CN102776186A

CN102776186A - Specificity expression promoter of plant root and expression carrier of promoter

Info

Publication number: CN102776186A
Application number: CN2012101737571A
Authority: CN
Inventors: 张家明; 汪萌; 谭德冠
Original assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Current assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Priority date: 2012-05-31
Filing date: 2012-05-31
Publication date: 2012-11-14
Anticipated expiration: 2032-05-31
Also published as: CN102776186B

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

A kind of plant root specificity expression's promotor and expression vector thereof

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, obtain to have the new variety that higher disease and insect resistance and nutrient efficient utilize type at root specificity.

Background technology

Root system of plant is the organ of plant absorbing moisture, mineral nutrient, and root system development is most important to vine growth and development.Root system of plant also is to receive the organ that the soil-borne disease insect pest is infected easily.Root through specific gene is expressed, and might cultivate the transgenic new variety of drought resisting, impoverishment tolerant, anti-saline and alkaline, anti-soil-borne disease insect pest.

Cress distributes extensively, and this advantage has benefited from its unique myrosin system of defense (sulfo-glucoside/myrosin system), is commonly called as " mustard seed bomb ".When plant receives physical abuse, attack of insect or receives fungi and during the infection of pathogenic agent; Enzyme and substrate (myrosin and sulfo-glucoside are stored in the different cells respectively) meet; The sulfo-glucoside is degraded to toxic compounds; Participation is to the defence of 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 than big-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 ' the end upper reaches, is made up of different regulatory elements, and these regulatory elements are operability ground integrated structure gene 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 the space and the timeliness of control gene are transcribed.Along with the development of functional genomics research, the more function gene is cloned, but will obtain better transfer-gen plant, and the problem that at first will consider is promptly selected suitable promotor and it is efficiently expressed at special position.

What use always in the plant genetic engineering at present is composition type expression promoter, and foreign gene each position in plant is all expressed.The specifically expressing different with existing phraseology then 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 broken up the plant tissue organ and gene regulating is significant; Simultaneously, as food plant, foreign gene preferably directly is expressed in other positions beyond the edible position, meets the security of transgenic plant more.From plant, isolated at present specific expressed polytype promotor in organ or tissues such as blade, root, pollen tapetum, embryo, endodermis, aleurone layer and phloem.Wherein root-specific promoter is less relatively; Mainly contain: tobacco RB7 root promotor (U.S.Pat.No.5459252); Corn MR7 (U.S.Pat.No.5837848), the promotor of yam 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 with growth and from soil, absorbs moisture and inorganic salt, and the growth of root plays crucial effects for plant.Root-specific promoter can drive the goal gene specifically expressing in its downstream at root.Through the transgenic means, can with separated and K+ absorb relevant transporter specifically overexpression will improve fertilising efficient effectively in root, the bioavailability of raising nutrient.With the cash crop of root,,, thereby improve its utility value in the special enhancing of the root expression of gene level relevant with starch and sugar metabolism like potato as storage organ.Root system carries out dietetic alimentation, transportation, storage and synthetic vitals as plant materials, also is simultaneously the important component part of depending on for existence under opposing disease and pest and the degeneration-resistant environment.The root that many phytopathogens and insect are destroyed plant causes serious crop damage and loss.The transgenic plant of only in the organ or tissue that influenced by specific insect or pathogenic agent, expressing deleterious peptide 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 be used for raw insecticide in the tissue site of selecting is changed into activity form.Hsu etc. have reported that the mosaic gene that comprises root-specific promoter TobRB7 and beta-glucuronidase gene is preferentially changing into the purposes aspect the activity form with the raw insecticide in the root; The raw insecticide (glucuronide of methylol oxamoyl) of deactivation is administered on the blade; Transport it into root through plant phloem then, convert it into activated nematocides form by glucuronidase at this.Therefore, plant gene specific expressed effective root system of plant generation, differentiation and developmental 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, and size is 1447 bp, and the similarity of the promotor of the myrosin gene TGG4 that expresses with another root is 43.3%; And has the sequence under < 400>1 in the sequence table, the Nucleotide of the sequence of this promotor shown in SEQ ID NO:1.

