CN106191068A - The promoter of Soybean Root specifically expressing and application thereof - Google Patents

The promoter of Soybean Root specifically expressing and application thereof Download PDF

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CN106191068A
CN106191068A CN201610830389.1A CN201610830389A CN106191068A CN 106191068 A CN106191068 A CN 106191068A CN 201610830389 A CN201610830389 A CN 201610830389A CN 106191068 A CN106191068 A CN 106191068A
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promoter
biomaterial
root
expression
application
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傅永福
张晓玫
程志远
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific

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  • Life Sciences & Earth Sciences (AREA)
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  • Wood Science & Technology (AREA)
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  • Biotechnology (AREA)
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  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention provides the promoter of a kind of Soybean Root specifically expressing, named GmPHT1.01, its nucleotide sequence, as shown in SEQ ID No.1, present invention also offers the application in regulation and control downstream gene expression of the described promoter.By Agrobacterium rhizogenes instantaneous conversion system, with GmPHT1.01 promoter expression gus gene, result shows that GmPHT1.01 promoter can be obviously promoted the expression of gus gene in plant roots, thus GmPHT1.01 promoter has specificity in plant roots, it is suitable in the root of the plants such as various crops, forest, vegetable, flowers, herbage specifically expressing target gene and improves the expression of genes of interest.

