CN102329795A - Cucumber violaxanthin decyclization oxidase gene promoter and application thereof - Google Patents

Abstract

The invention discloses a cucumber violaxanthin decyclization oxidase gene promoter and application thereof. The promoter is any one of the following 1)-3): 1) a DNA molecule shown as a sequence 1 in a sequence table; 2) a DNA molecule which is hybridized with the DNA sequence limited in the 1) under strict conditions and has promoter function; and 3) a DNA molecule which has over 90 percent of homology with the DNA sequence limited in the 1) or the 2) and has promoter function. The invention discloses a sequence of the cucumber violaxanthin decyclization oxidase gene promoter. Proved by experiments, the promoter has efficient and specific expression in a green plant tissue, mainly in the green plant tissue for photosynthesis, such as expression for promoting a target gene in a leaf, so the promoter has significance for further researching the biological information of related molecules of plant photosynthesis.

Description

Cucumber zeaxanthin diepoxide decylization oxidase gene promotor and application thereof

Technical field

The present invention relates to a kind of cucumber zeaxanthin diepoxide deoxidation cyclase gene promotor and application thereof.

Background technology

Plant always is faced with various abiotic stresses and coerces in growth and development process.Under the high light; The luminous energy that plant leaf can utilize only accounts for the sub-fraction of luminous energy that photosynthetic organ absorbs, and superfluous excitation energy is as can not in time being dissipated, and will cause light to suppress even photosynthetic mechanism is damaged; If there is other environment-stress simultaneously, the photo damage effect also can strengthen.Plant has formed the actual mechanism of a whole set of superfluous luminous energy that dissipates in the evolution of long period of time process.The xenthophylls circulation is exactly wherein a kind of protection mechanism, in the plant defense photo damage, plays an important role.

Xenthophylls circulation is by zeaxanthin diepoxide (V), antheraxanthin (A) and three kinds of pigments of ZXN (Z), and their two kinds of enzymes compositions transforming each other of catalysis.Research show Z and A can be from chlorophyll hyperabsorption excitation energy and dissipate with the form of heat energy, thereby protect photosynthetic device to avoid the destruction of high light.In higher plant, zeaxanthin diepoxide decylization oxydase (VDE) is an xenthophylls round-robin key enzyme in this process.

Cucumber high light when the strong outdoor cropping of illumination can cause its photosynthetic capacity to descend, and especially under low temperature stress, suppresses even the light intensity of medium tenacity also can make plant that light takes place.Therefore, to the clone of cucumber zeaxanthin diepoxide VDE gene and promotor thereof and the research tool ten minutes significance of function.

Summary of the invention

The purpose of this invention is to provide a kind of dna molecular with promoter function.

Dna molecular provided by the present invention derives from Curcurbitaceae Cucumis cucumber (Cucumis sativus L.) kind and wears many stars, is following 1)-3) in any:

1) dna molecular shown in the sequence 1 in the sequence table;

2) under stringent condition with 1) dna sequence dna hybridization that limits and dna molecular with promoter function;

3) with 1) or 2) dna sequence dna that limits has 90% above homology, and have the dna molecular of promoter function.Be preferably 95% above homology.

Sequence 1 is made up of 1983 Nucleotide in the sequence table.

The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain said dna molecular all belong to protection scope of the present invention.

Said in an embodiment of the present invention recombinant vectors inserts the recombinant plasmid that said dna molecular obtains for the MCS at pCAMBIA1391, and the MCS of said insertion dna molecular is specially restriction endonuclease sites Sal I and Nco I.

Said expression cassette can start the goal gene of expressing by said dna molecular by the said dna molecular with promoter function, and transcription termination sequence is formed; Said dna molecular is connected with said goal gene with functional mode, and said goal gene is connected with said transcription termination sequence.

Said dna molecular also belongs to protection scope of the present invention in the application of startup goal gene in expression of plants.In an embodiment of the present invention, said plant is specially Arabidopis thaliana; The said leaf specific expressino that is expressed as.

The method of utilizing said dna molecular to cultivate transgenic plant also belongs to protection scope of the present invention, and this method comprises the steps:

1) makes up the goal gene recombinant expression vector: goal gene is inserted the said recombinant vectors that carries said dna molecular (xanthin decylization oxidase gene promoter gene); Make said dna molecular start said destination gene expression, obtain the goal gene recombinant expression vector;

2) the goal gene recombinant expression vector that step 1) is made up imports in the purpose plant, obtains the transgenic plant of the said goal gene of specifically expressing in blade.In an embodiment of the present invention, said purpose plant is specially Arabidopis thaliana.

