CN102776186B - Specificity expression promoter of plant root and expression carrier of promoter - Google Patents
Specificity expression promoter of plant root and expression carrier of promoter Download PDFInfo
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Abstract
The invention discloses a specificity expression promoter of the plant root and an expression carrier of the promoter. The promoter is a promoter of the fifth myrosinase gene TGG5, the length is 1447bp, and the promoter has the sequence under the <400>1 item in a sequence table. The promoter substitutes a constitutive CaMV35S promoter on a plant expression carrier pBI121, and a new plant expression carrier is built and is named as pBITG5. The Arabidopsis is successfully guided in by an inflorescence soaking method, a transgenic plant is obtained, the specific efficient expression of the promoter at the root part is displayed through GUS (glucuronidase) histochemical staining, and the promoter is not expressed in other tissue cells. Through the promoter obtaining, conditions are created for obtaining disease and pest resistance, inverse resistance and nutrient efficient utilization type new varieties by utilizing genetic engineering, the foundation is provided for the specificity expression of the foreign gene in the root, and potential commercial value and theory and practice significance are realized.
Description
Technical field
The present invention relates to plant genetic engineering field, specifically, relate to a kind of acquisition of novel myrosin TGG5 gene promoter and the structure of plant expression vector, relate in particular to induction exogenous gene and express at root specificity, obtain and to have the new variety that higher disease and insect resistance and nutrient efficient utilize type.
Background technology
Root system of plant is the organ that plant absorbs moisture, mineral nutrient, and root system development is most important to vine growth and development.Root system of plant is also easily to be subject to the organ that the soil-borne disease insect pest is infected.Root by specific gene is expressed, and likely cultivates the transgenosis new variety of drought resisting, impoverishment tolerant, anti-saline and alkaline, anti-soil-borne disease insect pest.
Cress is widely distributed, and this advantage has benefited from its unique myrosin system of defense (glucosinolate/myrosin system), is commonly called as " mustard seed bomb ".When plant is subject to physical abuse, attack of insect or is subject to fungi and during the infection of pathogenic agent, enzyme and substrate (myrosin and glucosinolate are stored at respectively in different cells) meet, glucosinolate is degraded to toxic compounds, the defence of participation to disease and pest, the metabolism of sulphur, nitrogen and growth regulating etc.Six the myrosin gene TGG1, TGG2, TGG3, TGG4, TGG5, the TGG6 that in Arabidopis thaliana, have found at present, wherein known myrosin gene TGG1, TGG2, TGG3 and novel myrosin gene TGG4, TGG5, TGG6, they have larger difference at aspects such as DNA sequence, gene structure and proteins encoded characteristics, TGG1, TGG2, TGG4, TGG5 are proved to be the gene of function, and TGG3, TGG6 are the pseudogenes of complete myrosin of can not encoding.
Promotor is the section of DNA sequence that is positioned at structure gene 5 ' end upstream, is comprised of different regulatory elements, and these regulatory elements are integrated structure gene operably by different way, the time of origin that controlling gene is expressed and the degree of expression.In plant materials, promotor is the instrumentality that natural gene and recombination are transcribed, and space and the timeliness of control gene are transcribed.Along with the development of functional genomics research, more functional gene is cloned, but will obtain better transfer-gen plant, and the problem that at first will consider is namely selected suitable promotor and made it at the high efficient expression in special position.
What in plant genetic engineering, commonly use at present is composition type expression promoter, and foreign gene each position in plant is all expressed.The specifically expressing different from existing phraseology is that goal gene is expressed at the position that needs are arranged, and makes the more economical effective performance of effect of goal gene; In addition, the specific expressed control methods of research plant tissue are significant to plant tissue Organ Differentiation and gene regulating; Simultaneously, as food plant, foreign gene is preferably directly expressed in other positions beyond edible position, more meets the security of transgenic plant.At present from plant, isolating specific expressed polytype promotor in the organ or tissues such as blade, root, pollen tapetum, embryo, endodermis, aleurone layer and phloem.Wherein root-specific promoter is relatively less, mainly contain: tobacco RB7 root promotor (U.S.Pat.No.5459252), corn MR7(U.S.Pat.No.5837848), the promotor of potato PGT2 (MEIKE KOSTER-TOPFER, WOLF B FROMME, MARIO ROCHA-SOSA, et al. A Class II patatin promoter is under developmental control in both transgenic potato and tobacco plants [J]. Mol Gen Genet, 1989,219: 390-396.) etc.
