CN101892224B - Purple leaf fusion gene and use of purple leaf fusion gene as visible selective marker gene in plant transgenic technology - Google Patents

Abstract

The invention discloses a purple leaf fusion gene and use of the purple leaf fusion gene as a visible selective marker gene in plant transgenic technology. Particularly, the fusion gene is constructed by utilizing an arabidopsis vein-specific VS promoter and an adjustment gene for synthesizing tomato anthocyanin, is called a PL2 gene, and has a nucleotide sequence shown in a sequence table 1. The fusion gene can serve as the visible selective marker gene and substitute for conventional selective marker genes, such as antibiotics resistance genes and the like, in the plant transgenic technology to select transgenic plants by utilizing the characteristic that transformed buds have purple leaves; and compared with conventional resistant selective markers, the fusion gene has the advantages of visibility, stability, persistency and the like. In addition, after transgenic varieties with the visible marker are popularized and used, the purple leaves can serve as markers to distinguish the transgenic plants from non-transgenic plants, which can greatly reduce the cost of the environmental security detection of the transgenic plants. The invention also provides the embodiment of the use of the fusion gene in micro-tomato transformation.

Description

Purple vein fusion gene and as the application of visual selection marker gene in plant transgenic technology

[technical field]: the invention belongs to plant genetic engineering field, specifically with the leaf vein specific VS promotor of Arabidopis thaliana with the regulatory gene structure PL2 fusion gene of tomato cyanogen glycosides route of synthesis and as the application of visual selection marker gene in plant transgenic technology.

[background technology]: plant transgenic technology is that the good goal gene that utilizes recombinant DNA technology to clone imports vegetable cell or tissue, and expresses therein, thereby makes plant obtain new proterties.Genetically modified crops can be improved the output of farm crop, improve the content of beneficiating ingredient in the plant, change the color of plant, reduce the usage quantity of weedicide, agrochemical agricultural chemicals etc., and save a large amount of manpowers, thereby brought huge economic and social benefit to human society.But the security of transgenic plant has also caused heated argument and common concern in the world simultaneously, wherein because present transgenic technology is often carried out the screening of transgenic plant by use antiviral antibiotic class or antiweed class selective screening mark, these selective screening marker gene finally can together be incorporated in the genome of plant with goal gene, become one of principal element that affects transgenic plant safety.The resistant maker gene safety issue has two approach in the solution transgenic plant: still use resistant maker gene when (1) transforms, obtain the resistant maker gene in its genome to be deleted after the transgenic plant again.But this method steps is loaded down with trivial details, the cycle of acquisition three T-DNAs vector is longer; (2) the development safe maker gene is used for Genetic Transformation in Higher Plants, will be a very promising research direction and utilize color as security visual selection mark.On the other hand, various countries for guaranteeing that the domestic economy interests are not encroached on, are absolutely necessary to the detection of transgenic plant and product in world's trade activity.Equally, in to genetically modified organism security control process, producer drift etc. is the importance that ecological environment security detects.The transgene component analytical procedure is used in the at present detection of these aspects more, mainly contains DNA detection method and protein detection method two classes (Yang Jianxia, 2004).In addition, biochip technology has begun to be applied to the detection of transgenic plant.And these methods all need to drop into larger manpower and materials cost undoubtedly.If can in transgenic plant, introduce safe, lasting, visual selection markers, will be possible to use remarkable visual proterties and directly distinguish transgenosis and non-transgenic plant, and judge whether producer drift etc., can greatly reduce input and the cost of genetically modified organism safety detection.