The method that said goal gene recombinant expression vector is imported said purpose plant can be agrobacterium-mediated transformation, Ti-plasmids method, Ri plasmid method, plant viral vector method, direct DNA conversion method, microinjection, conductometric titration etc., and concrete employing is agrobacterium-mediated transformation in an embodiment of the present invention.

In addition, increase said dna molecular (xanthin decylization oxidase gene promoter gene) total length or arbitrary segmental primer also belongs to protection scope of the present invention.

The present invention has cloned the correlated series of cucumber zeaxanthin diepoxide decylization oxidase gene promotor from cucumber (Dai Duoxing); This promotor of experiment confirm is an efficient specific expressed promotor; Mainly in carrying out photosynthetic green plants tissue (like blade), start the expression of goal gene, this has very important significance for further research photosynthesis of plant associated molecule biological information.

Description of drawings

Fig. 1 is the GUS determination of activity histogram as a result of the root that contains CsVDEP promotor transgenic arabidopsis, stem, leaf, flower and fruit pod.X-coordinate is represented the position of plant, and A-E represents root, stem, leaf, flower and the fruit pod of transgenic arabidopsis respectively; Ordinate zou is represented the relative reactivity of GUS.

Fig. 2 contains CsVDEP promotor transgenic arabidopsis and changes Arabidopis thaliana root, the stem of pCAMBIA1391 empty carrier, the GUS coloration result figure of Ye Hehua.Wherein, first row is for containing the GUS coloration result figure of CsVDEP promotor transgenic arabidopsis root, stem, Ye Hehua, and second row is the GUS coloration result figure (negative control) of the Arabidopis thaliana root that changes the pCAMBIA1391 empty carrier, stem, Ye Hehua.A is the GUS coloration result of transgenic arabidopsis root; B is the GUS coloration result of transgenic arabidopsis leaf; C is the GUS coloration result of transgenic arabidopsis flower; D is the GUS coloration result of transgenic arabidopsis stem.

Fig. 3 is promotor (CsVDEP) PCR product agarose gel electrophoresis figure.Wherein, the 1st swimming lane is DL2000bpmarker, and the 2nd swimming lane is PCR product C sVDEP.

Embodiment

Below in conjunction with specific embodiment the present invention is described further.

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

Cucumber variety is worn the seed (closing seed ltd available from Beijing jade) of many stars

The pCAMBIA1391 carrier is (general like spit of fland biotechnology (Beijing) ltd, catalog number (Cat.No.): Biovector-104802)

The acquisition of embodiment 1, promotor (CsVDEP)

The planting seed of cucumber variety Dai Duoxing in the mixed-matrix that the peat composed of rotten mosses, vermiculite and field soil (volume ratio is 1: 1: 1) are housed, when treating that plant grows to 5-6 sheet blade, is chosen the tender blade of children fine noon, extract total DNA of blade.And be template with this DNA; Go out a possible cucumber zeaxanthin diepoxide decylization oxidase gene promoter sequence according to academy of agricultural sciences's cucumber gene group sequence alignment; And design following PCR primer: upstream primer is 5 '-TCTAGGATTAGTAGATCGATTGTTACC-3 '; Downstream primer is 5 '-GGCGCATCAGTGATTTGAAAAGAAG-3 ', through PCR method clone cucumber zeaxanthin diepoxide decylization oxidase gene promotor.Reaction system is following: 10 * PCR damping fluid, 5 μ l; DNTP Mixture (25mM) 4 μ l; Taq enzyme (5U/ μ l) 1 μ l; Each 2 μ l of upstream and downstream primer (concentration is 10 μ M); Template (DNA) 2 μ l; DdH ₂O is supplemented to 50 μ l.The PCR reaction conditions is following: 95 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30S, 58 ℃ of annealing 30s, 72 ℃ are extended 2min, totally 30 circulations, last 72 ℃ are extended 10min.