The growth of plant relies on root system from soil, absorbing moisture and inorganic salt with growth, and the growth of root plays vital effect for plant.Root-specific promoter can drive the goal gene specifically expressing in its downstream at root.By the transgenosis means, separated and K+ can be absorbed relevant transporter specifically overexpression in root, will effectively improve fertilising efficiency, the bioavailability of raising nutrient.Using root as the cash crop of storage organ, as potato, at the expression level of the special enhancing of the root gene relevant to starch and sugar metabolism, thereby improve its utility value.Root system, as the vitals that plant materials carries out dietetic alimentation, transportation, storage and synthesizes, is also simultaneously the important component part of depending on for existence under opposing disease and pest and degeneration-resistant environment.The root that many phytopathogens and insect are destroyed plant, cause serious crop damage and loss.The transgenic plant of only expressing poisonous peptide in the organ or tissue that affected by specific insect or pathogenic agent provide the method that reduces crop harm and loss.For example, the bacillus thuringiensis protein expression in transgenic corns provides the resistance to the European corn snout moth's larva.Tissue-specific promoter also can change into activity form for raw insecticide in the tissue site by selection.Hsu etc. have reported the purposes of mosaic gene aspect preferentially the raw insecticide in root being changed into to activity form that comprises root-specific promoter TobRB7 and beta-glucuronidase gene, the raw insecticide of deactivation (glucuronide of methylol oxamoyl) is administered on blade, then by plant phloem, transport it into root, by glucuronidase, convert it into activated nematocides form at this.Therefore, plant gene specific expressed effective root system of plant generation, Differentiation and development mechanism of having disclosed in root.
Summary of the invention
The object of the present invention is to provide a kind of plant root specificity expression's promotor.
To achieve these goals, technical scheme of the present invention is: the promotor that a kind of plant root specificity expression is provided, this promotor is the promotor of the 5th myrosin gene TGG5 of Arabidopis thaliana, size is 1447 bp, the similarity of the promotor of the myrosin gene TGG4 expressed with another root is 43.3%, and has in sequence table<sequence under 400 > 1 Nucleotide of the sequence of this promotor as shown in SEQ ID NO:1.
By in Arabidopsisecotype Col-0, extracting the myrosin TGG5 gene promoter that total DNA clone obtains, be 1447 bp through its length that checks order.By software analysis, this promotor has the controlling element that Plant Promoter should have.This promotor can import plant by useful goal gene (comprise disease and insect resistance gene, resistance gene, promote the gene of secondary substance metabolism, the gene of promotion mineral absorption etc.) effectively, the expression of differential high efficient in roots of plants, thus obtain having the new variety of utilizing type than high resistance root disease and pest and nutrient efficient.
Another object of the present invention is to provide a kind of plant root specificity expression carrier, wherein, proceeded to aforesaid Arabidopis thaliana root specificity expression's promotor.Utilize aforesaid Arabidopis thaliana specificity expression's promotor by the CaMV35S promotor with on Hind III and BamH I displacement plant expression vector pBI121, be built into the new plant expression vector that contains gus gene in the promotor downstream, called after pBITG5.By inflorescence infusion method transformation mode plant Arabidopis thaliana, successfully obtain transfer-gen plant.The RT-PCR result shows that the TGG5 gene expresses at root specificity, and the GUS histochemical stain identifies that the promotor of further confirmation TGG5 gene is the promotor that root specificity is expressed, and pBITG5 is a kind of root specificity expression's carrier.
One, the structure of the Cloning and Expression carrier of TGG5 gene promoter.
Adopt the precious biological total DNA extraction test kit in Dalian to extract Arabidopsisecotype Col-0 genomic dna.Sequence according to the TGG5 gene in database, design two specific primer AP17 (5'TGA GAA GCT TCC AGT TGG GTT TGG GTT AGT TTG 3', the 5' end has Bam HI site) and AP18 (5'TGT TGG ATC CGG TTT GTA TTT TCT TTA TTG ATG GGC T 3', the 5' end has Hind III site).By pcr amplification, obtain a product that is about 1500 bp, CaMV 35S promoter on double digestion rear substitution plant expression vector pBI121, obtain new plant expression vector, in Shanghai, gives birth to the work order-checking, long 1447 bp of this promotor of sequencing result, in sequence canonical sequence table<400 > 1.By software analysis, this promotor has the controlling element that Plant Promoter should have, as TATA box, CAAT box etc.New plant expression vector called after pBITG5.