Blade the nearly all plant of whole vegitabilia all shows green, flower and fruit then present colourful, pigment composition and content in the blade of this and plant, flower, the fruit are different relevant, chlorophyll is occupied an leading position in blade, and in flower and fruit, spend the content of cyanogen glycosides considerably beyond chlorophyll, thereby give plant flowers and the various colors of fruit.Flower cyanogen glycosides is that anthocyanidin is combined with various monose and the flavones polyphenolic compound that forms.It is a class secondary metabolite of phytosynthesis, can control plant from red to purple or even blue a series of variations, mainly is accumulated in the vacuole of plant epidermis cell.Early-stage Study shows, some transcription regulaton factors have regulating and controlling effect to plant flowers cyanogen glycosides synthetic.Such as: (the Elomaa et al. such as Elomaa, 2003) Herba Leibnitziae transcription regulaton factor gene GMYB10 is placed under the CaMV35S promotor change tobacco over to, transfer-gen plant footpath, leaf, calyx, pollen sac, ovary wall all contrast a large amount of anthocyanins of accumulation, and color changes.Quattrocchio etc. (Quattrocchio et al., 1999) change the C1 of corn and Lc gene after the petunia over to, obtain red callus and pink corolla.These results show that the synthetic controlling elements of colored cyanogen glycosides can the regulating and controlling effect of allos performance to spending the cyanogen glycosides to synthesize.

Tissue or organ specific promoters can be regulated and control goal gene and express at specific tissue or organ, in plant gene engineering technology, can utilize tissue or organ specific promoters targetedly, special, regulate and control the expression of goal gene efficiently.Vein is positioned at the blade of plant shoot, is comprised of the vascular tissue that is applied in the mesophyll, is transporting and the underwork of leaf.Our imagination: if obtain to drive goal gene specific expressed promotor in vein, utilize in plant gene engineering technology that this type of promotor is specific expressed in vein targetedly to be caused or promote the synthetic key gene of flower cyanogen glycosides, the vein colour-change feature (such as the purple vein) that then the specificity accumulation because of flower cyanogen glycosides can be presented is as a kind of visual selection markers in the plant transgenic technology.The novel plant transformation technology system of setting up accordingly, not only can solve the safety issue that traditional selection markers may be brought to transgenic plant, can also be applied to this visual selection markers in the testings such as environmental safety of transgenic plant, whether to have coloured vein as the separator of transgenosis and non-transgenic plant, simple, to greatly reduce the cost of transgenic plant detection, certainly will in the actual application of transgenic plant new variety, play a great role.

In addition, flower cyanogen glycosides belongs to the flavonoid class compound, also belongs to polyphenols, has very strong removing oxyradical, anti-oxidant function (Lin Xiaoxia etc., 2005), has the effect that improves stress resistance of plant.Simultaneously, flower cyanogen glycosides also is the focus in human food prods and the health research as a kind of natural anti-oxidation component substances.Colored cyanogen glycosides in a large amount of epidemiological study proof food has preventive and therapeutic action to human numerous disease, for example studies have shown that anthocyanin has the effect (fourth is sharp, 2004) that reduces the coronary heart disease sickness rate; And for example: the synthetic regulatory gene Del of the colored cyanogen glycosides of specific expressed Common Snapdragon and ROS1 in tamato fruit, can make the high-caliber colored cyanogen glycosides of accumulation in the tamato fruit, feed with the purple tomato of this accumulation flower cyanogen glycosides and easily to suffer from cancer mouse (Trp53-/-), life (Butelli et al., 2008) that can the significant prolongation mouse.Therefore, appropriateness accumulation flower cyanogen glycosides can also improve quality and the economic worth of transgenic plant product.

In sum, no matter the visual colour-change that produces based on flower cyanogen glycosides accumulation regulation and control as a kind of security selection markers in the Plant Transformation process or as the visual detection marks of a kind of transgenic plant, all has very high development and application and is worth.Simultaneously again can a certain amount of antioxidant component of render transgenic plant interior accumulation-flower cyanogen glycosides, reached the desired result of killing three birds with one stone.

[summary of the invention]: the present invention seeks to utilize the leaf vein specific VS promotor of Arabidopis thaliana and the regulatory gene gene fusion construct of tomato cyanogen glycosides route of synthesis, as the visual selection markers gene in the plant transgenic technology.

