CN1328385C

CN1328385C - High affinity ammonium transport factor fuse gene and its use in transgenic plant

Info

Publication number: CN1328385C
Application number: CNB2005100145444A
Authority: CN
Inventors: 王宁宁; 张茹; 林景瑜; 李鹏丽; 李晔; 崔孟祥
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2005-07-15
Filing date: 2005-07-15
Publication date: 2007-07-25
Anticipated expiration: 2025-07-15
Also published as: CN1737148A

Abstract

The present invention relates to a fuse gene of a high affinity ammonium transport factor and the use in transgenic plants, which belongs to the field of plant genetic engineering. In the present invention, the promoters of the specificity express genes AtPRP3 and the genes ATAMT1 of high affinity ammonium transport factors of an arabidopsis root system are used for constructing fuse genes AtPRP 3-AtAMT1; the fuse genes are transported into mode plant arabidopsis and mini-type tomatoes successfully so that the transgenic arabidopsis and tomatoes present high ability of low nitrogen stress resistance. The transportation of the fuse genes can improve the absorption rate of the transgenic plant to nitrogen fertilizer, reduce the application amount and pollution of chemical fertilizer, reduce production cost and protect environment. In addition, the transgenic tomatoes have the characteristics of senility delaying, obvious enhancement of chlorophyll and lycopene in fruits, etc.; the lycopene has strong oxidation resistance and prevention of multifunction cancers. The fuse genes can be transported into other crops to obtain new transgenic species with efficient absorption to nitrogen fertilizer and soil depletion resistance.

Description

High affinity ammonium transport factor fuse gene and the application in transgenic plant thereof

Technical field

The invention belongs to plant genetic engineering field, specifically use promotor and the high affinity ammonium transport factor AtAMT1 of Arabidopis thaliana root system different expression gene AtPRP3; 1 gene constructed AtPRP3-AtAMT1; 1 fusion gene and the application in transgenic plant thereof.

Background technology:

Modern agriculture production need be used a large amount of fertilizer in soil, not only need big economic investment, also can cause pollution and destruction to geologic media, has therefore greatly limited modern agricultural development.Simultaneously, the quick growth of world population has caused great pressure to the supply of agricultural byproducts.Expensive, poor efficiency that changes that the modern agriculture production model causes and geologic media destroy has become in the world wide key subjects that developing country especially needs to be resolved hurrily.The fast development of world population requires higher agricultural soil utilization ratio and more high-quality and agriculture production output.How to improve plant absorbs nutrient from soil ability with reduce agriculture production cost, the environment that conserves water and soil just becomes the important topic that we will face.Utilize genetic engineering technique to transform farm crop the receptivity of micronutrient in the soil and utilising efficiency have been become the new millennium important topic of research of agricultural science in the world.

Farm crop from soil to the absorption of nitrogen mainly by corresponding N O on the root cells plasma membrane ₃ ^-Transport factor and NH ₄ ⁺Transport factor is responsible for.Under two kinds of simultaneous situations of nitrogen form, NH ₄ ⁺Utilized by preferential absorption.At present root system of plant is inhaled ammonium from soil Physiologic Studies shows, this process presents tangible two-phase dynamic characteristic, promptly has high affinity ammonium movement system (high-affinity ammonium transport system simultaneously, HATS) and low affinity ammonium movement system (low-affinityammonium transport system, LATS).HATS brings into play the main film of striding when plant is in low ammonium concentration (being lower than 1mmol/L) change the ammonium effect, and demonstrate the saturation kinetics feature, the LATS function then is to be in high ammonium concentration plant to start when (being higher than lmmol/L), and demonstrates the unsaturation dynamic characteristic.No matter be HATS or LATS, mostly the translocator member that it comprises is constitutive expression, but under certain condition also can be by abduction delivering (Sohlenkamp C et al, Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants.FEBS Letters, 2000,467:273-278).

To studies show that of model plant Arabidopis thaliana: ammonium transport factor is mainly by AtAMT1 and AtAMT2 dpd gene family coding, the wherein ATAMT1 in the AtAMT1 family; The ammonium transport factor of 1 coded by said gene in the ammonium transport factor that all have been found to NH ₄ ⁺The highest (the Gazzarrini S et al of avidity, Three functional transporters for constitutive, diurnallyregulated and starvation-induced uptake of ammonium into Arabidopsis roots.Plant Cell, 1999,11:937-948), this means and effectively to absorb nitrogenous fertilizer from barren soil, therefore be chosen as the goal gene of this experiment.But, AtAMT1 in the wild-type Arabidopis thaliana; 1 only just has expression under the condition that coerced by low nitrogen, this gene is not expressed under normal cultivation and planting conditions.Plant absorbs ammonium fertilizer by low affinity ammonium movement system under normal cultivation and planting conditions, therefore need apply a large amount of chemical fertilizer in soil.