Through extracting the myrosin TGG5 gene promoter that total dna clone obtains among the environmental Col-0 of Arabidopis thaliana, be 1447 bp through its length of order-checking.Through software analysis, this promotor has the controlling element that the higher plant promotor should have.This promotor can import plant with useful goal gene (comprise disease and insect resistance gene, resistance gene, promote the metabolic gene of secondary substance, promote the gene of mineral substance absorption etc.) effectively; The expression of differential high efficient in roots of plants, thus obtain having the new variety that higher anti-root disease and pest and nutrient efficient utilize type.

Another object of the present invention is to provide a kind of plant root specificity expression carrier, wherein, changed aforesaid Arabidopis thaliana root specificity expression's promotor over to.The promotor of utilizing aforesaid Arabidopis thaliana specificity expression is built into the new plant expression vector that contains gus gene in the promotor downstream, called after pBITG5 through with the CaMV35S promotor on Hind III and the BamH I displacement plant expression vector pBI121.Through inflorescence infusion method transformation mode plant Arabidopis thaliana, successfully obtain transfer-gen plant.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 root specificity expression promoter, and pBITG5 is a kind of root specificity expression's a carrier.

One, the structure of the clone of TGG5 gene promoter and expression vector.

Adopt the precious biological total DNA extraction test kit in Dalian to extract the environmental Col-0 genomic dna of Arabidopis thaliana.Sequence according to the TGG5 gene in the DB; 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).Obtain a product that is about 1500 bp through pcr amplification; CaMV 35S promoter on the double digestion rear substitution plant expression vector pBI121 obtains new plant expression vector, gives birth to worker's order-checking in Shanghai; Long 1447 bp of this promotor of sequencing result are in the sequence canonical sequence table < 400>1.Through software analysis, this promotor has the controlling element that the higher plant promotor should have, like 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

Change above-mentioned expression vector over to Agrobacterium C58 through the electric shock conversion method, the laggard performing PCR of resistance screening is identified.Be accredited as the male transformant through inflorescence infusion method arabidopsis thaliana transformation, collect T0 and carry out resistance screening, transfer-gen plant is further carried out PCR identify for seed.Select PCR to be accredited as male plant continuation cultivation and obtain the transgenic pure lines.

2) GUS histological chemistry is detected

Get T1 and separate than carrying out the GUS histochemical stain, observe the expression of gus gene, take a picture at each plant organ of transgenic arabidopsis for the seedling of 3:1 for seed resistance.

Root specificity expression promoter provided by the invention and plant expression vector thereof replace the composing type CaMV35S promotor on the plant expression vector with this promotor, make up a new plant expression vector and in root, express.The acquisition of this promotor; Be foreign gene (root or root growth are grown 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 genetic engineering technique also can obtain the new variety that disease and insect resistance, degeneration-resistant and nutrient efficient utilize type simultaneously, the promotor of research therefore express to(for) root specificity has potential commercial value and important use value.

Description of drawings

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: in the cotyledon of plant, hypocotyl, detect expression, detect the expression of gus gene at root less than gus gene;

B: in true leaf, petiole and the stem of plant, detect expression, detect the expression of gus gene at root less than gus gene;

C: in the spending of plant, detect expression less than gus gene;

D: in the fruit of the angle of plant, all detect expression less than gus gene;

E: detect the expression (amplification) of gus gene at the tip of a root;

F: detect the expression of gus gene 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, is verified as root specifically expressing (Fig. 1) through PCR method by amplification acquisition among the total DNA of the environmental Col-0 of Arabidopis thaliana and through scientific experiment.