Description

The promoter of Soybean Root specifically expressing and application thereof
Technical field
The present invention relates to plant genetic engineering field, particularly relate to a kind of Soybean Root and root nodule specifically expressing GmPHT1.01 promoter and application thereof.
Background technology
Promoter is cis acting element important in gene, is normally at tens, transcriptional start site upstream base Place.Promoter can be divided into three kinds according to transcriptional profile difference: constitutive promoter, tissue or organ specific promoters and induction Type promoter.The activity of tissue or organ specific promoters is to be lured by specific tissue cellularity and chemistry, physical signalling Leading regulation, the expression of the most this kind of promoter has the most spatiotemporal.Under the driving of this kind of promoter, the expression of gene is past Toward being only limited to some specific organ or tissue position or specific developmental stage.Tissue or organ specific promoters can not only Make the expression product of genes of interest in the position accumulation of certain organ or tissue, improve Zonal expression amount, mesh can also be avoided simultaneously Mark gene expresses the adverse effect caused in other histoorgans.
Up to the present, existing substantial amounts of tissue or organ specific promoters are separated, special including blade Different promoter, flower specific promoter and seed-specific expression promoter and root-specific promoter etc..Root is that plant absorbs moisture and battalion Supporting the vitals of material, root specific expression systems can be used for studying the height of plant and oozes stress-tolerance, phytoremediation and rhizosphere and divide Secrete.Root-specific promoter mas2, GFP and tobacco calreticulin (calreticulin) the gene constructed fusion such as Borisjuk Expression vector.Transgene tobacco water planting result of study shows: root cells can not only efficiently produce GFP, and can be by purpose egg White matter is secreted in fluid medium (Borisjuk et al., 1999).Additionally, answer rare talent etc. to use pinaster root specificity to open Mover PmPgRP10 drives CMO/BADH bivalent gene and proceeds to Oryza sativa L..CMO enzyme, the BADH enzyme of transfer-gen plant root and leaf is lived Property and other physiological and biochemical index be determined, result shows: CMO/BADH bivalent gene can express at root specificity (should Rare talent, 2006).
GmPHT1.01 gene belongs to the family member of plant phosphorus transport protein, rises emphatically in the transport process of regulation and control phosphorus The effect wanted.It is therefore desirable to research and the promoter sequence of acquisition regulating and controlling soybean phosphorus transporter gene GmPHT1.01, thus it is deep Enter to study plant phosphorus transport process and effective means and approach are provided.
Summary of the invention
It is an object of the invention to provide the promoter sequence of regulating and controlling soybean phosphorus transporter gene GmPHT1.01, thus be In the root of the plants such as various crops, forest, vegetable, flowers, herbage, specifically expressing target gene provides effective way.
For reaching object above, the invention provides the GmPHT1.01 promoter of a kind of Soybean Root specifically expressing, its tool Have:
1) nucleotide sequence shown in SEQ ID NO.1；Or
2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks and/or adds one or several nucleotide and obtained Have equal function by 1) derivative nucleotide sequence.
Should be appreciated that those skilled in the art can opening according to Semen sojae atricolor phosphorus transporter gene GmPHT1.01 disclosed by the invention Mover (SEQ ID No.1), on the premise of not affecting its activity, replaces, lacks and/or increases one or several nucleotide, Obtain the mutant nucleotide sequence of described promoter.Such as at nonactive section, in the case of not changing promoter function, carry out following At least one nucleotide sequence obtained in replacement mode: the T of the 110th is substituted by A, replaces the A of the 2601st For T, the A of the 3004th is substituted by T.
Present invention also offers the carrier containing GmPHT1.01 promoter, host cell, conversion plant cell or expression Box.
Various carrier known in the art is can be selected in the present invention, as long as being suitable for the expression of GmPHT1.01 promoter, Can be such as commercially available carrier and plasmid.
Biomaterial of the present invention is carrier, host cell, conversion plant cell or expression cassette.Present invention also offers GmPHT1.01 promoter or the above-mentioned biomaterial containing it are regulating and controlling downstream gene answering in specifically expressing in plant roots With.
Present invention also offers GmPHT1.01 promoter or the above-mentioned biomaterial containing it is being planted at raising downstream gene Application in expression in thing root.
Present invention also offers GmPHT1.01 promoter or contain its above-mentioned biomaterial in preparing transgenic plant Application.
Present invention also offers GmPHT1.01 promoter or contain its above-mentioned biomaterial in plant roots Growth adjustment Application.
Present invention also offers GmPHT1.01 promoter or the above-mentioned biomaterial containing it is improved at plant germplasm resource In application.
Present invention also offers GmPHT1.01 promoter or above-mentioned biomaterial the answering in plant breeding containing it With.
Preferably, above-mentioned plant is crop, forest, vegetable, flowers, herbage.
It is highly preferred that above-mentioned plant is crop.Most preferably Semen sojae atricolor.
Present invention also offers clone from soybean gene group to obtain used by the GmPHT1.01 promoter sequence of 5240bp Specific primer, it includes forward primer: 5 '-attgGAGCTCGTGTCCCTTTGTATGATGGAATC-3 '；And reverse primer: 5’-gagtCTGCAGCACTCACTAACTCAGCTACCTGA-3’。
Test kit containing above-mentioned specific primer falls within protection scope of the present invention.
Above-mentioned specific primer can also be used for detecting the startup of Semen sojae atricolor phosphorus transporter gene GmPHT1.01 of the present invention Son.