For obtain normal cultivate and planting conditions under root also can continuous expression AtAMT1; 1 gene, thereby the transgenic plant that can continue efficient absorption soil nitrogenous fertilizer, we have chosen gene one AtPRP3 gene relevant with growth with the formation of root hair in the mouseearcress, this specific gene expression is in root hair portion, structural protein (the Bernhardt C et al of a proline rich on the coding root hair epidermic cell cell wall, Expression of AtPRP3, a proline-rich structural cell wall protein fromArabidopsis, is regulated by cell-type-specific developmental pathways involved in root hairformation.Plant Physiol, 2000,122:705-714).The function of these structural protein is participate in cell walls synthetic, but with root system the absorption of nitrogenous fertilizer is had no relation.In our experimental design, with goal gene AtAMT1; 1 merges with the promotor of AtPRP3 gene, and the fusion gene that is constituted changes recipient plant over to, and resulting transfer-gen plant will can both continue in whole root growth and development process and cross expression high affinity ammonium transport factor AtAMT1 specifically; 1, on the relative barren of nitrogen supply soil, plant, also can continuous and effective to absorb competent nitrogenous fertilizer used for growth and development of plants on ground, this specific character be the wild-type plant can't obtain.Simultaneously, in natural wild-type plant, the AtPRP3 promotor can only drive AtPRP3 genetic expression and generate the structural protein of proline rich and then synthesizing of involved in plant cell walls, and can't drive AtAMT1; 1 gene is expressed at root and is generated high affinity ammonium transport factor.

Plantation can efficient absorption nitrogen nutrition from soil the new variety crop can reduce amount of application of nitrogen fertilizer greatly, reduce production costs and water and soil pollutes.And this crop new variety of acquisition that develop into of genetic engineering technique provide possibility.But up to the present, also successfully do not cross the document and the patent report of expressing the Nitrogen Absorption associated protein and obtaining the transgenic crop of efficient absorption nitrogenous fertilizer in the soil both at home and abroad.

Summary of the invention

The present invention has overcome shortcoming of the prior art, difference clone plant root system specific expressing promoter and high affinity ammonium transport factor gene, and structure can be at the high affinity ammonium transport factor fuse gene of root system of plant specifically expressing.This fusion gene is changed in the Plant Genome, thereby can obtain root system cross express high affinity ammonium transport factor can be from the transgenic crop new variety of efficient absorption nitrogenous fertilizer under barren soil or the low nitrogen application condition, the water and soil that causes for the production cost that reduces agricultural planting and a large amount of nitrogen applications pollutes significant.

In order to solve the problems of the technologies described above, the present invention is achieved by the following technical solutions:

Utilize promotor and the high affinity ammonium transport factor AtAMT1 of Arabidopis thaliana root system different expression gene AtPRP3; 1 gene constructed AtPRP3-AtAMT1; 1 fusion gene.

The pcr amplification primer of the promotor of designed Arabidopis thaliana root system different expression gene AtPRP3 is as follows, and wherein downstream primer is introduced the BglII point of contact:

Upstream primer: 5 '-TAGAAGAGTTGTAACGCAAGCTGTG-3 '

Downstream primer: 5 '- AGATCTACCATTGCTGAGCGCTTGGC-3 '

Designed Arabidopis thaliana high affinity ammonium transport factor AtAMT1; The pcr amplification primer of 1 gene is as follows, and upstream primer is introduced the BglII point of contact, and downstream primer is introduced the BstEII point of contact:

Upstream primer: 5 '- AGATCTCAACATGTCTTGCTCGGCCA-3 '

Downstream primer: 5 '- GGTC4CCAAATCAAACCGGAGTAGGT-3 '

AtPRP3-AtAMT1; The construction step of 1 fusion gene is as follows:

(1) with the arabidopsis thaliana genomic dna is template,, reclaims amplified production and carry out the TA clone with the promotor of PCR method amplification AtPRP3 gene;

(2) with the arabidopsis thaliana genomic dna be template, with the PCR method AtAMT1 that increases; 1 gene reclaims amplified production and carries out the TA clone;

(3) contain the TA cloned plasmids of AtPRP3 gene promoter and contain AtAMT1 with BglII/EcoRI, BglII/BstEII double digestion respectively; The TA cloned plasmids of 1 gene reclaims AtPRP3 gene promoter fragment and AtAMT1; 1 gene fragment;

(4) with EcoR I/BstE II double digestion pCAMBIA1301 plasmid, reclaim big carrier segments;

(5) mix above-mentioned AtPRP3 gene promoter fragment, AtAMT1; 1 gene fragment and pCAMBIA1301 carrier segments are carried out ligation under ligase enzyme catalysis, finish the AtPRP3-AtAMT1 on the pCAMBIA1301 carrier; The structure of 1 fusion gene.

