CN1470636A - Ethylene-insenstiive plant expression vector pBinETR1 and its use - Google Patents

Abstract

The present invention relates to a plant expression vector pBinETR1 which is not sensitive to ethylene is constructed, and the trangenic plant containing said expression vector pBinETR1 has the functions of resisting senility. Said ivnention also can be used in transgenic flower plant, it can prolong flowering season and flower-inserting period of flower plant.

Description

A kind of ethylene-insenstiive plant expression vector pBinETR 1 and application thereof

Technical field

The invention belongs to plant genetic engineering field.Make up the segmental antisense plant expression vector of the cDNA pBinETR1 of ETR1 gene 797bp, comprised first and second hydrophobic region of ETR1.Transform petunia with the agrobacterium tumefaciens that contains this expression vector, transfer-gen plant is insensitive to ethene.This technology has very important practical value in transforming the ornamental plant quality.

Background technology

Ethene is regulating growth of plants, maturation and old and feeble important hormone.By the plant genetic engineering approach, the research of regulation and control ethene and plant senescence mainly comprises following two approach at present:

The one, the ethene biosynthetic pathway.Two key enzymes are arranged, oxidation of ethylene enzyme (ACO) and ethylene synthetase (ACS) in the ethene biosynthetic pathway.Justice or inverted defined gene by changing ACO, ACS over to can suppress the expression of these two key genes, reduce the resultant quantity of endogenous ethylene, delay senility, and postpone the fructescence.But exogenous ethylene still can promote The Plant Senescence.

The 2nd, the ethylene signaling approach both delayed senility by the susceptibility of change plant to ethene.Having isolated 5 ethylene receptor genes at present from Arabidopis thaliana, be divided into two gene families, is respectively ETR1, the ERS1 of ETR1 family and ETR2, EIN4 and the ERS2 of ETR2 family.That wherein research is more thorough is ETR1, and it is the gene that at first responds ethene in the ethylene signaling approach, has obtained the clone from many plants.

In plants such as tomato, Dianthus caryophyllus L., petunia, succeed by changing ETR1 gene alteration ethylene sensitivity over to.Binary vector plasmid pMON11063 and pMON26601 have been made up as (1997) such as Wilkinson.The former comprises Arabidopis thaliana etr1-1 (mutant receptors of a dominance ethylene sensitivity of coding) cDNA, CaMV 35S promoter and pea rbcs-E9 gene a 3 ' terminal upstream sequence; The latter comprises 3 ' end of the embedding of etr1-1/NR gene and body cDNA, FMV 35S promoter and a rouge alkali synthetase gene.The binary vector that builds is transformed into agrobacterium tumefaciens ABI from intestinal bacteria and makes up Ti-plasmids.Big large time delay of the fructescence of transgenic Fructus Lycopersici esculenti, the old and feeble and also delay that comes off of petunia flower.The expression of external source etr1-1 gene function in non-homogeneous plant, demonstrating ethene identification and response approach is high conservative in plant materials.

Utilize genetically engineered to change the ethylene sensitivity of flowers, can kind of blooming period prolonging and the bottle of cut-flower insert the life-span.The arabidopsis mutant body allelotrope etr1-1 that Clark etc. (1999) will have the CaMV 35S promoter changes in the petunia, the transfer-gen plant flower aging all has delay in various degree, but the percentage of fertile fruit of transfer-gen plant reduces compared with the control after the self-pollination, fruit maturation postpones, and the incidence of adventive root also significantly reduces.This shows the transgenic petunia that goes for the prolongation of flower life-span, also needs the tissue specificity of ethene insensitivity.Bovy etc. (1999) have made up the allelic double base plant expression vector of Arabidopis thaliana etr1-1; used promotor is respectively constitutive promoter, petunia specific promoter FBP1 and the composing type strong promoter CaMV 35S of the 2.7kb that the etr1-1 gene itself has, and binary vector transforms agrobacterium tumefaciens AGLO and makes up Ti-plasmids.After agrobacterium tumefaciens transformed Dianthus caryophyllus L., the slotting life-span of transfer-gen plant bottle all obtained prolonging.

The research of ethylene receptor gene-transformed plant at present, it mainly is full-length gene conversion plant with Arabidopis thaliana etr1-1 mutant, do not see that the ethylene receptor gene that clone from other plant is come out or the function of gene fragment study, and successfully transform the report of allos plant.The hydrophobic region of ETR1 is the ethene calmodulin binding domain CaM, mainly influences the response of plant to external source ETR1 gene.Chinese rose ETR1 cDNA fragment has just in time comprised this hydrophobic region.Therefore after this gene fragment transforms plant, can insert generation material impacts such as life-span to ethylene sensitivity, aging and the bottle of transfer-gen plant.