Get 10 μ L PCR products and carry out agarose gel electrophoresis.The result shows, obtains the purpose fragment (Fig. 3) of size about 2000bp.Downcut the purpose fragment; Be connected to pGEM-T easy carrier (Promega company after the purifying and recovering; Article number: A1360); To connect product transformed into escherichia coli DH5 α bacterial strain, scribble IPTG (sec.-propyl-β-D-sulfo-galactopyranoside) and X-gal (5-bromo-4-chloro-3-indoles-β-D-galactoside) on the surface, contain grow overnight on the LB flat board of penbritin (100 mcg/ml).Second day picking white colony shakes bacterium and spends the night in the LB liquid nutrient medium.Alkaline process extracts DNA, the sample presentation order-checking.The purpose fragment that after the homology comparative analysis, confirm to obtain promptly is the fragment of cucumber zeaxanthin diepoxide decylization oxidase gene promotor, with this fragment called after CsVDEP, is connected to the recombinant plasmid called after pGEM-T easy:CsVDEP behind the pGEM-T easy carrier.The gene order of CsVDEP is shown in sequence in the sequence table 1.

Embodiment 2, the checking of promotor (CsVDEP) functionally active

One, the plant expression vector construction that contains promoter sequence

Recombinant plasmid pGEM-T easy:CsVDEP with embodiment 1 gained is a template, and at the two ends of CsVDEP design primer, upstream primer is: 5 ' ACGCGTCGACTCTAGGATTAGTAGATCGATTGTTACC-3 ' (sequence of underscore part is restriction enzyme site Sal I and protection base thereof, and the later sequence of restriction enzyme site Sal I is corresponding to the 1-27 position of sequence 1); Downstream primer is: 5 '- CATGCCATGGGGCGCATCAGTGATTTGAAAAGAAG-3 ' (sequence of underscore part is restriction enzyme site Nco I and protection base, and the later sequence of restriction enzyme site Nco I is corresponding to the 1959-1983 position of sequence 1).Amplify the CsVDEP fragment that two ends are contained restriction enzyme site Sal I and Nco I respectively through the PCR reaction.After again the CsVDEP fragment that obtains and pCAMBIA1391 carrier being used above-mentioned two kinds of digestion with restriction enzyme respectively simultaneously; Big fragment after the enzyme of purpose fragment CsVDEP cut product and pCAMBIA1391 carrier enzyme and cut reclaims simultaneously; After the connection of T4 ligase enzyme, transformed into escherichia coli.The positive colony that obtains is identified with order-checking through PCR, obtains to make up successful plant expression vector, called after pCAMBIA1391:CsVDEP.

Two, the preparation of agrobacterium tumefaciens competent cell, conversion

(1) preparation of agrobacterium tumefaciens competent cell

Operate according to following steps:

(1) single bacterium colony of picking agrobacterium tumefaciens EHA105 is inoculated in that 5ml is additional to be had in the YEB substratum of 70mg/L Rifampin (Rif) liquid, and 28 ℃, shaking table concussion (rotating speed is 200rpm) overnight cultures.

(2) culture of getting 2ml step (1) continues to be cultured to OD to liquid YEB substratum ₈₀₀Be about 0.5.

(3) with the culture ice bath of step (2) 30 minutes, 4 ℃, 5000rpm, centrifugal 5 minutes, abandoning supernatant.

(4) use the NaCl suspension thalline of the concentration of 10ml precooling as 0.1mol/L.

(5) 4 ℃, 5000rpm, the suspension liquid of centrifugation step (4) 5 minutes, is abandoned supernatant.

(6) use the CaCl of the concentration of 1ml precooling as 20mmol/L ₂The suspension thalline is distributed into 200 μ l/ pipe, freezing-70 ℃ of preservations in back in the liquid nitrogen.

(2) freeze-thaw method transforms Agrobacterium

Operate according to following steps:

(1) competent cell for preparing of thawing step () on ice.

(2) add plant expression vector pCAMBIA1391:CsVDEP or the pCAMBIA1391 empty carrier that 1-2 μ l plasmid step 1 obtains, ice bath 45 minutes, in the liquid nitrogen freezing 1 minute, 37 ℃ of water-baths 3 minutes.

(3) the YEB liquid nutrient medium of adding 1ml antibiotic-free, 28 ℃, shaking table concussion (rotating speed is 200rpm) was cultivated 3 hours.

(4) the YEB liquid nutrient medium with the 1ml antibiotic-free returned lysosome to 12000rpm again to concentrate bacterium liquid in centrifugal 1 minute.

