CN101928707A - Purple spire fusion gene and use thereof as visual selection marker gene in plant transgenic technology - Google Patents

Abstract

The invention discloses a purple spire fusion gene and use thereof as a visual selection marker gene in plant transgenic technology. The arabidopsis spire specific promoter and tomato anthocyanin synthesis path regulator gene is utilized to construct the fusion gene which is named PL1 fusion gene, which has the nucleotide sequence shown in a sequence table 1. The fusion gene can be used as visual selection marker gene, replaces antibiotic resistance gene and other traditional selection marker genes in the plant transgenic technology, and selects transgenic plants by taking purple bud or spire as characteristics. Therefore, problems of food safety, environmental safety and the like possibly caused by the traditional selection marker genes in the plant gene engineering technique application process can be avoided, and defects of complicated steps, long experimental period, low deletion efficiency, species restriction and the like of the conventional method for obtaining antibiotic resistance marker-free transgenic plants are overcome. The invention also provides application of the fusion gene to mini-tomato transformation.

Description

Purple spire 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 make up the PL1 fusion gene and be marked at application in the transgenic plant as visual selection with the regulatory gene of Arabidopis thaliana spire specificity promoter and tomato cyanogen glycosides route of synthesis.

[background technology]: since the first strain transgenic plant were come out, the research of transgenic plant had obtained very big development with application.At present, existing a lot of transgenic plant enter commercially produces, the crop of multiple good character such as comprise pest-resistant, antiweed, antiviral, improvement quality, prolong storage period.But along with the continuous appearance of commercialization transgenic plant new variety, people have also proposed query to transgenic plant safety.

Because currently used plant transgenic technology is mainly undertaken by tissue culture, in its process, usually utilize selective markers such as antiviral antibiotic class or antiweed class to screen transformant, after screening process finishes, selectable marker gene and goal gene together are incorporated in the transgenic plant genome, the existence of these selectable marker genes become influence transgenic plant safety principal element it～.The security of transgenic plant mainly refers to food safety and two aspects of environmental safety, for fear of this potential safety issue, people attempt to obtain by several different methods the transgenic plant of safety, mainly comprise the utilization of safe marker gene and the deletion dual mode of traditional selectable marker gene.

When utilizing safe marker gene to carry out the transformed plant screening, its proteins encoded is not that non-cell transformed is killed, but transformant is under certain favourable metabolism or developmental condition, perhaps have some unique visual properties, thereby filter out transformed plant.The advantage of this safe marker gene is that selective agent has no side effect, and in most cases helps the regeneration of transformed plant and the raising of transformation efficiency.For example: the ipt gene of sequences encoding isopentenyl transferase is a phytokinin synthetic key gene, Ebinuma will carry out the tobacco conversion as safe marker gene from the ipt gene of Agrobacterium P022T-DNA, the result proves that transformant can grow on the substratum that does not contain phytokinin, and forms indefinite bud; Unconverted cell then can not normal growth and differentiation, finally death (Ebinuma, 1997).And adopt the visual selection mark, and then in the Plant Transformation process, need not to use selective agent, according to some unique visual properties of the coded generation of visual selection marker gene transformed plant/material and non-transformed plant/material are made a distinction.For example, Saika can produce the visual characteristics of green fluorescence by the coded product of GFP gene, filters out the transformed calli (Saika, 2009) of paddy rice fast.

The traditional marker-free or the method for tag delete are to utilize traditional selective marker to filter out elementary transfer-gen plant earlier, utilize the reorganization of transfer-gen plant descendant inheritting that selectable marker gene is separated with goal gene then, or utilize the locus specificity enzyme that the marker gene deletion is cultivated and do not had the transfer-gen plant of selecting the border note.Mainly contain four kinds of systems: cotransformation system, homologous recombination system, locus specificity recombination system and transposon system.For example: Chakraborti is in the research work of the transgene tobacco that utilizes Cre/lox recombination system acquisition marker-free, designed two different structures: the one, be goal gene with the anti-aphides gene ASAL of CaMV35S promoters driven, kalamycin resistance gene NPT II is a selectable marker gene, makes up the lox site of Cre recombinase identification at this gene flank; Second structure is that the Cre gene with the CaMV35S promoters driven is a goal gene, and herbicide resistance gene bar is the selection markers gene.These two make up transformation of tobacco respectively, the transgenic plant in T0 generation of single copy of obtaining are respectively hybridized, effect deletion NPT II resistant gene by the Cre recombinase, the deletion rate is 19.2%, filial generation separates the anti-aphid transgenic plant (Chakraborti, 2008) that obtain not have the antibiotics resistance mark.But these class methods exist complex steps to some extent, experimental period is long, deletion efficiency is low, be subjected to problems such as species restriction.