Leaf vein specific promoter of the present invention (vein-specific promoter), be called for short the VS promotor and be us with a kind of vascular bundle specificity promoter of the promoter engineering initiative of many organs of Arabidopis thaliana expressing gene, it is specific expressed in the vein of different plants that it can drive goal gene.The present invention utilizes the synthetic genes involved of this promoters driven control flower cyanogen glycosides specific expressed in vein, and the vein of render transgenic plant presents purple.When utilizing fusion gene provided by the invention to carry out Plant Transformation, can the purple vein as the selection markers of transgenic plant, the screening of render transgenic plant has reached visual, compare with traditional antibiotics resistance screening method, have easy and simple to handle, low-cost, secure visual, and need not to carry out the follow-up advantages such as tag delete, significant for the potential safety issue that the traditional resistant maker gene of solution may bring.Simultaneously because the activity of VS promotor is special in the leaf development process, stable, lasting, it drives flower cyanogen glycosides in vein syntheticly has stability, can not subdue with the increase of leaf age, can utilize whether have the purple vein as the separator of transgenosis and non-transgenic plant in most of period of growth and development of plants.This will bring great convenience for the environmental safety in the transgenic plant popularizing planting process detects, whether the staff can be directly judges transgenic plant from the color of plant leaf vein, this will save a large amount of human and material resources and financial resources undoubtedly, greatly reduce the cost of transgenic plant detection.

The invention provides a kind of new purple vein fusion gene, its nucleotide sequence is shown in sequence table 1.The present invention utilizes the leaf vein specific promoter of Arabidopis thaliana abbreviation VS promotor and tomato cyanogen glycosides route of synthesis regulatory gene to make up to form, with its called after PL2 fusion gene (from purple leaf initial).The present invention also comprises the nucleic acid molecule that the homology more than 85% is arranged with above-mentioned nucleotide sequence.

Utilize the method for the invention, any vein or vascular bundle specificity promoter can also be merged with the functional gene that causes or promote purple to produce, but obtain the fusion gene that the render transgenic plant produces the purple vein.

The present invention provides a kind of described purple vein fusion gene as the visual application of Security selection marker gene in plant transgenic technology simultaneously, that is: utilize this fusion gene to carry out Plant Transformation as visual selection marker gene, but the vein of render transgenic plant presents purple, it as the visual selection mark, can be obtained positive transgenic plant.Simultaneously, whether have the visual separator that the purple vein also can be used as transgenosis and non-transgenic plant, decrease detects relevant testing cost to the transgenic plant environmental safety.

As the example of concrete application, the invention provides a kind of construction process of this purple vein fusion gene and as the application of visual selection marker gene in plant transgenic technology.Operating process is as follows:

The construction process of the PL2 fusion gene in the described application comprises the steps:

The first, take arabidopsis thaliana genomic dna as template, with PCR method amplification VS promotor part, reclaim amplified production and carry out the TA clone;

The second, the TA cloned plasmids that contains the VS promotor and the pCAMBIA1301 plasmid that obtain with the above-mentioned the first step of Kpn I/Bgl II double digestion reclaim respectively promoter fragment and large carrier segments;

Three, above-mentioned two fragments are mixed, under ligase enzyme catalysis, carry out ligation, make up " VS promotor-GUS " fusion gene in the middle of obtaining;

Four, take tomato dna group DNA as template, with PCR method amplification PL2 fusion gene structure gene part, reclaim amplified production and carry out the TA clone;

Five, the TA cloned plasmids that contains PL2 fusion gene structure gene and the above-mentioned the 3rd that obtains with above-mentioned the 4th step of Sal I/BstE II double digestion goes on foot the middle plasmid that makes up that contains " VS promotor-GUS " fusion gene that obtains, and reclaims respectively this structure gene fragment and large carrier segments;

Six, two fragments that above-mentioned the 5th step reclaimed are mixed, and carry out ligation under ligase enzyme catalysis, finish the structure of the PL2 fusion gene on pCAMBIA1301 (available from CAMBIA company, being commercialization carrier commonly used at present) carrier.