(5) the bacterium liquid after will transforming is applied to and contains 50mg/L kantlex (Kan), and on the solid LB substratum of 70mg/L Rifampin (Rif), 28 ℃, constant incubator was cultivated 2-3 days.

With the Agrobacterium EHA105 called after EHA105/pCAMBIA1391:CsVDEP that changes recombinant vectors pCAMBIA1391:CsVDEP over to; Change the Agrobacterium EHA105 called after EHA105/pCAMBIA1391 of pCAMBIA1391 empty carrier over to.

Three, the acquisition of transgenic arabidopsis

(1) Agrobacterium-mediated Transformation Arabidopis thaliana

Arabidopis thaliana transforms with reference to [Clough SJ such as Clough; Bent AF. (1998) Floral dip:a simplified methodfor Agrobacteriym-mediated transtormation of Arabidopsis thaliana.The Plant Journal; 16 (6), 735～743] the colored method of dipping in is carried out.Transformation receptor is Arabidopis thaliana (Arabidopsis thaliana).With Agrobacterium (EHA105/pCAMBIA1391:CsVDEP or EHA105/pCAMBIA1391) the bacterium liquid of incubated overnight, with dip-dyeing solution dilution, be immersed in the During Agrobacterium liquid 2 minutes to the bud that will bloom, 25～28 ℃ of dark cultivations 20 hours.Then, after plants transformed yielded positive results, collect seed.

(2) screening of transgenic arabidopsis and detection

With the seed of collecting, carry out surface sterilization after, be planted in the enterprising row filter of MS substratum that contains Totomycin.Will be on the hygromycin resistance substratum can normal growth the Arabidopis thaliana plant be transferred to flowerpot, it is yielded positive results.Extract plant leaf DNA with the SDS-CTAB improved method, to the CsVDEP gene fragment, design primer (upstream primer: 5 '-ACTAAACATTTGGTTCACCAATA-3 '; Downstream primer: 5 '-GGCGCATCAGTGATTTGAAAAGAAG-3 '; Carry out pcr amplification; Screening obtains changing over to the positive plant (obtaining the PCR product about 350bp) of CsVDEP gene from the Arabidopis thaliana plant that imports EHA105/pCAMBIA1391:CsVDEP, collects the seed of positive plant.Simultaneously with the Arabidopis thaliana plant that imports EHA105/pCAMBIA1391 as contrast.

Four, the functional verification of promotor (CsVDEP)

The functional verification of promotor (CsVDEP) is to carry out through the gus gene expression analysis of transgenic arabidopsis and tissue chemical analysis's experiment.Specific as follows said:

(1) activation analysis of the gus gene of transgenic arabidopsis

(1) making of methyl umbelliferone (MU) typical curve

With stop buffer (0.2mol/L Na ₂CO ₃) MU is store the liquid dilution, be made into a series of MU reference liquids of concentration range at 0-10 μ mol/L, make a typical curve through the fluorescence intensity of measuring them.

(2) GUS extracts

1) gets fresh vegetable material (changeing root, stem, leaf, flower, the fruit pod of CsVDEP gene Arabidopis thaliana), put into 1.5ml Effendof pipe, add 300 μ l GUS extracting solutions (prescription: 0.1mol/L phosphoric acid buffer (pH=7.0) 50ml; 10% sarcosyl 1ml; 0.5mol/L EDTA (pH=8.0) 2ml; Methyl alcohol 20ml; Triton X-100 100 μ l; Beta-mercaptoethanol 100 μ l; Use H ₂The O constant volume is to 100ml), tissue is ground with refiner on ice.

2) 4 ℃, the centrifugal 10min of 8000rpm changes supernatant in the new pipe over to, and is subsequent use.

(3) GUS is active detects

1) each sample of measuring get 3 parallel.

When 2) sample is measured, prepare 160 μ l in advance, 4-methyl umbelliferone-β of 1mmol/l-D-glucuronide (4-Methylumbellifery-β-D-Glucuronide is called for short MUG) solution is in Eppendorf tube, at 37 ℃ of following water-bath 5min.

3) to above-mentioned 2) in add 40 μ l cell extracts, abundant mixing rapidly, and take out reaction terminating liquid (the 0.2mol/L Na that 100 μ l mixed solutions join 900 μ l at once ₂CO ₃) in, with 0 point of this pipe as enzymatic reaction.