1, Arabidopis thaliana is environmental and to plant method for planting be with from Nottingham Arabidopsis Stock Center; The environmental Col-0 planting seed of the Arabidopis thaliana of England is on standard nutrition soil; Place for 4 ℃ and forward culturing room's cultivation after 3 days to, temperature is 22 ℃ of daytimes, 20 ℃ of evenings; Illumination 16 hours/day, intensity of illumination 5000lx.The Arabidopis thaliana seed disinfection is in the Eppendorf of 1.5ml pipe, to add small quantities of seed, with 75% alcohol immersion several seconds of 1ml, sucking-off supernatant; Add the 1ml sterilized water, turn upside down for several times, leave standstill the several seconds, the sucking-off supernatant; The chlorine bleach liquor who adds 1ml 5% again puts upside down mixing 6～8min, with aseptic water washing 5 times; Seed after the sterilization drips on the substratum with aseptic dropper with 0.1% agarose mixing of 1ml sterilization; Vernalization is 3 days in 4 ℃ of refrigerators, puts into the growth cabinet of illumination 8h/d and sprouts, and temperature is 22 ℃ of daytimes, changes the 14h/d illumination box over to after 20 days 20 ℃ of evenings, and temperature is the same; Sprouted back 30 days, and chose the consistent transplantation of seedlings of stalwartness, growth in the compost of prior sterilization, cover preservative film, wait the normal back of seedling growth (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 a blade of getting the environmental Col-0 plant of Arabidopis thaliana, 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) with 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.It is synthetic that primer dodges brilliant biotech firm by Shanghai, and archaeal dna polymerase LA Taq is from adopting the precious biotech firm in Dalian.

The segmental pcr amplification of purpose: in the PCR pipe, add successively

Mixing is instantaneous centrifugal gently, and the Eppendorf pipe is put in the pcr amplification appearance, covers 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, behind the electrophoresis 30min, observe and photograph under the 5V/cm constant voltage condition through gel imaging system.Adopt the precious biological small pieces segment DNA purification kit purified product in Dalian.Again with the purified product double digestion.The endonuclease reaction system is following:

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 subsequent use.

With plant expression vector pBI121 double digestion, reaction system is following:

Enzyme is cut product carry out agarose gel electrophoresis, photograph, inspection clip size, cut glue and reclaim big fragment, concentrate, carry out ligation.Reaction system is following:

Flick mixing instantaneous centrifugal after, 16 ℃ of reaction overnight, next day is all as transformed into escherichia coli (E. co1i) DH5 α, after enzyme is cut evaluation, gives birth to the order-checking of worker biotech firm by Shanghai and identifies.Each fragment is surveyed 3 clones at least, to get rid of the influence of PCR mistake to sequence.Sequencing result is long 1447 bp of this promotor, in the sequence canonical sequence table < 400>1.Through software analysis, this promotor has the controlling element that the higher plant promotor should have, like 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 1 μ l recombinant plasmid and 100 μ l Agrobacterium competent cells respectively in centrifuge tube, and fully mixing is put 10min on ice; Above-mentioned mixed liquid is transferred in the pole cup of uv irradiating sterilization the impulsive discharge of shocking by electricity (voltage 1.8kv, electric capacity 25 μ f, impedance 400 Ω); Electric shock adds 500 μ l liquid YEP substratum respectively after finishing, and behind the mixing, the mixed liquid of sucking-off is in the 10ml centrifuge tube gently, and 28 ℃, 200rpm cultivates 4～6h; Draw 200 μ l bacterium liquid, be applied to and contain on the corresponding antibiotic solid YEP substratum, cultivate 1～2 d for 28 ℃.Single bacterium colony is carried out bacterium liquid PCR evaluation and screening positive transformant.