Wherein the pcr amplification primer of designed VS promotor part is as follows, and wherein upstream primer is introduced Kpn I restriction enzyme site, and downstream primer is introduced BglII and SalI restriction enzyme site:

Upstream primer: 5 '-GGTACCTCGACGCGTGAGAGTTTCCGGC-3 '

Downstream primer: 5 '-AGATCTGTCGACATTTGCTGTGTCAATTCTCACTTC-3 '

The pcr amplification primer of designed PL2 fusion gene structure gene part is as follows, and upstream primer is introduced Sal I restriction enzyme site, and downstream primer is introduced BstE II restriction enzyme site:

Upstream primer: 5 '-GTCGACCATGAACAGTACATCTATGTCTTCAT-3 '

Downstream primer: 5 '-GGTCACCTTAATCAAGTAGATTCCATAAGTCA-3 '

The application of PL2 fusion gene in plant transgenic technology in the described application, operating process is as follows:

Transform Micro-Tom with the PL2 fusion gene: with the Agrobacterium of common YEP liquid nutrient medium cultivation with the PL2 fusion gene, to OD ₆₀₀Be the centrifugal collection thalline in 1.0 left and right sides, then use liquid MS medium (containing the 100mmol/L Syringylethanone) the Eddy diffusion thalline with volume; Get the blade of Micro-Tom aseptic seedlings, in above-mentioned bacterium liquid, infected 10 minutes, then put to common culture medium and secretly cultivated 2 days; Above-mentioned explant moved to take off on the bacterium culture medium illumination and cultivated 3 days, move on the differentiation adventitious buds substratum, induce and sprout, utilize the purple vein to filter out positive bud as visable indicia; When positive bud grows to approximately 2 centimetres high, change on the adventive root division culture medium and cultivate root induction; The plant that will take root preferably moves soil and is cultured to and blossoms and bears fruit.

Advantage of the present invention and positively effect:

The present invention utilizes the VS promotor of Arabidopis thaliana and the regulatory gene of tomato cyanogen glycosides route of synthesis to make up the PL2 fusion gene; With this fusion gene conversion of plant, as visable indicia, filter out positive transfer-gen plant with the purple vein, and the phenotype of this purple vein is stable lasting in the growth and development process of transfer-gen plant.This fusion gene is compared with resistant genes such as traditional antiviral antibiotic or antiweeds, has advantages of safety, visual, compares with traditional safe maker gene, and it is lasting, stable to have advantages of.This fusion gene is applied in the plant transgenic technology as the selection markers gene of secure visual, not only avoided using the potentially dangerous of the aspects such as food safety that traditional selectable marker gene may bring and environmental safety, and avoided using the problems such as that safe maker gene commonly used may occur is unstable, easy silence, the detection of render transgenic plant is convenient, quick.The purple vein is applied to as visable indicia can also greatly reduce the cost of detection in the testings such as transgenic plant environmental safety.Therefore, the present invention will provide for the practical application of plant transgenic technology strong support.

[description of drawings]:

Fig. 1 is the application of PL2 fusion gene in the mini-tomato conversion process, and goal gene is hygromycin gene hpt.Fig. 1 a: induce the stage of sprouting after the conversion, the conversion bud with purple vein is containing vigorous growth on the substratum of sprouting of Totomycin; Fig. 1 b: the growing state of conversion bud after continuing growth, root induction with purple vein; Fig. 1 c: the conversion bud with purple vein moves the autochthonal long initial stage after root induction, and the blade vein purple of transfer-gen plant is high-visible; Fig. 1 d: wild-type induced bud (non-transformed bud) can only be grown at the substratum of sprouting that does not contain Totomycin, can see that non-transformed bud does not have the phenotype of purple vein; Fig. 1 e: transfer-gen plant moves the autochthonal long later stage, can observe the transfer-gen plant that grown up to by the conversion bud with purple vein in the earth culture process of growth, and the purple sustainable existence is in vein, and vine growth and development is normal.Fig. 1 f: the wild-type plant moves the growing state behind the soil, does not have the phenotype of purple vein.