The sequence of this fusion gene is as follows:

tagaagagtt gtaacgcaag ctgtggttgt gggtgacgat cttccttatt tcgatattgg

ctatccattc aagtttaggt tgttgctttg ttctactatt aatatagctc ggccttgtga

ttatctagtt acgtttattg ctgtgtcttt tgttcatgca tattatttca ttgctttata

atgatacata atatcatact ttggtaatta cttagggatg tctttttgaa attattaatc

agtcatagaa catatatatt catccaactc taatagatgg tatatgatat gaatgtttac

cataccacac aacatacgat ggaaaaatga tgagttgatg acaggtcata tattattatg

tatcttggca tgccatatgt tttgggctca caattatatt tcaattctta accaaattgg

gcttcttaaa cattcaccag gcccattgac ataatcattt cgattcagtt ttcagaaggg

tttttgactc taggatccct ttctctaaag ctcattgcca ttaacgttaa acaaaaacac

tagaaatcaa caaaaactat ttacctaaac caaaacaatc acagtttata cagagtacaa

aatagtgtca taaaacccaa gtaaattaga gaattgatta gggaatatat atgtaagcag

agttactaat aaaacacctt ctgggataaa agagaagaag gtattatcag cgaaactcgc

agctttagtt taggcctcac atgaatcaga taagtacttc atcaaactta gctgggatta

agaaaggccc ttccatctct ctaatttctc ttctctattt gaaaacaact tttaaactat

gaatcaaact gcgagcctta aacattctct aagcatatga gaaaacaaag acgaagaacc

ctaattctaa gatccaacat aagaaaataa acaaaaaccg aagagaaaaa tatatcgccg

aatatatata acaaatctcc gatcattagt caatcgccgc ttgagcttgg atttgaggca

aagacaaata tataggattt gcttttttca tcattagggt ttccagtttt caatgttttg

ggcttatatt atcgggcctt taggtttaat tggcccatta gttgaaacta tacacggatg

gttttcttac tttctaattg ctacaaacaa cagactaatc tgtttttaca tatttaatgg

ttcagatgtc atcaaagatt atctgtccaa atatttatat tataagatac attttaatat

ttaattatga tttagtagag cggccgatcc gttttgatat gtttagaact aaaaattgaa

atatactaaa atcaaaattg gcaaacattt gaacgagtct catttatatg tcctctgacc

atattattta atgggcctat cacatttccc gagcccagat aacgtctttt cagttctcct

atccttttgg tcagcttgtg catggttgtt acttgttaat gatggccaac atgtttttga

tcgtgcatta cacttaggta tgagaatctc aaacatacat gattatcatc aattactcac

ttttaagata acgtttcttt atttcatatt caaattttta ataccccaaa cattttctta

taaagcccca tttggtgaaa tcgttttcct catcacaatt taaatacaaa gaacacaaca

aacgaactag cttaaggcaa tatacaagcc aagcgctcag caatggtaga tctcaacatg

tcttgctcgg ccaccgatct cgccgtcctg ttgggtccta atgccacggc ggcggccaac

tacatatgtg gccagctagg cgacgtcaac aacaaattca tcgacaccgc tttcgctata

gacaacactt acctcctctt ctccgcctac cttgtcttct ctatgcagct tggcttcgct

atgctctgtg ccggttccgt gagagccaag aatactatga acatcatgct taccaacgtc

cttgacgctg cagccggtgg tctcttctat tatctgtttg gctacgcctt tgcctttgga

tctccgtcca atggtttcat cggtaaacac tactttggtc tcaaagacat ccccacggcc

tctgctgact actccaactt tctctaccaa tgggcctttg caatcgctgc ggctggaatc

acaagtggct cgatcgctga acggacacag ttcgtggctt acctaatcta ttcctctttc

ttaaccgggt ttgtttaccc ggtcgtctct cactggttct ggtcagttga tggatgggcc

agcccgttcc gtaccgatgg agatttgctt ttcagcaccg gagcgataga tttcgctggg

tccggtgttg ttcatatggt cggaggtatc gctggactct ggggtgcgct catcgaaggt

ccacgacttg gccggttcga taacggaggc cgtgccatcg ctcttcgtgg ccactcggcg

tcacttgttg tccttggaac attcctcctc tggtttggat ggtacggatt taaccccggt

tccttcaaca agatcctagt cacgtacgag acaggcacat acaacggcca gtggagcgcg

gtcggacgga cagctgtcac aacaacgtta gctggctgca ccgcggcgct gacaacccta

tttgggaaac gtctactc tc gggacattgg aacgtcactg atgtatgcaa cggcctcctc

ggagggtttg cagccataac tggtggctgc tctgtcgttg agccatgggc tgcgatcatc

tgcgggttcg tggcggccct agtcctcctc ggatgcaaca agctcgctga gaagctcaaa

tacgacgacc ctcttgaggc agcacaacta cacggtggtt gcggtgcgtg gggactaata

ttcacggctc tcttcgctca agaaaagtac ttgaaccaga tttacggcaa caaacccgga

aggccacacg gtttgtttat gggcggtgga ggaaaactac ttggagctca gctgattcag

atcattgtga tcacgggttg ggtaagtgcg accatgggga cacttttctt catcctcaag

aaaatgaaat tgttgcggat atcgtccgag gatgagatgg ccggtatgga tatgaccagg

cacggtggtt ttgcttatat gtactttgat gatgatgagt ctcacaaagc cattcagctt

aggagagttg agccacgatc tccttctcct tctggtgcta atactacacc tactccggtt

tgattt

The present invention can also utilize the promoters driven Arabidopis thaliana ammonium transport factor gene A tAMT1 of Arabidopis thaliana AtPRP3 gene; 1 expresses in transgenic plant, is used to improve the specific absorption of crop to nitrogenous fertilizer.Operating process is as follows:

(1) transforms mini-tomato with fusion gene: have AtPRP3-AtAMT1 with common YEP liquid nutrient medium cultivation; The Agrobacterium of 1 fusion gene is 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 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 and cultivate, induce and sprout; When regeneration bud grows to about 2 centimetres high, change on the adventive root division culture medium and cultivate root induction; To take root in the soil of plant immigration preferably, and continue to be cultured to and blossom and bear fruit.