Two, the acquisition of transfer-gen plant and GUS histochemical stain.
1) acquisition of transfer-gen plant
By the electric shock conversion method, above-mentioned expression vector is proceeded to Agrobacterium C58, the laggard performing PCR of resistance screening is identified.Be accredited as positive transformant by inflorescence infusion method arabidopsis thaliana transformation, collect T0 and carry out resistance screening for seed, transfer-gen plant is further carried out to the PCR evaluation.Select PCR to be accredited as positive plant continuation cultivation and obtain the transgenosis pure lines.
2) GUS histological chemistry is detected
Get T1 and separate than for the seedling of 3:1, carrying out the GUS histochemical stain for seed resistance, observe the expression of gus gene at each plant organ of transgenic arabidopsis, take a picture.
Promotor and plant expression vector thereof that root specificity provided by the invention is expressed, replace the composing type CaMV35S promotor on plant expression vector by this promotor, builds a new plant expression vector and express in root.The acquisition of this promotor, for foreign gene (root or root growth are grown to influential gene) specific expressed providing the foundation in root, foreign gene can be the gene of the gene (comprising the gene that can strengthen or modify the nutritive value of root), the coding that change root tissue function gene, antiweed or the unsuitable environmental condition characteristic of killing insect or pathogenic agent toxin (insect or the pathogenic agent of target preference attack root) etc.Utilize simultaneously genetic engineering technique also can obtain the new variety that disease and insect resistance, degeneration-resistant and nutrient efficient utilize type, the promotor of research therefore express to(for) root specificity has potential commercial value and important using value.
The accompanying drawing explanation
Fig. 1 is the colored graph (the GUS cell of expression dyed is blue) of TGG5 promoters driven gus gene at the root specifically expressing;
A: can't detect the expression of gus gene in the cotyledon of plant, hypocotyl, the expression of gus gene detected at root;
B: can't detect the expression of gus gene in true leaf, petiole and the stem of plant, the expression of gus gene detected at root;
C: can't detect the expression of gus gene in the spending of plant;
D: all can't detect the expression of gus gene in the fruit of the angle of plant;
E: the expression (amplification) of gus gene detected at the tip of a root;
F: the expression of gus gene detected in the root-hair zone of plant.
Embodiment
The invention provides a kind of Arabidopis thaliana root specificity expression's promotor and plant expression vector thereof.This promotor is that the methods analyst that utilizes information biology is tentatively confirmed, increased in the total DNA of Arabidopsisecotype Col-0 by PCR method and obtains and be verified as root specifically expressing (Fig. 1) by scientific experiment.
1, Arabidopsisecotype and implantation methods are by from Nottingham Arabidopsis Stock Center, the Arabidopsisecotype Col-0 planting seed of England is on the standard Nutrition Soil, place after 3 days for 4 ℃ and forward culturing room's cultivation to, temperature is 22 ℃ of daytimes, 20 ℃ of evenings, illumination 16 hours/day, intensity of illumination 5000lx.The Arabidopis thaliana seed disinfection is to add a small amount of seed in the Eppendorf of 1.5ml pipe, with the 75% alcohol immersion several seconds of 1ml, and the sucking-off supernatant liquor; Add the 1ml sterilized water, turn upside down for several times, standing several seconds, sucking-off supernatant liquor; The chlorine bleach liquor who adds again 1ml 5%, put upside down and mix 6～8min, uses aseptic water washing 5 times; Seed after sterilization mixes with 0.1% sterilizing agarose of 1ml, drips on substratum with aseptic dropper; In 4 ℃ of refrigerators, vernalization is 3 days, puts into the growth cabinet of illumination 8h/d and sprouts, and temperature is 22 ℃ of daytimes, in 20 ℃ of evenings, proceeds to the 14h/d illumination box after 20 days, and temperature is the same; Sprouted latter 30 days, and chose stalwartness, grow consistent transplantation of seedlings in advance sterilizing compost, cover preservative film, wait seedling growth normal rear (generally needing 3～5 d) to go preservative film to cultivate (temperature is 22 ℃ of daytimes, 20 ℃ of evenings, 14h/d illumination).