Flower cyanogen glycosides is that anthocyanidin combines with various monose and the flavones polyphenolic compound that forms.It is a class secondary metabolite of phytosynthesis, and the energy controlling plant, mainly is accumulated in the vacuole of plant epidermis cell to purple or even blue a series of variations from red.At present, from corn (Ludwig, 1989), Common Snapdragon (Martin, 1991), petunia (Quattrocchio, 1999), tobacco (Pattanaik, 2010) etc. separate in the plant and cloned part structure gene and the regulatory gene relevant with the anthocyanidin biosynthesizing, myb transcription factor is the regulatory factor of the colored cyanogen glycosides of the very important adjusting of class genetic expression.Human composing types such as Kim, tissue specificity and inducible promoter purple can occur when driving the IbMYB1 gene instantaneous conversion tobacco of sweet potato to be changed, predict that this gene can be used as a potential visable indicia gene (Kim, 2010) in the genetic transformation process of sweet potato.LeANT1 is a MYB class transcriptional regulator, can regulate and control the biosynthesizing of flower cyanogen glycosides in the tomato.People such as Mathews cross and express the transgenic Fructus Lycopersici esculenti that the LeANT1 gene has obtained a series of purples, illustrate to express the accumulation that the ANT1 gene can cause flower cyanogen glycosides.The same transformation of tobacco that makes up, transgene tobacco also shows the colour-change from green to grey violet even intense violet color, shows LeANT1 also can play a role in other dicotyledons (Mathews, 2000).

Can predict that from forefathers' above-mentioned result of study the colour-change feature that the specificity accumulation of flower cyanogen glycosides is presented has the potentiality as a kind of visual selection markers in the plant transgenic technology.Utilize this visual selection markers can set up a kind of safer transformation technology system, this system is used in the genetic transformation of plant, to solve the safety issue that traditional selection markers may be brought to transgenic plant, certainly will in the actual application of transgenic plant new variety, play a great role.

[summary of the invention]: the present invention seeks to solve the potential safety issue because of using traditional resistance screening marker gene to bring in the plant transgenic technology application process, and complex steps, the experimental period of transgenic plant methods such as acquisition antibiotic-free resistance marker commonly used are long, cost is high, deletion efficiency is low, be subjected to defective such as species restriction, provide a kind of visual selection markers gene that is used for plant transgenic technology, i.e. purple spire fusion gene.When utilizing this fusion gene to carry out Plant Transformation, can the purple young shoot or spire as visable indicia, filter out positive transfer-gen plant, thereby obtain not have the transgenic line of traditional selection markers such as antibiotics resistance.

The present invention at first provides a kind of new purple spire fusion gene, and its nucleotide sequence is shown in sequence table 1.The present invention utilizes Arabidopis thaliana spire specificity promoter and tomato cyanogen glycosides route of synthesis regulatory gene to make up to form, with its called after PL1 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 spire specificity promoter and the functional gene that causes or promote purple to produce can also be merged, but obtain the fusion gene that the render transgenic plant produces purple spire or young shoot.

The present invention provides visual selection marker gene the application in plant transgenic technology of a kind of described purple spire fusion gene as safety simultaneously, that is: utilize this fusion gene to carry out Plant Transformation as visual selection marker gene, but young shoot or the spire of render transgenic plant present purple, it as the visual selection mark, can be obtained the male transgenic plant.