(2) with fusion gene arabidopsis thaliana transformation dish: when first scape of mouseearcress extracted out, sub-scape is cut above the leaf first of scape, scape new after 4-10 days is extracted out, the long transformation experiment that is used for during to 2-10 centimetre; Have AtPRP3-AtAMT1 with common YEP liquid nutrient medium cultivation; The Agrobacterium of 1 fusion gene is to OD ₆₀₀Be the centrifugal collection thalline in 1.8 left and right sides, 1/2MS substratum (containing 5% sucrose and the 0.02%Silwet L-77) thalline that suspends again with liquid makes OD ₆₀₀Be about 0.8; Get 200ml bacterium liquid and put into beaker, plant is stood upside down put into bacterium liquid immersion 10-15 minute, lie against then in the disk,, secretly cultivated one day with the preservative film sealing; Preservative film was taken off in second day, plant is uprightly cultivated, until fruit pod overstrike time results seed fully.

Beneficial effect

This fusion gene is changed in the Plant Genome, thereby can obtain root system cross express high affinity ammonium transport factor can be from the transgenic crop new variety of efficient absorption nitrogenous fertilizer under barren soil or the low nitrogen application condition, the water and soil that causes for the production cost that reduces agricultural planting and a large amount of nitrogen applications pollutes significant.

We utilize the method for transformation of soil Agrobacterium mediation, with AtPRP3-AtAMT1; 1 fusion gene has successfully changed among model plant Arabidopis thaliana and the mini-tomato Micro-Tom.Compare with wild-type, transgenic arabidopsis and transgenic Fructus Lycopersici esculenti all show extremely strong low nitrogen resisting and coerce ability, the transgenosis mini-tomato also show aging delay with fruit in the characteristics that significantly improve of content of lycopene.The resistance of oxidation of Lyeopene is 100 times of vitamin-E, and multiple cancer is had preventive and therapeutic effect, have the title of " gold in the tomato ".AtPRP3-AtAMT1; 1 fusion gene changes other crop over to, also can obtain efficient absorption nitrogenous fertilizer, reduces applying quantity of chemical fertilizer and water and soil pollution, resist histie genetically modified crops new variety.

Description of drawings

Fig. 1: have AtPRP3-AtAMT1; The binary vector of 1 fusion gene makes up schema

Fig. 2: the fusion gene PCR of transgenosis mini-tomato detects

Fig. 3: the low nitrogen resisting of non-transgenic (A) and transgenosis (B) mini-tomato is coerced ability

Fig. 4: the fusion gene PCR of transgenic arabidopsis dish detects

Fig. 5: the low nitrogen resisting of non-transgenic (A) and transgenosis (B) mouseearcress is coerced ability

Fig. 6: the Lyeopene in the tamato fruit is measured

Fig. 7: unit leaf area measuring chlorophyll content in the tomato leaf

Embodiment

Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail.

Embodiment 1: Arabidopis thaliana root system specific expressing promoter AtPRP3 promotor clone

With the arabidopsis thaliana genomic dna is the AtPRP3 gene promoter that template is utilized PCR method amplification 1733bp, reclaims amplified production and carries out the TA clone.

(1) pcr amplification purpose fragment

The sequences Design special primer of the specific expressed AtPRP3 gene promoter area of having delivered according to GenBank of mouseearcress root system is introduced the BglII restriction enzyme site in downstream primer.

Upstream primer: 5 '-TAGAAGAGTTGTAACGCAAGCTGTG-3 '

Downstream primer: 5 '- AGATCTACCATTGCTGAGCGCTTGGC-3 ' (introducing the BglII 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 AtPRP3 gene promoter fragment.

The PCR reaction system:

Reagent	Add-on
Reagent	Add-on	Sterilization distilled water Taq Plus DNA Polymerase 10 * Reaction Buffer dNTP (10mmol/L) upstream primer (10 μ mol/L) downstream primer (10 μ mol/L) Taq Plus DNA Polymerase (5U/ μ L) template DNA	37.5μL 5μL 1μL 2μL 2μL 0.5μL 2μL

The PCR response procedures:

94 ℃ 5 minutes,

94 ℃ 30 seconds, 70 ℃ 3 minutes, 7 circulations;

94 ℃ 30 seconds, 65 ℃ 3 minutes, 30 circulations;

72 ℃ 10 minutes;

4 ℃ of preservations.