4) behind the 15min, take out the reaction solution of 100 μ l, change the reaction terminating liquid of 900 μ l over to.

5) with spectrophotofluorometer under excitation wavelength 365nm, emission wavelength 455nm, measure the fluorescent value of different time points.

6) get the supernatant of 100 μ l, with Xylene Brilliant Cyanine G method working sample protein content.

7) calculate GUS activity in each sample unit time.Wherein method of calculation by: according to step 5) the typical curve of survey fluorescent value and step 1); The MU concentration change of the unit time that calculates; The sample protein content of the participation reaction that records divided by step 6) with the MU concentration change of unit time again; Obtain the MU concentration change of the albumen unit time of unit mass, the GUS that is in the unit time is active.

GUS active level analytical results is shown in Fig. 1 (data among Fig. 1 are three repeated experiments results' MV).The GUS activity mainly shows changes in the CsVDEP gene Arabidopsis leaf, and the GUS activity is lower in root, stem, flower and the fruit pod.And there is not the GUS activity as the Arabidopis thaliana plant of the commentaries on classics pCAMBIA1391 empty carrier of negative control.

(2) the GUS tissue chemical analysis of transgenic arabidopsis

(1) [prescription is: 200mM PBS (PH7.0) vegetable material root to be detected, stem, leaf, flower and negative control material (wild-type Arabidopis thaliana) all to be added the GUS staining fluid; 100mM K ₃Ife (CN) ₆100mM K ₄Ife (CN) ₆0.5mM EDTA (PH8.0); X-Gluc (10mg/ml); Polysorbas20] in, 37 ℃ of insulations several hours or spend the night.

(2) sample (comprising green materials such as blade) is with 70% ethanol decolorization 2-3 time, is white in color to negative control material.

(3) blueness under the microscopic examination, white background is GUS and expresses the site.

The result is as shown in Figure 2, and in the Arabidopis thaliana that has transformed the CsVDEP promotor, GUS dyeing is obvious blue in blade, do not observe obvious blue in the petal, but dyeing is obvious in the calyx.Can observe faint blueness in the plant root.And change the Arabidopis thaliana plant of pCAMBIA1391 empty carrier root, stem, leaf, spend all and be not colored.

The gus gene expression analysis of above-mentioned transgenic arabidopsis and the experimental result of tissue chemical analysis show that the CsVDEP promotor is an efficient specific expressed promotor in the blade, and this is significant for further research photosynthesis of plants.

Claims (10)

1.DNA molecule is following 1)-3) in any:

1) dna molecular shown in the sequence 1 in the sequence table;

2) under stringent condition with 1) dna sequence dna hybridization that limits and dna molecular with promoter function;

3) with 1) or 2) dna sequence dna that limits has 90% above homology, and have the dna molecular of promoter function;

Said 3) gene in is with 1) or 2) gene that limits preferably has the homology more than 95%.

2. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain the said dna molecular of claim 1.

3. recombinant vectors according to claim 2 is characterized in that: said recombinant vectors inserts the recombinant plasmid that the said dna molecular of claim 1 obtains for the MCS at pCAMBIA1391.

4. expression cassette according to claim 2 is characterized in that: said expression cassette starts the goal gene of expressing by the said dna molecular with promoter function by said dna molecular, and transcription termination sequence is formed; Said dna molecular is connected with said goal gene with functional mode, and said goal gene is connected with said transcription termination sequence.

5. the said dna molecular of claim 1 is starting the application of goal gene in expression of plants.

6. application according to claim 5 is characterized in that: said plant is an Arabidopis thaliana.

7. according to claim 5 or 6 described application, it is characterized in that: the said leaf specific expressino that is expressed as.

8. the method for utilizing the described dna molecular of claim 1 to cultivate transgenic plant comprises the steps:

1) makes up the goal gene recombinant expression vector: goal gene is inserted claim 2 or 3 said recombinant vectorss, make the described dna molecular of claim 1 start said destination gene expression, obtain the goal gene recombinant expression vector;

2) the goal gene recombinant expression vector that step 1) is made up imports in the purpose plant, obtains the transgenic plant of the said goal gene of specifically expressing in blade.

9. method according to claim 8 is characterized in that: said purpose plant is an Arabidopis thaliana.

10. the said dna molecular total length of claim 1 or arbitrary segmental primer increase.

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