As the example of concrete application, the invention provides a kind of construction process of this purple spire 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 PL1 fusion gene in the described application comprises the steps:

The first, with the arabidopsis thaliana genomic dna be template,, reclaim amplified production and carry out the TA clone with PCR method amplification PL1 fusion gene promotor part;

The second, contain the TA cloned plasmids and the pCAMBIA1301 plasmid of PL1 fusion gene promotor with the KpnI/NcoI double digestion, reclaim promoter fragment and big carrier segments respectively;

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

Four, be template with tomato dna group DNA,, reclaim amplified production and carry out the TA clone with PCR method amplification PL1 fusion gene structure gene part;

Five, the TA cloned plasmids that contains PL1 fusion gene structure gene and the above-mentioned the 3rd that obtains with above-mentioned the 4th step of SalI/BstE II double digestion goes on foot the middle plasmid that makes up that contains " spire specificity promoter-GUS " fusion gene that obtains, and reclaims this structure gene fragment and big carrier segments respectively;

Six, above-mentioned two fragments are mixed, under ligase enzyme catalysis, carry out ligation, finish the structure of the PL1 fusion gene on pCAMBIA1301 (, being commercialization carrier commonly used at present) carrier available from CAMBIA company.

The pcr amplification primer of wherein designed PL1 fusion gene promotor part is as follows, and wherein upstream primer is introduced the KpnI restriction enzyme site, and downstream primer is introduced SalI and NcoI restriction enzyme site:

Upstream primer: 5 '-GGTACCATGTGATTATTCCAAATACATACTT-3 '

Downstream primer: 5 '-CCATGGTCGACATGGAGCTAGTTTCTCTCTCTCT-3 '

The pcr amplification primer of designed PL1 fusion gene structure gene part is as follows, and upstream primer is introduced the SalI restriction enzyme site, and downstream primer is introduced BstE II restriction enzyme site:

Upstream primer: 5 '-GTCGACCATGAACAGTACATCTATGTCTTCAT-3 '

Downstream primer: 5 '-CTCGAGTTAATCAAGTAGATTCCATAAGTCA-3 '.

The application of PL1 fusion gene in plant transgenic technology in the described application, operating process is as follows:

Transform mini-tomato Micro-Tom with the PL1 fusion gene: cultivate the Agrobacterium that has the PL1 fusion gene with common YEP liquid nutrient medium, to OD ₆₀₀Be the centrifugal collection thalline in 1.0 left and right sides, thalline again suspends to use liquid MS medium (containing the 100mmol/L Syringylethanone) with volume then; Get the blade of mini-tomato Micro-Tom aseptic seedlings, in above-mentioned bacterium liquid, infected 10 minutes, put then to secretly cultivating 2 days on the culture medium altogether; 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 young shoot to filter out positive bud as visable indicia; When positive bud grows to about 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 regulatory gene of Arabidopis thaliana spire specificity promoter and tomato cyanogen glycosides route of synthesis to make up the PL1 fusion gene; In the Plant Transformation process, this fusion gene can substitute resistant genes such as traditional antiviral antibiotic or antiweed, as visable indicia, filters out positive transfer-gen plant with purple spire or young shoot.This fusion gene is applied to plant transgenic technology as the selection markers gene of secure visual, not only avoided using the potentially dangerous of aspects such as food safety that traditional selectable marker gene may bring and environmental safety, and compare with the method for commonly used acquisition non selecting sign transgene plant, have easy and simple to handle, flow process short, low cost and other advantages.Therefore, the present invention will provide strong support for the practical application of Plant Transformation technology.

[description of drawings]:

Fig. 1 is the application of PL1 fusion gene in the mini-tomato conversion process, and goal gene is hygromycin gene hpt.Figure a: after the conversion, hygromycin resistance screening visible purple bud of initial stage and green bud also deposited; Figure b: in the resistance screening later stage, visible green bud flavescence gradually, withered to dead illustrates that it is non-conversion bud; And the purple bud has hygromycin resistance, can continue vigorous growth, is illustrated as hpt and transforms bud.Figure c: purple transforms bud through continuing to move autochthonal length after growth, the root induction.As seen the mature leaf purple of transfer-gen plant fades away, and can carry out continuing vigorous growth normally, normally blossoming and bearing fruit.

Fig. 2 is that the PCR of PL1 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 2-8:PL1 fusion gene transgenic Fructus Lycopersici esculenti plant is the PCR product of template.Swimming lane 9:PL1 fusion gene plasmid be template PCR over against photograph; Swimming lane 10:H ₂O is the negative contrast of the PCR of template.

[embodiment]:

Embodiment 1:PL1 fusion gene promotor is partly cloned

The first, be the promotor that template is utilized the PL1 fusion gene of PCR method amplification 1593bp with the arabidopsis thaliana genomic dna, reclaim amplified production and carry out the TA clone.