(2) evaluation of segmental clone of purpose and positive colony

1. the segmental recovery of purpose (trapping method)

Preparation contains the sepharose of two stock layout product slotted eyes, and the parallel two row's combs of inserting during glue are respectively as point sample hole and collection hole; The PCR product is added in the point sample hole, carries out electrophoresis; When purpose fragment electrophoresis the place ahead, and when being about to enter to collection hole, powered-down, blot in the collection hole electrophoretic buffer and with new buffer solution for cleaning 3-4 time, fill with power-on again, continuation electrophoresis then with new damping fluid; After the purpose fragment all enters collection hole, stop electrophoresis, with the whole damping fluid sucking-offs in the collection hole, transfer in the 1.5mL centrifuge tube; Add the dehydrated alcohol of 2 times of volumes and the 3mol/L sodium-acetate (pH5.2) of 1/10 volume, mixing was placed 2 hours for-20 ℃; 13, centrifugal 15 minutes of 000r/min abandons supernatant; Precipitation is washed 1-2 time with 75% ethanol, and vacuum is drained, and is dissolved in the 6 μ L aseptic double-distilled waters.

2. add the A tail

Because the fragment that obtains through the polymerization of pfu high-fidelity polysaccharase is flat terminal, therefore, the dna fragmentation that the recovery purifying obtains only after adding the A tail, could better be connected with the T carrier.

The reagent that adds following reaction system, 72 ℃ are reacted half hour, finish to add the A tail.

Reagent	Add-on
Reagent	Add-on	Taq DNA Polymerase 10×Reaction Buffer MgCl ₂(25mmol/L) target DNA fragment of dATP (2mmol/L) Taq DNA Polymerase (5U/ μ L) recovery	1μL 1μL 1μL 1μL 6μL

3. connect

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

Reagent	Add-on
Reagent	Add-on	The PCR product T4 DNA Ligase that added the A tail behind 10 * Ligation Buffer 50%PEG pGEM-T-Easy carrier purifying	1μL 1μL 1μL 6μL 1μL

4. the evaluation of conversion and positive colony

CaCl routinely ₂Induce and method for transformation, preparation E.coli DH5 α 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 1742bp fragment that comprises the AtPRP3 gene promoter of 3kb with BglII and EcoR I double digestion.By foregoing PCR primer and amplification condition, be template with the plasmid extract, carry out pcr amplification, detect the AtPRP3 gene promoter fragment that produces 1733bp through agarose gel electrophoresis, be the positive colony that contains the AtPRP3 promotor.

5. sequence verification

Cut the positive colony of identifying with PCR through enzyme, deliver the bacterium stab culture and carry out dna sequencing, prove with the sequence of GenBank accession No.AL162651 in full accord.

Embodiment 2: Arabidopis thaliana high affinity ammonium transport factor AtAMT1; 1 gene clone

The Arabidopis thaliana AtAMT1 that has delivered according to GenBank; The sequence information of 1 gene cDNA (GenBank accession numberX75879) design special primer is introduced Bg1 II and BstE II restriction enzyme site respectively at 5 ' end.The size of PCR product should be 1526bp.

Upstream primer: 5 '- AGATCTCAACATGTCTTGCTCGGCCA-3 ' (introducing Bgl II point of contact)

Downstream primer: 5 '- GGTCACCAAATCAAACCGGAGTAGGT-3 ' (introducing the BstEII point of contact)

Extracting arabidopsis thaliana genomic dna according to a conventional method, is template with the genomic dna, with the PCR method AtAMT1 that increases; 1 gene carries out the TA clone with the pUCm-T carrier then.PCR reaction system, reaction conditions, the segmental recovery of purpose, clone and order-checking are with embodiment 1.

Embodiment 3: utilize pCAMBIA1301 vector construction AtPRP3-AtAMT1; 1 fusion gene

(1) from intestinal bacteria, extracts vector plasmid pCAMBIA1301 (this bacterial strain can obtain from other laboratory or buy from reagent company), reclaim big carrier segments with EcoR I/BstE II double digestion.

(2) from embodiment 1 prepared TA clones, extract plasmid,, reclaim the AtPRP3 promoter fragment by agarose gel electrophoresis (trapping method is with embodiment 1) with Bgl II/EcoR I double digestion.

(3) from embodiment 2 prepared TA clone, extract plasmid,, reclaim AtAMT1 by agarose gel electrophoresis with Bgl II/BstE II double digestion; 1 gene fragment.

(4) above-mentioned three fragments are spent the night in 16 ℃ of connections under ligase enzyme catalysis, finish the AtPRP3-AtAMT1 on the pCAMBIA1301 carrier; 1 fusion gene makes up (see figure 1).

Linked system:

Reagent	Add-on
Reagent	Add-on	T4 DNA Ligase T4 DNA Ligase Buffer pCAMBIA1301 carrier recovery Segment A tPRP3 promotor reclaims Segment A tAMT1 gene and reclaims fragment	1μL 1μL 5μL 1.5μL 1.5μL

(5) with connecting mixture Transformed E .coli DH5 α, select the white colony that transforms on the flat board, method is with embodiment 1.

(6) plasmid of extraction white colony produces two fragments with BstEII and EcoR I double digestion, and one is the pCAMBIA1301 carrier segments of 8975bp, and another is the AtPRP3-AtAMT1 of 3256bp; 1 fusion gene.