Fig. 2 is that the PCR of PL2 fusion gene transgenosis mini-tomato plant identifies; Swimming lane M:plus2000Marker; Swimming lane 1: the genomic dna with wild-type tomatoes is the negative contrast of template; The genomic dna of swimming lane 2-5:PL2 fusion gene Transgenic Tomato Plants is the PCR product of template.Swimming lane 6:PL2 fusion gene plasmid is the positive control of the PCR of template; Swimming lane 7:H ₂O is the negative contrast of the PCR of template.

[embodiment]:

The clone of the VS promotor of embodiment 1:PL2 fusion gene

The first, utilize the VS promotor of PCR method amplification 1109bp take arabidopsis thaliana genomic dna as template, reclaim amplified production and carry out the TA clone.

(1) pcr amplification purpose fragment

According to the sequences Design special primer of the VS promoter region of Arabidopis thaliana, in upstream primer, introduce Kpn I restriction enzyme site, in downstream primer, introduce BglII and SalI restriction enzyme site.

Upstream primer: 5 ' GGTACCTCGACGCGTGAGAGTTTCCGGC-3 ' (introducing Kpn I point of contact)

Downstream primer: 5 '- AGATCTGTCGACATTTGCTGTGTCAATTCTCACTTC-3 ' (introducing BglII and SalI point of contact)

Extract according to a conventional method arabidopsis thaliana genomic dna, take genomic dna as template, utilize above-mentioned primer to carry out pcr amplification, preparation VS promoter fragment.

The PCR reaction system:

The PCR response procedures:

94 ℃ 5 minutes;

94 ℃ 30 seconds, 60 ℃ 1 minute 30 seconds, 30 circulations;

72 ℃ 10 minutes;

4 ℃ of insulations.

(2) evaluation of the clone of purpose fragment and positive colony

1. the recovery of purpose fragment

Reclaim target DNA fragment by agarose gel electrophoresis, recovery method adopts the DNA sepharose of precious biological (TaKaRa) company in Dalian to reclaim test kit, and concrete operation step is seen catalogue.

2. connect

The reagent that adds following reaction system, 16 ℃ of reactions are spent the night, and realization purpose fragment is connected with pGEM-T-Easy (available from Promega company) carrier.

3. the evaluation of conversion and positive colony

CaCl routinely ₂Induce and method for transformation, preparation bacillus coli DH 5 alpha competent cell connects product transformed competence colibacillus cell with 10 μ L, then evenly is applied on the flat board that contains Amp, X-gal and IPTG, is inverted for 37 ℃ and cultivates 12-14 hour.Select the white colony that transforms on the flat board, extract according to a conventional method plasmid, with Kpn I and Bal II double digestion, produce the pGEM-T-Easy carrier segments and the 1109bp fragment that comprises the VS promotor of PL2 fusion gene of 3kb.By foregoing PCR primer and amplification condition, take the plasmid extraction thing as template, carry out pcr amplification, detect the VS promoter fragment that produces 1109bp through agarose gel electrophoresis, be the positive colony that contains this promoter sequence.

4. sequence verification

Through the positive colony of identifying, deliver the bacterium stab culture and carry out dna sequencing to Sangon (worker Bioisystech Co., Ltd is given birth in Shanghai).

Embodiment 2: the VS promotor of being cloned with embodiment 1 substitutes the CaMV 35S promoter on pCAMBIA 1301 carriers, makes up " VS promotor-GUS " fusion gene in the middle of obtaining

(1) from intestinal bacteria, extracts vector plasmid pCAMBIA 1301 (available from CAMBIA companies), with reclaiming large carrier segments (wherein including the gus reporter gene sequence) behind the Kpn I/Bgl II double digestion.

(2) from embodiment 1 prepared TA clones, extract plasmid, with Kpn I/Bgl II double digestion, reclaim (with embodiment 1) VS promoter fragment by agarose gel electrophoresis.