(1) pcr amplification purpose fragment

According to the sequences Design special primer in Arabidopis thaliana spire specificity promoter district, in upstream primer, introduce the KpnI restriction enzyme site, in downstream primer, introduce SalI and NcoI restriction enzyme site.

Upstream primer: 5 '- GGTACCATGTGATTATTCCAAATACATACTT-3 ' (introducing the KpnI point of contact)

Downstream primer: 5 '- CCATGGTCGACATGGAGCTAGTTTCTCTCTCTCT-3 ' (introducing SalI and NcoI point of contact)

Extracting arabidopsis thaliana genomic dna according to a conventional method, is template with the genomic dna, utilizes above-mentioned primer to carry out pcr amplification, preparation PL1 fusion gene 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 segmental clone of purpose and positive colony

1. the segmental recovery of purpose

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 the concrete operations 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, evenly is applied to then 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 plasmid according to a conventional method,, produce the pGEM-T-Easy carrier segments and the 1593bp fragment that comprises PL1 fusion gene promotor of 3kb with KpnI and SalI double digestion.By foregoing PCR primer and amplification condition, be template with the plasmid extract, carry out pcr amplification, detect the PL1 fusion gene promoter fragment that produces 1593bp through agarose gel electrophoresis, be the positive colony that contains this promoter sequence.

4. sequence verification

Through the positive colony of identifying, to deliver the bacterium stab culture and carry out dna sequencing to Sangon (worker Bioisystech Co., Ltd is given birth in Shanghai), its nucleotide sequence is shown in sequence table 1.

Embodiment 2: the CaMV 35S promoter with on alternative pCAMBIA 1301 carriers of cloned spire specificity promoter makes up " spire specificity promoter-GUS " fusion gene in the middle of obtaining

(1) from intestinal bacteria, extracts vector plasmid pCAMBIA 1301 (available from CAMBIA companies), with reclaiming big carrier segments (wherein including the gus reporter gene sequence) behind the KpnI/NcoI double digestion.

(2) from embodiment 1 prepared TA clones, extract plasmid, use the KpnI/NcoI double digestion, reclaim (with embodiment 1) PL1 fusion gene promoter fragment by agarose gel electrophoresis.

(3) above-mentioned 2 fragments are spent the night in 16 ℃ of connections under ligase enzyme catalysis, " spire specificity promoter-GUS " fusion gene of finishing on pCAMBIA 1301 carriers makes up.

Linked system:

(4) with connecting mixture transformed into escherichia coli DH5 α competent cell, method is with embodiment 1.

(5) select the white colony that transforms on dull and stereotyped (Kan resistance), extract plasmid according to a conventional method, produce two fragments with Kpn I/ and BstEII double digestion plasmid DNA, one is pCAMBIA 1301 carrier segments of 8991bp, and another is 3644bp " spire specificity promoter-GUS " fusion gene.

(6) be that template is carried out the PCR reaction with the plasmid, identify " spire specificity promoter-GUS " fusion gene in the plasmid, the size of amplified fragments is 1866bp.The primer is as follows:

Upstream primer: 5 '-GGTACCATGTGATTATTCCAAATACATACTT-3 '

Downstream primer: 5 '-TCGCGATCCAGACTGAATGCC-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:PL1 fusion gene

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

Upstream primer: 5 '-GTCGACCATGAACAGTACATCTATGTCTTCAT-3 '

Downstream primer: 5 '-CTCGAGTTAATCAAGTAGATTCCATAAGTCA-3 '

Extracting tomato dna group DNA according to a conventional method, is template with the genomic dna, with PCR method amplification PL1 fusion gene structure gene, carries out the TA clone with the pGEM-T-Easy carrier then.PCR reaction system, reaction conditions, the segmental recovery of purpose, clone and order-checking are with embodiment 1.

Embodiment 4: make up the PL1 fusion gene on the pCAMBIA1301 carrier

(1) extracts the plasmid that contains " spire specificity promoter-GUS " fusion gene that embodiment 2 prepares, with reclaiming big carrier segments (wherein not containing the gus reporter gene sequence) behind the SalI/BstE II double digestion.

(2) from embodiment 3 prepared TA clone, extract plasmid, use the SalI/BstEII double digestion, reclaim (with embodiment 1) PL1 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, the PL1 fusion gene of finishing on pCAMBIA 1301 carriers makes up.The method of linked system and transformed into escherichia coli is with embodiment 2.