(7) plasmid with white colony is that template is carried out the PCR reaction, identifies the AtPRP3-AtAMT1 in the plasmid; 1 fusion gene, the size of amplified fragments is 1608bp.The primer is as follows:

5’-AGATCTCAACATGTCTTGCTCGGCCA-3’

5’-TAATCATCGCAAGACCGGCAACA-3’

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

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

Embodiment 4: the preparation of transgenosis mini-tomato

Use AtPRP3-AtAMT1; The step that 1 fusion gene transforms mini-tomato is as follows:

(1) Agrobacterium is cultivated: picking has AtPRP3-AtAMT1 from the flat board; The single bacterium colony of the Agrobacterium of 1 fusion gene is inoculated in 3mL and contains in the common YEP liquid nutrient medium of 50mg/L kantlex and 25mg/L Streptomycin sulphate, 28 ℃ of following shaking culture 1 day; Get 1mL bacterium liquid and add in the 20mL fresh liquid YEP substratum, continue shaking culture to OD ₆₀₀Be about 1.0; Bacterium liquid is changed in the 50mL centrifuge tube, and 3000r/min collected thalline in centrifugal 20 minutes; Again thalline suspends with the liquid MS medium (contain the 100mmol/L Syringylethanone, be called for short AS) with volume.

The composition of MS substratum (unit: mg/L):

NH4NO3 (ammonium nitrate) 1650

KNO3 (saltpetre) 1900

KH2PO4 (potassium primary phosphate) 170

MgSO47H2O (sal epsom) 370

CaCl22H2O (calcium chloride) 440

Iron salt solutions: the FeSO47H2O (ferrous sulfate) of 5.57g and the Na2-EDTA (disodium ethylene diamine tetraacetate) of 7.45g are dissolved in the 1L water.

MnSO44H2O (manganous sulfate) 22.3

ZnSO47H2O (zinc sulfate) 8.6

H3BO3 (boric acid) 6.2

KI (potassiumiodide) 0.83

NaMoO42H2O (Sodium orthomolybdate) 0.25

CuSO45H2O (copper sulfate) 0.025

CoCl26H2O (cobalt chloride) 0.025

Glycine 2

Vitamin 0.4

Pyridoxine hydrochloride 0.5

Nicotinic acid 0.5

Inositol 100

Sucrose 30000

Agar 10000

pH 5.8

(2) agriculture bacillus mediated conversion: with 10% peace tiformin sterilization mini-tomato seed 10 minutes, plant then on the MS substratum, cultivate aseptic seedling; Get the aseptic seedlings of 8 days seedling ages, excise the cotyledon end, and every cotyledon is cut into two, place the bacterium liquid of having got ready to infect 10 minutes; Take out blade, blot with aseptic filter paper, secretly cultivated 2 days towards transferring to being total on the culture medium (MS+ vitamin B5+1mg/L indolylacetic acid+1mg/L zeatin+100mmol/L Syringylethanone) on the blade face.

(3) plant regeneration and cultivation: above-mentioned explant moved to take off on the bacterium culture medium (MS+ vitamin B5+1mg/L indolylacetic acid+1mg/L zeatin+400mg/L cephamycin), light was cultivated 3 days down; Explant is moved on the differentiation adventitious buds substratum (Totomycin of MS+1.5mg/L indolylacetic acid+1mg/L+0.1mg/L zeatin+proper concn), every two all subcultures once, induce and sprout; When regeneration bud grows to about 2 centimetres high, it is changed on the adventive root division culture medium (Totomycin of MS+0.6mg/ml indolebutyric acid+proper concn), every two all subcultures once, root induction; The plant that will take root preferably moves in the soil, continue to be cultured to bloom, solid.

(4) PCR of transfer-gen plant detects: a blade of every transformed plant of difference clip, extract total DNA, and be template with total DNA, use primer:

5’-AGATCTCAACATGTCTTGCTCGGCCA-3’(+)

5’-AAGCTGCATA GAGAAGACAAGGT-3’(-)

Carry out the PCR reaction, carry out agarose gel electrophoresis then, the AtPRP3-AtAMT1 of 184bp occurs; 1 fusion gene band carries out the PCR reaction with total DNA of wild-type mini-tomato for negative contrast, the fusion gene band do not occur, proves that the purpose fragment has been incorporated into (see figure 2) in the Plant Genome.

Object tape is that (from left to right, swimming lane is followed successively by Marker DL2000 to 184bp., and the negative contrast of-wild-type, the cracking of CB03 Agrobacterium bacterium are over against photograph, and the CB03 transgenic arabidopsis reaches 3 CB03 transgenic strains in Fig. 2.)

(5) low nitrogen resisting of the transgenosis mini-tomato ability of coercing detects

Cultural method in the low nitrogen water planting liquid:

Nitrogenous complete water planting liquid is Hoagland water planting liquid, and the ammonium root concentration in the low nitrogen water planting liquid is 1/40 of nitrogenous complete water planting liquid, and total nitrogen concentration is 1/80 of a nitrogenous complete water planting liquid.15 days T of growth in the low nitrogen water planting liquid ₁Normal for transgenic seedling growth, and wild-type tomatoes can not normal growth under the isometric growth condition, blade is the withered (see figure 3) of flavescence gradually.