2, the structure of the clone of TGG5 gene promoter and carrier is the blade of getting Arabidopsisecotype Col-0 plant, and liquid nitrogen grinding adopts the precious biotech firm in Dalian total DNA extraction test kit to extract total DNA.With gene specific primer AP17 (5'TGA GAA GCT TCC AGT TGG GTT TGG GTT AGT TTG 3', 5' end has Bam HI site) and the promoter fragment of AP18 (5'TGT TGG ATC CGG TTT GTA TTT TCT TTA TTG ATG GGC T 3', the 5' end has Hind III site) the TGG5 gene that increases.Primer is synthetic by Shanghai Shan Jing biotech firm, and archaeal dna polymerase LA Taq is from adopting the precious biotech firm in Dalian.
The pcr amplification of purpose fragment: add successively in the PCR pipe
Mix gently, instantaneous centrifugal, the Eppendorf pipe is put in the pcr amplification instrument, cover heating cap.Response procedures: 94 ℃ of sex change 2min; 94 ℃ of sex change 40s, 62 ℃ of annealing 40s, 72 ℃ are extended 1min 30 sec, 35 circulations; 72 ℃ are extended 7min and finish reaction.Get 5 μ l pcr amplification reaction liquid and carry out 1.0% agarose gel electrophoresis, after electrophoresis 30min, observe and take a picture by gel imaging system under 5V/cm constant voltage condition.Adopt the precious biological small pieces segment DNA purification kit purified product in Dalian.Again by the purified product double digestion.The endonuclease reaction system is as follows:
30 ℃ of reaction 3h, adopt the precious biological small pieces segment DNA purification kit in Dalian to reclaim purifying enzyme and cut product, with 40 μ l ddH
2The O wash-out obtains each enzyme, and to cut product standby.
By plant expression vector pBI121 double digestion, reaction system is as follows:
Enzyme is cut to product and carry out agarose gel electrophoresis, photograph, inspection clip size, cut glue and reclaim large fragment, concentrated, carry out ligation.Reaction system is as follows:
Flick mix instantaneous centrifugal after, 16 ℃ of reaction overnight, all as transforming intestinal bacteria (E. co1i) DH5 α, after enzyme is cut evaluation, identified by the order-checking of Shanghai Sheng Gong biotech firm next day.Each fragment is surveyed 3 clones at least, to get rid of the impact of PCR mistake on sequence.Sequencing result is long 1447 bp of this promotor, in sequence canonical sequence table<400 > 1.By software analysis, this promotor has the controlling element that Plant Promoter should have, as TATA box, CAAT box etc.The carrier called after pBITG5 that checks order correct.
3, expression vector imports Agrobacterium C58 and adopts the electric shock conversion method, draws respectively 1 μ l recombinant plasmid and 100 μ l Agrobacterium competent cells in centrifuge tube, fully mixes, and puts 10min on ice; Above-mentioned mixed liquid is transferred in the sterilizing pole cup of uv irradiating to the impulsive discharge of shocking by electricity (voltage 1.8kv, electric capacity 25 μ f, impedance 400 Ω); Electric shock adds respectively 500 μ l liquid YEP substratum after finishing, and after mixing gently, the mixed liquid of sucking-off is in the 10ml centrifuge tube, and 28 ℃, 200rpm, cultivate 4～6h; Draw 200 μ l bacterium liquid, be applied to and contain on corresponding antibiotic solid YEP substratum, cultivate 1～2 d for 28 ℃.Single bacterium colony is carried out to bacterium liquid PCR evaluation and screening positive transformant.
4, the agrobacterium-mediated transformation arabidopsis thaliana transformation is to infect Arabidopsisecotype Col-0 by the method that inflorescence soaks, and the 35S promoter of introducing constitutive expression is as a reference.Choose that stalwartness, growth are consistent, the seedling of bolting, water the previous day and irrigate; Agrobacterium first shakes 30ml, then gets 25ml and put into 500ml YEP+Km (corresponding microbiotic), shakes the above (OD of bacterium 24h
600Value is 1.8～2.0); The centrifugal 15min(room temperature of 4000rpm) collection bacterium; With 2 times of volumes (1L), transform Buffer (1000ml 1/2MS+10 μ l BA (1mg/mL)+Tween20 400 μ l+5% sucrose, adjust PH5.8 with KOH) and dissolve, after mixing, be used for transforming; Get the Arabidopis thaliana plant of inflorescence 15 about cm, the flower of cutting the fruit pod and having opened, whole rachis are immersed in and transform in solution 10min or whole rachis is immersed in and transforms solution and be placed in vacuum unit and vacuumize 5min; Take out the relief plant and lie low, cover preservative film, be placed in dark, erect again every other day; Can sowing after about one and a half months (22 ℃/20 ℃, 16h/8h light/dark); After the transformed plant sowing, according to transforming plasmid resistance screening transfer-gen plant, further the plantation screening obtains the transfer-gen plant pure lines.