The present invention has separated the GmPHT1.01 promoter of Soybean Root specifically expressing first, utilizes GmPHT1.01 to start sublist Reaching gus gene, result shows that GmPHT1.01 promoter can be obviously promoted the expression of gus gene in plant roots, thus, GmPHT1.01 promoter has specificity in plant roots, is suitable for plants such as various crops, forest, vegetable, flowers, herbages Specifically expressing target gene the expression of target gene can be improved in root.
Accompanying drawing explanation
Fig. 1 is GmPHT1.01-GUS expression of results in Soybean Root.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
The clone of embodiment 1 Semen sojae atricolor GmPHT1.01 promoter
Utilize forward primer forward primer: 5 '-attgGAGCTCGTGTCCCTTTGTATGATGGAATC-3 ' and reversely draw Thing: 5 '-gagtCTGCAGCACTCACTAACTCAGCTACCTGA-3 ' PCR from soybean gene group expands and checks order and obtains length For the GmPHT1.01 promoter of 5240bp, its nucleotide sequence is as shown in SEQ ID NO.1.PCR primer two ends are respectively provided with Sac I and the restriction enzyme site of Pst I and protection base.
Above-mentioned PCR amplification system is: (20 μ l system)
PCR program: 95 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 3min totally 25 circulations；72℃10min.
The expression of embodiment 2 Semen sojae atricolor GmPHT1.01 promoter regulation downstream gene GUS
Obtain GmPHT1.01 promoter according to PCR clone in embodiment 1, cut glue and reclaim product Sac I and Pst I pair Enzyme action.Carrier pCAMBIA3301 (pCAMBIA3301 is purchased from Cambia company of Australia) is first double with Hind III and Nco I Enzyme action becomes flat end with Klenow polishing after falling 35S promoter, and Solution I converts expanding propagation to DH5 α after connecting.Upgrading grain After again with Sac I and Pst I double digestion, after recovery, the recovery fragment Solution I with GmPHT1.11 promoter is connected.Obtain Obtain binary expression vector pGmPHT1.01::GUS, plant expression vector pGmPHT1.01::GUS is converted to bacillus coli DH 5 alpha Middle expanding propagation.
The conversion of competent escherichia coli cell, including: (1) preparation LB solid medium containing kanamycin；(2) take out It is stored in the competent cell of-80 DEG C, after ice bath melts, adds 5 μ l connection products and mix gently, ice bath places 30 points Clock；(3) 42 DEG C of heat shocks 30 seconds, are placed in ice bath 3 minutes immediately after；Add the 300 μ l LB fluid medium without antibiotic, 37 DEG C of shaking table shaken cultivation 1 hour (160rpm)；(4) take appropriate culture fluid to be uniformly applied on selective medium, 37 DEG C of inversions Cultivate 16 hours, with sterilizing toothpick picking 5-10 (or more) bacterium colony, be placed in 200 μ l selected liq LB culture medium and shake bacterium Cultivate；(5) Agrobacterium EHA105 is converted after the positive colony expanding propagation upgrading grain that PCR detection screens.It is situated between by Agrobacterium rhizogenes Lead method for transformation, pGmPHT1.01::GUS is proceeded to grand No. one of Semen sojae atricolor sky, it is thus achieved that convert the root of hair 50 of pGmPHT1.01::GUS Bar.
GUS activity analysis shows, GmPHT1.01 promoter is specifically expressing in the root (Fig. 1) of plant, can be used for including respectively Plant the specifically expressing target gene in the root of interior various plants such as crop, forest, vegetable, flowers, herbage.
The method of the transformation of soybean of Agrobacterium rhizogenes mediation:
(1) by Semen sojae atricolor with after chlorine fumigation and steaming method sterilizing 12 hours, take out and be placed in after super-clean bench blows off residue chlorine, with going out The ultra-pure water of bacterium soak 16 hours standby；
(2) the Agrobacterium K599 with pGmPHT1.01::GUS is chosen monoclonal activation after, test tube is little shake after big with YEP Shake to bacterium solution OD between 0.8-1.0,4000 turns, after 22 DEG C of centrifugal 10min, be resuspended in LCCM (1/10X Gamborg B5Salt, 30g/L sucrose, adds 40mg/L acetosyringone after 3.9g/L MES, pH 5.4. sterilizing) in；
(3) radicle of soaked Semen sojae atricolor is cut, be outer implant with hypocotyl, outer implant is dipped in resuspended bacterium solution 30min completes to infect, and blots dip-dyeing solution on filter paper, and the outer implant of the Semen sojae atricolor after infecting is placed in CCM (1/10X Gamborg B5 Salt, 30g/L sucrose, 3.9g/LMES, 4.25g/L agar, add Cysteine 400mg/L, and after pH 5.4. sterilizing 40mg/L acetosyringone) go up lucifuge cultivation 3 days；
(4) hypocotyl of the outer implant of the Semen sojae atricolor after co-culturing inserts induction of hairy roots culture medium (1X Gamborg B5Salt, 30g/L sucrose, and 0.59g/L MES, 7g/L agar, add Cefotaxime 100mg/L after pH5.7. sterilizing) in, long day Cultivate 10-14 days induction root of hairs under illumination, during hairy root growth to 2cm, draw materials；
The GUS dyeing of transformant:
(1) tissue sample is added in 90% acetone, be placed in 20～30 minutes on ice；
(2) after acetone treatment, with 50mM phosphate buffer (pH7.2), 2mMK4Fe[CN]6And 2mM K3Fe[CN]6Molten Liquid rinse tissue；
(3) sample is placed on (50mM phosphate buffer (pH7.2), 1mM X-gluc, 2mM in X-Gluc dyeing liquor K4Fe[CN]6,2mM K3Fe[CN]6), bleed 10 minutes, then 37 DEG C of stained over night；
Within (4) second days, use 70% ethanol decolorization；
(5) on microscope slide, add several HCG solution, the sample after decolouring is placed in one；
(6) tabletting, transparent 15 minutes or the most transparent (depending on the developmental stage according to tissue).
GUS prescription of its dyeing liquor:
20mM X-gluc (MW=521.8)
Dissolving with DMSO or DMF, 100mg X-gluc need to dissolve with 9.58mlDMSO, final concentration of 20mM。
50mM phosphate buffer (pH7.0)
A liquid: NaH2PO4·2H2O 3.12g is dissolved in sterile distilled water, is settled to 100ml；
B liquid: Na2HPO4·12H2O 7.17g is dissolved in sterile distilled water, is settled to 100ml；
Take 39mlA liquid to mix with 61mlB liquid.
Dyeing liquor is prepared: the 5mM potassium ferricyanide；5mM potassium ferrocyanide；10mMEDTA；50mM phosphate buffer；1mM X- gluc。
Although, the present invention is described in detail the most with a general description of the specific embodiments, but On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