(3) above-mentioned 2 fragments are spent the night in 16 ℃ of connections under ligase enzyme catalysis, finish " VS promotor-GUS " Fusion gene construction on pCAMBIA 1301 carriers.

Linked system:

(4) transform the bacillus coli DH 5 alpha competent cell with connecting mixture, method is with embodiment 1.

(5) select the white colony that transforms on dull and stereotyped (Kan resistance), extract according to a conventional method plasmid, produce two fragments with Kpn I and BstE II double digestion plasmid DNA, one is pCAMBIA 1301 carrier segments of 8979bp, and another is 3179bp " VS promotor-GUS " fusion gene.

(6) carry out the PCR reaction take plasmid as template, identify " VS promotor-GUS " fusion gene in the plasmid, the size of amplified fragments is 1405bp.The primer is as follows:

Upstream primer: 5 '-GGTACCTCGACGCGTGAGAGTTTCCGGC-3 '

Downstream primer: 5 '-TCG CGATCC AGACTGAAT GCC-3 '

(7) cut the positive colony of identifying with PCR through enzyme, deliver the order-checking of order-checking company.

The clone of the structure gene of embodiment 3:PL2 fusion gene

According to the sequence information design special primer of tomato cyanogen glycosides route of synthesis regulatory gene cDNA, introduce respectively Sal I and BstE II restriction enzyme site at 5 ' end, the size of PCR product should be 825bp.

Upstream primer: 5 '-GTCGACCATGAACAGTACATCTATGTCTTCAT-3 '

Downstream primer: 5 '-GGTCACCTTAATCAAGTAGATTCCATAAGTCA-3 '

Extract according to a conventional method tomato dna group DNA, take genomic dna as template, with the structure gene of PCR method amplification PL2 fusion gene, then carry out the TA clone with the pUCm-T carrier.PCR reaction system, reaction conditions, the recovery of purpose fragment, Cloning and sequencing are with embodiment 1.

Embodiment 4: make up the PL2 fusion gene at the pCAMBIA1301 carrier

(1) extracts the plasmid that contains " VS promotor-GUS " fusion gene that embodiment 2 prepares, with reclaiming large carrier segments (wherein not containing the gus reporter gene sequence) behind the SalI/BstEII double digestion.

(2) from embodiment 3 prepared TA clone, extract plasmid, with Sal I/BstE II double digestion, reclaim (with embodiment 1) PL2 fusion gene structure gene fragment by agarose gel electrophoresis.

(3) above-mentioned 2 fragments are spent the night in 16 ℃ of connections under ligase enzyme catalysis, finish the PL2 Fusion gene construction on pCAMBIA 1301 carriers.Linked system and transform colibacillary method with embodiment 2.

(4) select the white colony that transforms on dull and stereotyped (Kan resistance), extract according to a conventional method plasmid, produce two fragments with Kpn I and BstE II double digestion plasmid DNA, one is pCAMBIA 1301 carrier segments of 8979bp, and another is the PL2 fusion gene of 1933bp.

(6) carry out the PCR reaction take plasmid as template, identify the PL2 fusion gene in the plasmid, the size of amplified fragments is 1933bp.The primer is as follows:

Upstream primer: 5 '-GGTACCTCGACGCGTGAGAGTTTCCGGC-3 '

Downstream primer: 5 '-GGTCACCTTAATCAAGTAGATTCCATAAGTCA-3 '

(7) cut the positive colony of identifying with PCR through enzyme, deliver the order-checking of order-checking company, its nucleotide sequence is shown in sequence table 1.

(8) from positive colony, extract plasmid, transform Agrobacterium LBA4404 with ordinary method, obtain the through engineering approaches Agrobacterium, be used for Plant Transformation.