(4) select the white colony that transforms on dull and stereotyped (Kan resistance), extract plasmid according to a conventional method, produce two fragments with KpnI and BstE II double digestion plasmid DNA, one is pCAMBIA 1301 carrier segments of 8991bp, and another is the PL1 fusion gene of 2415bp.

(6) be that template is carried out the PCR reaction with the plasmid, identify the PL1 fusion gene in the plasmid, the size of amplified fragments is 2415bp.The primer is as follows:

Upstream primer: 5 '-GGTACCATGTGATTATTCCAAATACATACTT-3 '

Downstream primer: 5 '-CTCGAGTTAATCAAGTAGATTCCATAAGTCA-3 '

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

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

The application of embodiment 5:PL1 fusion gene in the mini-tomato conversion process

(1) the PL1 fusion gene with embodiment 4 preparations transforms mini-tomato Micro-Tom, with the hygromycin gene hpt that is had on the pCAMBIA1301 carrier is the goal gene of conversion, detects feasibility and the practicality of PL1 fusion gene as the visual selection mark.Concrete method for transformation adopts agriculture bacillus mediated genetic transforming method (Meissner, 1997), existing green bud produces in the conversion process, also there is the purple bud to produce, but after the screening of 10mg/L hygromycin resistance, green bud is all dead by sieve, and the purple bud all has hygromycin resistance, can contain lasting vigorous growth (Fig. 1 a and 1b) on the hygromycin resistance substratum, be illustrated as the conversion bud of hpt gene.With these purple bud root inductions, move soil subsequently and be cultured to and blossom and bear fruit.Can observe the transfer-gen plant that become by the purple blastogenesis in the earth culture process of growth, the mature leaf purple fades away, vine growth and development normal (Fig. 1 c).

(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 the blade genomic dna, carries out PCR with following primer and reacts, and reaction system is with embodiment 1:

Upstream primer: 5 '-GGTACCATGTGATTATTCCAAATACATACTT-3 '

Downstream primer: 5 '-CTCGAGTTAATCAAGTAGATTCCATAAGTCA-3 '

The PCR product carries out agarose gel electrophoresis then, and the PL1 fusion gene band of 2408bp 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 into (see figure 2) in the Plant Genome.

(3) the PL1 fusion gene is expressed at the phasic specificity of sprouting of inducing that mini-tomato transforms

Being contained the mini-tomato explant that the Agrobacterium of PL1 fusion gene infects is inducing on the substratum of sprouting, existing green bud produces, also there is the purple bud to produce, but through behind the antibiotic-screening, green bud is all dead by sieve, and the purple bud all has resistance, the purple bud by root induction after, particularly plant moves after the soil purple of the mature leaf (see figure 1) that disappears gradually, and vine growth and development is normal.This has fully proved when having the carrier transformed plant of PL1 fusion gene, with purple spire or young shoot feasibility and the practicality as the visual selection mark of transfer-gen plant.

With the visual selection marker gene of PL1 fusion gene as the plant conversion, potentially dangerouss such as food that selective markers such as antiviral antibiotic or antiweed may bring and environmental safety have not only been avoided using, also overcome traditional acquisition non selecting sign transgene plant method such as complex steps, experimental period is long, deletion efficiency is low, be subjected to defectives such as species restriction, have easy and simple to handle, flow process is short, low cost and other advantages, this fusion gene also can be used for the conversion of other plant, equally obtains the transgenosis new variety with purple young shoot or spire as the selection markers of secure visual.Therefore, the present invention will provide strong support for the practical application of Plant Transformation technology.

[reference]:

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Sequence table

Sequence table SEQ UENCE LISTING

＜110〉Nankai University

＜120〉purple spire fusion gene and as the application of visual selection marker gene in plant transgenic technology

<160>1

<170>PatentIn?version?3.3

<210>1

<211>2408

<212>DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana (L.) Heynh.) tomato (Lycopersicon esculentum)

<220>

<221>primer_bind

<222>(1)...(25)

<223>atgtgattat?tccaaataca?tactt

<220>

<221>primerbind

<222>(1)...(25)