5, the screening of transfer-gen plant is according to transforming plasmid resistance screening transfer-gen plant, and the transfer-gen plant that resistance screening (kantlex 50 μ g/ml) obtains carries out PCR by the SDS method and identifies the preliminary plant of determining that conversion is successful.Concrete grammar is, gets 1, newborn blade (about 10mg), is placed in 1.5ml Epppendorf pipe, under room temperature, with the little pestle of plastics, blade ground to form rapidly to homogenate (within 20s); Add extracting solution (200mmol/L Tris-HCl pH8.0; 250mmol/L NaCl; 25mmol/L EDTA; 0.5% SDS; 121 ℃, high pressure steam sterilization 20min) 400 μ l, acutely shake concussion and mix; On microcentrifuge, 12000rpm, centrifugal 2min; Get supernatant liquor 300 μ l, transfer in another 1.5mlEpppendorf pipe, add the equal-volume Virahol, after mixing gently, standing 2min under room temperature; Room temperature, 12000rpm, centrifugal 5min, abandon supernatant, uncaps and place the 15min left and right, makes the Virahol volatilization, long but the time is difficult for, otherwise the DNA of precipitation is difficult to back dissolving; Add 50 μ l ddH
20, dissolution precipitation, obtain the DNA crude extract; Centrifugal sample 1min, supernatant liquor is for the PCR reaction; Get 5 μ l sample electrophoresis, detect the DNA extraction quality; Getting 1 μ l DNA is template, and other composition carries out the PCR reaction by standard program; Get 5 μ l PCR product electrophoresis detection.Continue plantation results T
1For the mature seed on the Arabidopis thaliana plant, get after 100～200 sterilizations to be seeded in and contain on corresponding antibiotic substratum, illumination cultivation after vernalization, after 10～20 days, count respectively the number of resistance seedling and non-resistance seedling, calculate the ratio (separate and compare) of resistance seedling and non-resistance seedling, 3:1 namely meets mendel's law.
It is that transfer-gen plant is carried out to the GUS staining analysis that the GUS of transfer-gen plant detects.Get T
1For seed resistance, separate than for the seedling of 3:1, directly adding X-gluc solution (the 50 mmol/L sodium phosphate buffers of proper amount of fresh preparation, pH 7.0,1 mmol/L X-gluc, 0.1% Triton X-100,0.1 mmol/L yellow prussiate of potash, 0.1mmol/L the Tripotassium iron hexacyanide, 20% methyl alcohol) in, 37 ℃ of incubation 1h are to spending the night; Then decolouring: use respectively 30%, 50%, 70% and 100% ethanol rinsing, each 5min, continue with 100% ethanol decolorization to background white or colourless till; Finally will take off lustful material and be placed on preservation or photograph in 70% ethanol.
Above disclosed is only preferred embodiment of the present invention, certainly can not limit with this interest field of the present invention, and the equivalent variations of therefore doing according to the claims in the present invention, still belong to the scope that the present invention is contained.
Claims (2)
1. a plant root specificity expression promotor, it is characterized in that: this promotor is the promotor of the 5th myrosin gene TGG5 of Arabidopis thaliana, and size is 1447 bp, the nucleotide sequence of its nucleotide sequence as shown in SEQ ID NO:1.
2. plant root specificity expression carrier, it is characterized in that: use Hind III and BamH I to the CaMV35S promotor on plant root specificity expression's claimed in claim 1 promotor double digestion rear substitution plant expression vector pBI121, be built into the plant root specificity expression carrier that contains gus reporter gene in the promotor downstream; Utilize the inflorescence infusion method to import Arabidopis thaliana, obtain transfer-gen plant.
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| CN101914539A (en) * | 2010-08-05 | 2010-12-15 | 中国热带农业科学院热带生物技术研究所 | Root specificity expression promoter and plant expression vector thereof |
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| US20060183137A1 (en) * | 2000-08-24 | 2006-08-17 | The Scripps Research Institute | Stress-regulated genes of plants, transgenic plants containing same, and methods of use |
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