The application of embodiment 5:PL2 fusion gene in the mini-tomato conversion process

(1) the PL2 fusion gene with embodiment 4 preparations transforms Micro-Tom, on the pCAMBIA1301 carrier with the goal gene of hygromycin gene hpt as transforming, detect the PL2 fusion gene as feasibility and the practicality of visual selection marker gene.Concrete method for transformation adopts agrobcterium-mediated transformation (Meissner, 1997), existing bud with purple vein produces in the conversion process, also there is the bud with purple vein to produce, but after the screening of 10mg/L hygromycin resistance, the bud that does not have the purple vein is all dead by sieve, and the bud with purple vein all has hygromycin resistance, can continue vigorous growth (Fig. 1 a) is illustrated as the conversion bud of hpt gene containing the hygromycin resistance substratum.With these conversion bud root inductions (Fig. 1 b) with purple vein, move subsequently soil and be cultured to and blossom and bear fruit.Can observe the transfer-gen plant that grown up to by the conversion bud with purple vein in the earth culture process of growth, vein purple sustainable existence, vine growth and development normal (Fig. 1 c and 1e).

(2) PCR of transfer-gen plant detects: the blade of difference clip transfer-gen plant and wild-type plant, with reference to " molecular cloning experiment guide (third edition) " (Huang Peitang etc., 2002) method is extracted leaves genomic DNA, carry out PCR reaction with following primer, reaction system is with embodiment 1:

Upstream primer: 5 '-GGTACCTCGACGCGTGAGAGTTTCCGGC-3 '

Downstream primer: 5 '-GGTCACCTTAATCAAGTAGATTCCATAAGTCA-3 '

Then the PCR product carries out agarose gel electrophoresis, and the PL2 fusion gene band of 1933bp appears in transfer-gen plant, and the fusion gene band does not appear in the non-transgenic plant, proves that the purpose fragment has been incorporated in the Plant Genome (to see Fig. 2)

(3) PL2 fusion gene continuous expression in the growth and development process of transgenosis mini-tomato

Being contained the mini-tomato explant that the Agrobacterium of PL2 fusion gene infects is inducing on the substratum of sprouting, existing bud with purple vein produces, also there is the bud with purple vein to produce, but through behind the antibiotic-screening, the bud that does not have the purple vein is all dead by sieve, and the bud with purple vein all has resistance, after bud with purple vein is induced to take root, the purple of vein slightly weakens but still high-visible (Fig. 1 b), vine growth and development is normal after moving soil, and the vein of mature leaf presents stable lavender (Fig. 1 c and 1e).This sufficient proof when using carrier conversion of plant with the PL2 fusion gene, with feasibility and the practicality of purple vein as the visual selection mark of transgenic plant.

The present invention utilizes the leaf vein specific VS promotor of Arabidopis thaliana and the regulatory gene of tomato cyanogen glycosides route of synthesis to make up the PL2 fusion gene; With the visual selection marker gene of PL2 fusion gene as Plant Transformation, compare with traditional safe maker gene, it is lasting, stable to have advantages of.This fusion gene is as the selection markers of secure visual, not only avoided using the potentially dangerous of the aspects such as food safety that traditional selective marker may bring and environmental safety, and avoided using the problems such as that security sign commonly used may occur is unstable, easy silence, the detection of render transgenic plant is convenient, quick.The purple vein is applied to as visable indicia can also greatly reduce the cost of detection in the testings such as transgenic plant environmental safety.Therefore, the present invention will provide for the practical application of plant transgenic technology strong support.

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Claims (2)

1. a purple vein fusion gene is called for short the PL2 fusion gene, and its nucleotide sequence is shown in sequence 1; This fusion gene is to utilize the leaf vein specific promoter abbreviation VS promotor of Arabidopis thaliana and the regulatory gene structure of tomato cyanogen glycosides route of synthesis to form.

2. purple vein fusion gene according to claim 1 is as the application of visual selection marker gene in the mini-tomato transgenic technology, it is characterized in that, use the carrier with the PL2 fusion gene to transform mini-tomato, as the visual selection mark, can obtain to contain the transgenic plant of other purpose fragments that contain simultaneously on described fusion gene sequence and the carrier with the purple vein.

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