<223>tgacttatgg?aatctacttg?attaa

<400>1

atgtgattat?tccaaataca?tactttggat?gtttaaactt?aatcttgttt?cttcctacgt 60

ataaatatta?atcatcgagg?taaaaaaagt?tttgtcttat?tttcgcgatg?catgaaggat 120

aaacctaatg?actttaattt?tttgaaaatg?taaccctttt?actcatagat?taattaccgt 180

atgtttttgt?tgccataatg?acagcctcta?caactgtgat?agtcaatttt?ttctgcaaat 240

attaaattag?gaattcaatg?ctactatcaa?tagaagaaac?agctgagtat?tacattttaa 300

tttaaagaca?aaatttttga?aaaatgttat?aatttctaac?aatattatta?aaatatgatg 360

cctataatgt?atttcctatg?ttcttaaaat?attttttttt?atatttagtt?ataaatacat 420

tatgaaccaa?taatagttgg?tgaattcaaa?tatctccatt?aatatttttt?gaaatctaca 480

aattattaat?atttagtcaa?taacaatgca?tagaaagttc?caaaaaaaat?tttgttaaca 540

gaaacttcca?aatttttttt?ttttatggaa?caagaaataa?cagatagaaa?actattttgt 600

tgtggaatgg?aagtagtaat?atacattaag?caaattttaa?aaaattatat?aagcctatac 660

gcgctcaaag?tatgttatct?agtaggtgta?attaataatg?catggtgcga?ttcagaattg 720

ggacaacaat?gaaaacggaa?ttaaaatatt?aactttaaaa?taaataaaaa?tttgagtaaa 780

tgtgttttct?gactattgag?gggcaaaaaa?aagacaatgc?caaaagtcta?cgggtttgac 840

tgtccagttc?ggtaataatc?taataactct?gtctttgacc?gcacgctcgt?gtaggggtcc 900

ttctgacatt?ttcactgttc?tacccctact?cgtgagccca?cccttttccc?atatcctaag 960

ggtaattttg?gaaatcccaa?tttaaaccga?ttgagaccgt?accggacttc?ctgggattct 1020

gctggagcat?ttatcaaaaa?ttattagcac?gaatgggttt?attaatttaa?aaactcacaa 1080

cttgatcaga?taaaatttca?taaacacttt?tacgatggat?tcgtacgatc?tatctaatga 1140

cttttttttt?tctaccacgg?tggatgaaag?ttatagtact?attagccaga?gacaattgat 1200

tatagatata?tccattaatc?catgatattt?atgatataaa?tagctgttaa?actatttcag 1260

catcgcagct?ttctgcaact?tttgttttta?atttaagagt?ttaataaata?aaagtattaa 1320

aaggagcata?acgaggcaac?aaaagtaatg?aacacggaga?aacaaaagcc?atgaagctca 1380

ttggttaggt?ttaagcttaa?taagaagatt?ttattaaatt?ttaatgacga?tgataacaat 1440

tatattttct?gacttcttta?aaaccccctc?ttacaaacag?aagctccctt?tttcagtaga 1500

agtccgattc?ccaatcttaa?agacaaagcc?attagaaaga?gaaagtgagt?gagagagaga 1560

gagaaactag?ctccatgtcg?accatgaaca?gtacatctat?gtcttcattg?ggagtgagaa 1620

aaggttcatg?gactgatgaa?gaagattttc?ttctaagaaa?atgtattgat?aagtatggtg 1680

aaggaaaatg?gcatcttgtt?cccataagag?ctggtctgaa?tagatgtcgg?aaaagttgta 1740

gattgaggtg?gctgaattat?ctaaggccac?atatcaagag?aggtgacttt?gaacaagatg 1800

aagtggatct?cattttgagg?cttcataagc?tcttaggcaa?cagatggtca?cttattgctg 1860

gtagacttcc?cggaaggaca?gctaacgatg?tgaaaaacta?ttggaacact?aatcttctaa 1920

ggaagttaaa?tactactaaa?attgttcctc?gcgaaaagat?taacaataag?tgtggagaaa 1980

ttagtactaa?gattgaaatt?ataaaacctc?aacgacgcaa?gtatttctca?agcacaatga 2040

agaatgttac?aaacaataat?gtaattttgg?acgaggagga?acattgcaag?gaaataataa 2100

gtgagaaaca?aactccagat?gcatcgatgg?acaacgtaga?tccatggtgg?ataaatttac 2160

tggaaaattg?caatgacgat?attgaagaag?atgaagaggt?tgtaattaat?tatgaaaaaa 2220

cactaacaag?tttgttacat?gaagaaatat?caccaccatt?aaatattggt?gaaggtaact 2280

ccatgcaaca?aggacaaata?agtcatgaaa?attggggtga?attttctctt?aatttaccac 2340

ccatgcaaca?aggagtacaa?aatgatgatt?tttctgctga?aattgactta?tggaatctac 2400

ttgattaa 2408

Claims (7)