4, the agrobacterium-mediated transformation arabidopsis thaliana transformation is to infect the environmental Col-0 of Arabidopis thaliana through the method that inflorescence soaks, and the 35S promoter of introducing constitutive expression is as a reference.Choose the seedling of stalwartness, growth unanimity, bolting, watering the previous day irrigates; Agrobacterium shakes 30ml earlier, gets 25ml again and puts into 500ml YEP+Km (corresponding microbiotic), shakes the above (OD of bacterium 24h ₆₀₀Value is 1.8～2.0); The centrifugal 15min of 4000rpm (room temperature) collects bacterium; Transform Buffer (1000ml 1/2MS+10 μ l BA (1mg/mL)+Tween20 400 μ l+5% sucrose transfers PH5.8 with KOH) dissolving with 2 times of volumes (1L), be used for behind the mixing transforming; Get the Arabidopis thaliana plant about inflorescence 15 cm, cut fruit pod and the flower opened, whole rachis is immersed in to transform in the solution 10min or whole rachis is immersed in and transforms solution and be placed on and vacuumize 5min in the vacuum unit; Take out the relief plant and lie low, cover preservative film, place dark, erect again every other day; Can receive seed (22 ℃/20 ℃, 16h/8h light/dark) behind about one and a half months; After transformed plant was received and planted, according to transforming plasmid resistance screening transfer-gen plant, further plantation screening obtained transfer-gen plant and is sheerly.

5, the screening of transfer-gen plant is according to conversion plasmid resistance screening transfer-gen plant, and the transfer-gen plant that resistance screening (kantlex 50 μ g/ml) obtains carries out PCR through the SDS method and identifies the preliminary successful plant of confirming to transform.Concrete grammar is, gets 1 in newborn blade (about 10mg), places 1.5ml Epppendorf pipe, under the room temperature, with the little pestle of plastics blade ground to form homogenate (within the 20s) rapidly; 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 the concussion mixing; On microcentrifuge, 12000rpm, centrifugal 2min; Get supernatant 300 μ l, transfer in another 1.5mlEpppendorf pipe, add the equal-volume Virahol, behind the mixing, leave standstill 2min under the room temperature gently; Room temperature, 12000rpm, centrifugal 5min abandons supernatant, uncaps and places about 15min, makes the Virahol volatilization, and is long but the time is difficult for, otherwise sedimentary DNA is difficult to Hui Rong; Add 50 μ l ddH ₂0, dissolution precipitation obtains the DNA crude extract; Centrifugal sample 1min, supernatant are used 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 to be seeded in after 100～200 sterilizations and contain on the corresponding antibiotic substratum; Illumination cultivation after the vernalization; Count the number of resistance seedling and non-resistance seedling after 10～20 days respectively, calculate the ratio (separate and compare) of resistance seedling and non-resistance seedling, 3:1 promptly meets mendel's law.

It is that transfer-gen plant is carried out the GUS staining analysis that the GUS of transfer-gen plant detects.Get T ₁Separate than X-gluc solution (the 50 mmol/L sodium phosphate buffers that directly add the proper amount of fresh preparation for the seedling of 3:1 for seed resistance; 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; Decolouring then: use 30%, 50%, 70% and 100% ethanol rinsing respectively, each 5min, continue with 100% ethanol decolorization to background white or colourless till; Lustful material be will take off at last and preservation or photograph in 70% ethanol will be placed on.

Above disclosedly be merely preferred embodiment of the present invention, can not limit the present invention's interest field certainly with this, the equivalent variations of therefore doing according to claim of the present invention still belongs to the scope that the present invention is contained.

Claims

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, has the sequence under < 400>1 in the sequence table.

2. plant root specificity expression's as claimed in claim 1 promotor is characterized in that: the Nucleotide of the sequence of this promotor shown in SEQ ID NO:1.

3. plant root specificity expression carrier; It is characterized in that: to the CaMV35S promotor on the specific expressed promotor double digestion rear substitution plant expression vector pBI121 of the described plant root of claim 1, be built into the root specific expression carrier that contains gus reporter gene in the promotor downstream with Hind III and BamH I; Utilize the inflorescence infusion method to import Arabidopis thaliana, obtain transfer-gen plant.