 3 of different development stage, carry out GUS tissue staining, detect tissue expression.Method is as follows:
(1) different sites of getting the transgenic arabidopsis plant that fresh evaluation is correct is placed in 15mL centrifuge tube.
(2) in each pipe, add GUS staining fluid to make its submerged plant sample;
(3) open centrifuge tube lid, vacuumize 30min;
(4) cover centrifuge tube lid, with masking foil, wrap up centrifugal tube wall and make its lucifuge, be placed in 37 ℃ of water-baths and spend the night, sample is fully dyeed;
(5) with dehydrated alcohol decolouring for several times, until the green of sample disappears;
(6) with Stereo microscope, observe the painted situation of sample and take pictures.
GUS coloration result shows (Fig. 4), and in seedling stage, GUS mainly expresses in the tip of a root, limb edge and the vegetative point of plant, and in the ripening stage, GUS is specifically expressing in axillalry bud and young tender bud mainly.Above experimental result shows, GhTCP2 gene is except seedling stage great expression, in the axillalry bud of maturation plant and the tender bud of children, has stronger expression, in blade and stem stalk, do not express.So those skilled in the art can provide promotor by specific expressed anti insect gene according to GhTCP2 gene promoter in cotton axillalry bud and bud, reduce the negative impact that anti insect gene produces cotton at other tissue expressions, do not affecting under the prerequisite of output of cotton, the insect resistance capacity that improves cotton, is on purpose applied to GhTCP2 gene promoter the research and development of transgenic cotton against pests.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. cotton GhTCP2 gene promoter, is characterized in that, its nucleotides sequence is classified as: the nucleotide sequence shown in SEQ ID No.1.
2. the carrier that contains gene promoter described in claim 1.
3. carrier as claimed in claim 2, it is pBI121-GhTCP2.
4. the application of gene promoter claimed in claim 1 in preparation transgenic plant, described plant is Arabidopis thaliana or cotton.
5. the application of gene promoter claimed in claim 1 expression pattern in plant tissue, described plant is Arabidopis thaliana or cotton.
6. application as claimed in claim 5, described plant tissue is axillalry bud or bud.
7. application as claimed in claim 5, described expression pattern is for expressing anti insect gene.
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