Summary of the invention

The invention is characterized in that used gene is the cDNA fragment (Gene Bank No AF380127) of the ETR1 gene 797bp that clones of our laboratory from cut rose flower, comprise first and second hydrophobic region, is ETR1 and ethene bonded position.With the antisense plant expression vector that this cDNA makes up, restriction enzyme site is less, with the carrier ease of connection, does not need to carry out repeatedly enzyme and cuts and be connected.Behind the plant expression vector construction, transform petunia with agrobacterium tumefaciens.The PCR that transfer-gen plant has been carried out total DNA detects and the RT-PCR detection of RNA always, and has carried out the Biological Detection of ethylene sensitivity.

The result shows, the expression vector that makes up with the cDNA fragment of the 797bp of Chinese rose ETR1 gene transforms the beyond thought effect that petunia produces, obtained the transfer-gen plant that ethylene sensitivity reduces, the conversion of this explanation antisense ETR1 gene fragment, can suppress the effect of endogenous ETR1, reduce the ethylene sensitivity of plant, thereby prolong the life-span of flower.So the insensitive plant expression vector of ethene of the present invention is in the plant modification quality, the application that especially prolongs in the plant genetic engineering at flower plant florescence has crucial meaning.

Description of drawings

Fig. 1 ETR1 cDNA plant expression vector pBinETR1 collection of illustrative plates

Fig. 2 transgenic petunia tissue cultured seedling

Fig. 3 transgenic petunia and wild-type petunia are to the detection of differing ethylene concentration susceptibility:

Ethylene concentration 1,10ppm; 2,20ppm; 3,30ppm

Embodiment one, embodiment

The structure of embodiment 1 ethylene-insenstiive plant expression vector pBinETR 1

ETR1 cDNA is cloned among the carrier pGEM T-Easy-Vector, extract plasmid, carry out pcr amplification with two Auele Specific Primers that designed SalI and BamHI restriction enzyme site, the amplified production electrophoresis detection is connected with pGEM T-Easy-Vector after determining correctly again, connect the product electricity and swash transformed into escherichia coli DH5 α or JM109, at the dull and stereotyped enterprising row filter of the LB of the adding 50mg/L AMP that scribbles X-gal and IPTG, the single bacterium colony upgrading grain of picking white, use SalI and BamHI double digestion then, according to the fragment that reclaims the step recovery 800bp in the test kit, with SalI and BamHI double digestion, also reclaim behind the double base plant expression vector pBin438 upgrading grain by the step in the test kit.The target gene fragment that reclaims is connected 24 hours with binary vector with the T4 ligase enzyme under 14 ~ 16 ℃, connect the product electricity and swash transformed into escherichia coli DH5 α or JM109, be added with the enterprising row filter of LB culture medium flat plate of kantlex, picking list bacterium colony upgrading grain, identify with SalI and BamHI double digestion, if obtain two bands of 13kb and 800bp, the illustration purpose gene fragment with the binary vector successful connection.Because two restriction enzyme sites on the primer of design are opposite with the direction on the binary vector, thus the present invention's structure for to contain the antisense double base plant expression vector of ETR1 gene fragment cDNA shown in (Fig. 1).

Embodiment 2 (conversion)

The binary expression vector electricity swashs the agrobacterium tumefaciens lba4404 that transforms Rifampin and kalamycin resistance, converted product spreads upon on the YEP plate culture medium that adds Rifampin and kantlex then, cultivate after 3 days for 28 ℃, can grow single bacterium colony, the single bacterium colony of picking part carries out bacterium colony PCR then, can obtain the band about 800bp.PCR male list bacterium colony is drawn on the YEP plate culture medium again, and 28 ℃ are cultured to and grow bacterium colony.Single colony inoculation that picking grows is to the YEP liquid nutrient medium that adds Rifampin and kantlex, and 28 ℃ are shaken bacterium and spend the night on the 250rpm shaking table.Next day, the Agrobacterium nutrient solution is diluted 30-50 doubly with being total to culture medium MS+BA1.0mg/L+NAA0.01mg/L.Petunia tissue cultured seedling blade with sterile culture is an explant.Young leaflet tablet is cut the edge, and with blade at the more standardized wound of blade surface, put into common culture medium, infected 5 minutes.Take out blade, blot remaining bacterium liquid with aseptic filter paper, put on the flat board that does not contain antibiotic callus inducing medium MS+BA1.0mg/L+NAA0.01mg/L+Kan100mg/L+Cab 200mg/L+ agar 7g/L, the blade edge otch is fully contacted with substratum, seal with the Parafilm film then, 25 ℃ of light were cultivated 3 days.