1. a purple spire fusion gene is called for short the PL1 fusion gene, and its nucleotide sequence is shown in sequence table 1; This fusion gene is to utilize the regulatory gene structure of Arabidopis thaliana spire specificity promoter and tomato cyanogen glycosides route of synthesis to form.

2. a purple spire fusion gene is characterized in that, is the nucleic acid molecule that the homology more than 85% is arranged with the described nucleotide sequence of claim 1.

3. purple spire fusion gene, it is characterized in that, utilize method according to claim 1, but spire specificity promoter and the functional gene that causes or promote purple to produce are merged the fusion gene that the render transgenic plant that is obtained produces purple spire or young shoot.

4. purple spire fusion gene according to claim 1 is characterized in that, the pcr amplification primer of designed PL1 fusion gene promotor part is as follows, and wherein upstream primer is introduced the KpnI restriction enzyme site, and downstream primer is introduced SalI and NcoI restriction enzyme site:

Upstream primer: 5 '-GGTACCATGTGATTATTCCAAATACATACTT-3 '

Downstream primer: 5 '-CCATGGTCGACATGGAGCTAGTTTCTCTCTCTCT-3 '

Wherein upstream and downstream restriction enzyme site that primer is introduced can be changed according to the multiple clone site on the selected binary expression vector.The selected binary expression vector of present embodiment is pCAMBIA1301, is commercialization carrier commonly used at present.

5. purple spire fusion gene according to claim 1 is characterized in that, the pcr amplification primer of the structure gene part of designed PL1 fusion gene is as follows, and upstream primer is introduced the SalI restriction enzyme site, and downstream primer is introduced the BstEII restriction enzyme site:

Upstream primer: 5 '-GTCGACCATGAACAGTACATCTATGTCTTCAT-3 '

Downstream primer: 5 '-CTCGAGTTAATCAAGTAGATTCCATAAGTCA-3 '

Wherein upstream and downstream restriction enzyme site that primer is introduced can be changed according to the multiple clone site on the selected binary expression vector.The selected binary expression vector of present embodiment is pCAMBIA1301, is commercialization carrier commonly used at present.

6. purple spire fusion gene according to claim 1 is characterized in that, the concrete operations step that makes up the PL1 fusion gene is as follows:

(1) with the arabidopsis thaliana genomic dna is template,, reclaims amplified production and carry out the TA clone with PCR method amplification PL1 fusion gene promotor part;

(2) contain the TA cloned plasmids and the pCAMBIA1301 plasmid of PL1 fusion gene promotor with the KpnI/NcoI double digestion, reclaim promoter fragment and big carrier segments respectively;

(3) above-mentioned two fragments are mixed, under ligase enzyme catalysis, carry out ligation, make up " spire specificity promoter-GUS " fusion gene in the middle of obtaining;

(4) be template with tomato dna group DNA,, reclaim amplified production and carry out the TA clone with PCR method amplification PL1 fusion gene structure gene part;

(5) the TA cloned plasmids that contains PL1 fusion gene structure gene and the middle plasmid that makes up that contains " spire specificity promoter-GUS " fusion gene of above-mentioned (3) step acquisition, recovery structure gene fragment and the big carrier segments respectively that obtains with SalI/BstE II double digestion above-mentioned (4) step;

(6) above-mentioned two fragments are mixed, under ligase enzyme catalysis, carry out ligation, finish Arabidopis thaliana spire specificity promoter-tomato cyanogen glycosides route of synthesis regulatory gene, the i.e. structure of PL1 fusion gene on the pCAMBIA1301 carrier.

7. a claim 1,2 or 3 described purple spire fusion genes are as the application of visual selection marker gene in plant transgenic technology, it is characterized in that, transform plant with the carrier that has the PL1 fusion gene, obtain to contain the transgenic plant of fusion gene sequence of the present invention with purple young shoot or spire as the visual selection mark.

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