Claims (10)

1. the promoter of Soybean Root specifically expressing, for GmPHT1.01 promoter, it is characterised in that its sequence is:
1) nucleotide sequence shown in SEQ ID NO.1；Or
2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks and/or adds the tool that one or several nucleotide is obtained Have equal function by 1) derivative nucleotide sequence.
Promoter the most according to claim 1, it is characterised in that described promoter is by following replacement mode extremely Few a kind of obtained nucleotide sequence: the T of the 110th is substituted by A, the A of the 2601st is substituted by T, by the 3004th A be substituted by T.
3. containing the biomaterial of promoter described in claim 1 or 2, described biomaterial is that carrier, host cell, conversion are planted Thing cell or expression cassette.
4. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 are regulating and controlling downstream gene at root or root Application in tumor specifically expressing.
5. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 are improving downstream gene in plant roots Or the application in root nodule expression.
6. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 answering in preparing transgenic plant With.
7. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 regulate in plant roots and Radical extension In application.
8. the promoter described in claim 1 or 2 or the biomaterial described in claim 3 are in plant germplasm resource is improved Application.
9. for cloning the specific primer of the promoter described in claim 1 or 2, it is characterised in that including:
Forward primer: 5 '-attgGAGCTCGTGTCCCTTTGTATGATGGAATC-3 '；Reverse primer: 5 '- gagtCTGCAGCACTCACTAACTCAGCTACCTGA-3’。
10. contain the test kit of specific primer described in claim 9.
CN201610830389.1A 2016-09-07 2016-09-18 The promoter of Soybean Root specifically expressing and application thereof Pending CN106191068A (en)

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CN2016108098613 2016-09-07

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110468135A (en) * 2019-08-29 2019-11-19 河南大学 Soybean rhythmic expression's promoter GmPRR9b1 and its application
CN114507666A (en) * 2022-03-03 2022-05-17 江苏省农业科学院 Soybean-derived root-specific promoter pro-GmPRlike and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LU QIN等: "Functional Characterization of 14 Pht1 Family Genes inYeast and Their Expressions in Response to NutrientStarvation in Soybean", 《PLOS ONE》 *
吴乃虎等: "《基因工程原理（下册）（第二版）》", 30 April 2014, 科学出版社 *
宋海娜: "大豆磷效率相关基因GmACP1和GmPht1;1的克隆与功能研究", 《中国博士学位论文全文数据库农业科技辑》 *
康丹等: "染色体步移技术克隆已知序列侧翼启动子的研究进展", 《农业生物技术学报》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110468135A (en) * 2019-08-29 2019-11-19 河南大学 Soybean rhythmic expression's promoter GmPRR9b1 and its application
CN110468135B (en) * 2019-08-29 2021-04-20 河南大学 Soybean rhythmicity expression promoter GmPRR9b1 and application thereof
CN114507666A (en) * 2022-03-03 2022-05-17 江苏省农业科学院 Soybean-derived root-specific promoter pro-GmPRlike and application thereof
CN114507666B (en) * 2022-03-03 2023-01-24 江苏省农业科学院 Soybean-derived root-specific promoter pro-GmPRlike and application thereof

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Application publication date: 20161207