When the bacterium colony of white appears in blade edge, the explant of cultivating altogether can be taken out, use aseptic water washing 3 times, wash remaining agrobacterium tumefaciens off, aseptic filter paper blots, and is transferred to select on the substratum 25 ℃ of illumination cultivation.The little sprout of renewable resistance on the callus of all backs, 2 week-4 takes place in explant visible callus in 1 week-2 week back on the selection substratum.Per 3 weeks are selecting on the substratum succeeding transfer culture once.To grow thickly and change root media MS+BA1.0mg/L+NAA0.1mg/L+Kan100mg/L+Cab 200mg/L+ agar 7g/L root induction over to after the bud plant division.The transformation tissue culture seedling as shown in Figure 2.

Embodiment 3 (PCR detection)

Detect when regeneration bud grows to the PCR that 3-4cm carries out DNA when high, positive strain system expands numerous.The CTAB method is adopted in the extraction of petunia, gets the about 50mg of petunia blade, grinds in 200 μ L CTAB extracting solutions, adds 300 μ L extracting solutions again, beat gently even after, 65 ℃ of water-bath 30min add the chloroform of 4 ℃ of precoolings of 500 μ L then, slightly beat to spare and vibrate.The centrifugal 5min of 10000rpm draws supernatant to another centrifuge tube, adds the Virahol of 4 ℃ of precoolings of equal-volume, slightly puts upside down until DNA and precipitates.The centrifugal 5min of 10000rpm abandons supernatant, adds 70% ethanol of 4 ℃ of precoolings, breaks up precipitation gently with the rifle head.The centrifugal 5min of 10000rpm abandons supernatant.After treating the liquid in pipe finish-drying, with 20-50 μ L ddH ₂The O dissolving DNA, after 1% agarose gel electrophoresis detected, it was standby to place-20 ℃ of refrigerators to preserve.

The DNA ddH that extracts ₂The template of 0.5 μ L (about 25ng) as the PCR reaction got in 10 times of O dilutions.A pair of primer and the segmental a pair of reverse primer of amplification ETR1 cDNA with amplification NPTII gene carry out the PCR reaction respectively, and the PCR reaction volume is 10 μ L, establish water contrast, positive control and wild-type simultaneously and contrast each one.The reaction conditions of NPTII gene PCR is 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 50s, 60 ℃ of annealing 50s, 72 ℃ are extended 1min, totally 28 circulations; 72 ℃ are extended 10min.The reaction conditions of ETR1 cDNA fragment PCR is 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 1min, 53 ℃ of annealing 1min30s, 72 ℃ are extended 1min, totally 30 circulations; 72 ℃ are extended 10min.After the PCR reaction is finished, get 8 μ L products electrophoresis detection on 1% agarose gel.Positive transfer-gen plant has the band of 850bp with the product of NPTII primer amplification, with the product of ETR1 primer amplification the band of 800bp is arranged, and wild-type does not have band.

Embodiment 4 (RT-PCR detection)

The transfer-gen plant positive to the PCR result of total DNA carries out the detection of the RT-PCR of total RNA.At first extract the RNA of plant tissue.Draw the 15mL extraction buffer in the 50mL centrifuge tube, 65 ℃ of heating in water bath.The liquid nitrogen precooling of mortar and pestle.Take by weighing transgenic petunia blade 1g, after adding liquid nitrogen and fully grinding, change in the centrifuge tube of above-mentioned 65 ℃ of preheatings, fully mixing.Add 14.4mL chloroform and 0.6mL primary isoamyl alcohol, mixing.The centrifugal 5min of room temperature, phase-splitting.Suct clearly in another 50mL centrifuge tube, add 12mL chloroform and 0.5mL primary isoamyl alcohol again, mixing.Suct clearly in another 50mL centrifuge tube, add the 10mmol/l LiCl of 1/4 volume, put upside down mixing, 4 ℃ of precipitations are spent the night.