(6) Lyeopene of transgenosis mini-tomato is measured

Lyeopene in the high effective liquid chromatography for measuring tomato:

1) experiment material medicine instrument

1. fresh sophisticated transgenic Fructus Lycopersici esculenti fruit sample of sample and wild-type fruit of the same period (contrast) sample go the seed peeling, carry out freezing treatment rapidly, pulverize, and put in the brown bottle, and sealing is kept in Dark Place in the vacuum drying oven, and measures as early as possible.

2. reagent acetone, dehydrated alcohol, sherwood oil (30 ℃～60 ℃ of boiling ranges), ethyl acetate, trichloromethane etc. are analytical pure; Acetonitrile, methyl alcohol are chromatographically pure; Standard substance: be Sigma company product (purity is 90%～95%, extracts in the tomato); Ultrapure water (greater than 17M Ω).Mobile phase solvent all is the HPLC chromatographically pure.

3. Tianjin, instrument island 10Avp high performance liquid chromatograph; Ultrospec 4300pro ultraviolet spectrophotometer; Rotary Evaporators; The semi-automatic photoelectric analytical balance of TG-328 type.

2) specimen preparation and analysis

The preparation of standard specimen is settled to 50ml after the 1mg Pure Lycopene is dissolved with moving phase, is mixed with the standardized solution of 20 μ g/ml.

Specimen preparation takes by weighing the about 100g of above-mentioned sample, after smashing to pieces with stamp mill, accurately takes by weighing in the 10 μ g what iodine flasks, add 0.1gBHT, with sherwood oil, acetone mixed solution (1: 1, V/V) extract repeatedly, anhydrous sodium sulfate dehydration is settled to 50 mL with mixed solution, and entire operation is answered lucifuge.Get said extracted liquid, evaporated under reduced pressure or nitrogen dry up, and use the 1mL dissolve with methanol, as last machine liquid, inject stratographic analysis behind 0.3 μ m filtering with microporous membrane.

Chromatographic condition chromatographic column C18 post 416mmid * 250mm, 5 μ m, Tianjin, island company.Mobile phase dichloromethane: methyl alcohol: acetonitrile (volume ratio 0125: 1: 1); Flow: 016～018ml/min.Detector UV-VIS detects wavelength 472nm, flow velocity 110ml/min, column temperature room temperature; Sample size: 20 μ l.

(test is handled and data determination is 3 repetitions, uses the mean value ecbatic at last, and data statistics is adopted Microsoft Excel with drawing.)

The measurement result of Lyeopene shows: AtPRP3-AtAMT1; Content of lycopene in the 1 transgenic Fructus Lycopersici esculenti fruit is 1.72 times of wild-type under the isometric growth condition.Measurement result is seen Fig. 6.

(7) chlorophyll measuring of transgenosis mini-tomato

The fresh blade of wild-type tomato (negative contrast) of getting a certain amount of positive transgenic seedling fresh leaf and a small amount of same growth period is material, punching, get some amount punching wafer grinding, 95% ethanol lixiviate, behind the constant volume under 665nm and 649nm wavelength colorimetric (colorimetric with 752 ultraviolet-visible spectrophotometers), basis then:

C (mg.L ^-1)=20.31 OD649+8.04 OD665 (OD is for dividing light value) calculates chlorophyll concentration,

Use formula at last:

Y (mgdm ^-2)=(concentration (mgL-1) * extracting solution total amount (mL))/(leaf area (dm ²) * 100) be converted into the unit leaf area chlorophyll content.

(test is handled and data determination is 3 repetitions, uses the mean value ecbatic at last, and data statistics is adopted MicrosoftExcel with drawing.)

Measurement result shows: the chlorophyll content of transfer-gen plant is 1.25 times of wild-type under the isometric growth condition.Measurement result is seen Fig. 7.

Embodiment 5: the preparation of transgenic arabidopsis

Use AtPRP3-AtAMT1; The step of 1 fusion gene arabidopsis thaliana transformation is as follows:

(1) preparation of vegetable material: in before around transformation experiment Arabidopis thaliana seed kind being buried, when first scape extraction, above the leaf scape is cut first of scape; Scape new after 4-10 days is extracted out, carries out transformation experiment when growing to 2-10 centimetre.

(2) Agrobacterium is cultivated and transforms: the single bacterium colony of picking Agrobacterium from the flat board is inoculated in 3mL and contains in the common YEP liquid nutrient medium of 50mg/L kantlex and 25mg/L Streptomycin sulphate, 28 ℃ of following shaking culture 1 day; Get 1mL bacterium liquid and add in the 20mL fresh liquid YEP substratum, continue shaking culture to OD ₆₀₀Be about 1.8;

Bacterium liquid is changed in the 50mL centrifuge tube, and 3000r/min collected thalline in centrifugal 20 minutes; With liquid 1/2MS substratum (containing 5% sucrose, 0.05%Silwet L-77) the Agrobacterium thalline that suspends again, be diluted to OD ₆₀₀Be about 0.8; Get 200ml bacterium liquid and put into beaker, plant is stood upside down put into bacterium liquid, petal was soaked 1-15 minute; Plant is taken out from bacterium liquid, unnecessary bacterium liquid is wiped, lie against in the disk,, secretly cultivated one day with the preservative film sealing; Take off preservative film the next morning, and plant is uprightly cultivated, and soil waters after all becoming dry, and gathers in the crops seed fully during overstrike until the fruit pod.