The mixed solution that spend the night precipitation next day is abandoned supernatant, the RNA of collecting precipitation in the centrifugal 20min of 10000rpm.Precipitation is dissolved among the SSTE of 300 μ L, changes the 1.5mL centrifuge tube over to; Wash tube wall with 200 μ LSSTE again, change same centrifuge tube over to.Add 480 μ L chloroforms and 20 μ L primary isoamyl alcohol respectively, mixing, extracting.The centrifugal 5min of 10000rpm, phase-splitting.Draw supernatant in another 1.5mL centrifuge tube, add the dehydrated alcohol of 2 times of volumes, mixing, more than-70 ℃ of precipitated rna 30min or-20 ℃ place 2h.10000 centrifugal 20min, precipitated rna is abandoned supernatant; 70% ethanol is washed 2 times, is dissolved in the ddH of twice sterilization of 40 μ L after the drying ₂Among the O.Get the total RNA of 2 μ L, add the ddH of twice sterilization of 1000 μ L ₂Among the O, mixing.Measure the optical density value of 230nm, 260nm, 280nm.Calculate A260nm/A280nm, the ratio of A260nm/A230nm, and by 1% agarose gel electrophoresis, the RNA quality of Detection and Extraction.

The DNAase that RNA after the detection carries out total RNA handles.Add RNA 20 μ L, Rnasin 1 μ L, 10 * buffer, 5 μ L, ddH respectively ₂ O 2 μ L, DNase 2 μ L, the reaction cumulative volume is 30 μ L, keeps 30min in 37 ℃ behind the mixing.Add isopyknic chloroform: primary isoamyl alcohol (24: 1) extracting, centrifugal 5min gets supernatant.Add the 3mol/L NaAc of 1/10 volume and the dehydrated alcohol of 2 times of volumes, place-70 ℃ of 2h precipitated rnas.The centrifugal 10min of 12000rpm abandons supernatant.Wash precipitation 2 times with 70% ethanol, be dissolved in the ddH of twice sterilization of 20 μ L ₂Among the O.

RNA after the processing can carry out reverse transcription after electrophoresis detection on 1% sepharose.Get RNA5 μ L, add Oligo dT (0.5mol/L) 1 μ L and ddH respectively ₂O 6 μ L place 10min for 70 ℃, place cooled on ice 2min fast, and are centrifugal.Add 5 * First Strand buffer, 4 μ L, DTT (0.1mol/L) 2 μ L and dNTP (10mmol/L) 1 μ L more respectively, mixing is placed on 42 ℃ and places 2min.Add 1 μ L Superscript II ThermoScript II, centrifugal behind the mixing, 42 ℃ keep 50min, and 70 ℃ keep 15min with termination reaction.

The first chain cDNA that produces with reverse transcription is that template is carried out the PCR reaction, compares with the used total RNA template of reverse transcription simultaneously.Reaction system 50 μ L are comprising 10 * PCR buffer, 5 μ L, MgCl ₂3 μ L, dNTP (2.5mmol/L) 4 μ L, primer 12.5 μ L, primer 2 2.5 μ L, template DNA 2 μ L, Taq enzyme (5Units/ μ L) 0.5 μ L, deionized water 30.5 μ L.Reaction conditions is with embodiment 3.Reaction finishes back electrophoresis detection on 1% sepharose.Transfer-gen plant can obtain the band of 800bp, and wild-type does not have band.This explanation Chinese rose ETR1 cDNA can transcribe in the petunia body.

Embodiment 5 (ethylene sensitivity detection)

RT-PCR male transfer-gen plant and wild-type plant are carried out the test of ethylene sensitivity.With pure ethylene (10 ⁶Ppm) being diluted to concentration is 10 ³The employed vessel volume of petunia group training is 100mL, and therefore, three concentration of the present invention handle 10ppm, 20ppm and 30ppm gets 10 respectively ³Ethene 1mL, 2mL and the 3mL of concentration, and be expelled in wild-type and the transgenic petunia group culture container.To seal film behind the ethene processing 24h and open (noting not causing tissue culture seedling pollution), discharge an ethene in the bottle, behind the 6h, again bottleneck be sealed, cultivate observations after 2 days with sealing film.The result shows that as shown in Figure 3, after ethene was handled, wilting in various degree appearred in the wild-type plant, did not have phenotypic difference before transfer-gen plant and the processing.This explanation transfer-gen plant is insensitive to exogenous ethylene, and the Chinese rose ETR1 cDNA that the present invention changes over to has possessed the biological function of anti-ethylene sensitivity in the petunia transfer-gen plant.

Claims (4)

1. the plant expression vector pBinETR1 of an anti-ethylene sensitivity, its structure is as follows:

2. expression vector according to claim 1 is in the transgenic plant application in engineering.

3. expression vector according to claim 1, the application in anti-ageing transgenic plant.

4. expression vector according to claim 1, the application in flower plant kind of blooming period prolonging transgenic plant.

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