(3) PCR of transformed plant detects: the blade of difference clip transfer-gen plant and non-transgenic plant, and extract total DNA and carry out, use primer:

5’-TAGAAGAGTTGTAACGCAAGCTGTG-3’

5’-AAGCTGCATA GAGAAGACAAGGT-3’

Carry out the PCR reaction, carry out agarose gel electrophoresis then, the AtPRP3-AtAMT1 of 1911bp appears in transfer-gen plant; 1 fusion gene band, the fusion gene band does not appear in the non-transgenic plant, proves that the purpose fragment has been incorporated into (see figure 4) in the Plant Genome.

Object tape is a 1911bp. (swimming lane+for making up plasmid PCR over against photograph in Fig. 4; One is the negative contrast of system; The negative contrast of wt wild-type mouseearcress; 1～15 is transgenic arabidopsis vegetable system.)

(4) low nitrogen resisting of the transgenic arabidopsis ability of coercing is measured

The detection of transgenic arabidopsis nitrogen receptivity and growth and development state under low nitrogen stress conditions:

The ability that the transgenic arabidopsis low nitrogen resisting is coerced all far is better than wild-type, the normal nitrogenous source of wild-type seedling is transferred on the 1/10MS nitrogenous source substratum after cultivating a week, lateral root takes place and obvious inhibition takes place all to be subjected to the rosette state young leaves, blade curls dorsad, dead successively in general 30 days, and most of transfer-gen plant can be grown more than 60 days on 1/10MS nitrogenous source substratum.(Fig. 5)

Sequence list

SEQUENCE LISTING

＜110〉Nankai University

＜120〉high affinity ammonium transport factor fuse gene and the application in transgenic plant thereof

<130>20050712

<160>5

<170>PatentIn version 3.1

<210>1

<211>25

<212>DNA

＜213〉Arabidopis thaliana

<220><221>primer_bind

<222>(1)..(25)

<223>

<400>1

tagaagagtt gtaacgcaag ctgtg 25

<210>2

<211>26

<212>DNA

＜213〉Arabidopis thaliana

<220>

<221>primer_bind

<222>(1)..(26)

<223>

<400>2

agatctacca ttgctgagcg cttggc 26

<210>3

<211>26

<212>DNA

＜213〉Arabidopis thaliana

<220>

<221>primer_bind

<222>(1)..(26)

<223>

<400>3

agatctcaac atgtcttgct cggcca 26

<210>4

<211>26

<212>DNA

＜213〉Arabidopis thaliana

<220>

<221>primer_bind

<222>(1)..(26)

<223>

<400>4

ggtcaccaaa tcaaaccgga gtaggt 26

<210>5

<211>3246

<212>DNA

＜213〉Arabidopis thaliana

<220>

<221>gene

<222>(1)..(3246)

<223>

<400>5

tagaagagtt gtaacgcaag ctgtggttgt gggtgacgat cttccttatt tcgatattgg 60

ctatccattc aagtttaggt tgttgctttg ttctactatt aatatagctc ggccttgtga 120

ttatctagtt acgtttattg ctgtgtcttt tgttcatgca tattatttca ttgctttata 180

atgatacata atatcatact ttggtaatta cttagggatg tctttttgaa attattaatc 240

agtcatagaa catatatatt catccaactc taatagatgg tatatgatat gaatgtttac 300

cataccacac aacatacgat ggaaaaatga tgagttgatg acaggtcata tattattatg 360

tatcttggca tgccatatgt tttgggctca caattatatt tcaattctta accaaattgg 420

gcttcttaaa cattcaccag gcccattgac ataatcattt cgattcagtt ttcagaaggg 480

tttttgactc taggatccct ttctctaaag ctcattgcca ttaacgttaa acaaaaacac 540

tagaaatcaa caaaaactat ttacctaaac caaaacaatc acagtttata cagagtacaa 600

aatagtgtca taaaacccaa gtaaattaga gaattgatta gggaatatat atgtaagcag 660

agttactaat aaaacacctt ctgggataaa agagaagaag gtattatcag cgaaactcgc 720

agctttagtt taggcctcac atgaatcaga taagtacttc atcaaactta gctgggatta 780

agaaaggccc ttccatctct ctaatttctc ttctctattt gaaaacaact tttaaactat 840

gaatcaaact gcgagcctta aacattctct aagcatatga gaaaacaaag acgaagaacc 900

ctaattctaa gatccaacat aagaaaataa acaaaaaccg aagagaaaaa tatatcgccg 960

aatatatata acaaatctcc gatcattagt caatcgccgc ttgagcttgg atttgaggca 1020

aagacaaata tataggattt gcttttttca tcattagggt ttccagtttt caatgttttg 1080

ggcttatatt atcgggcctt taggtttaat tggcccatta gttgaaacta tacacggatg 1140

gttttcttac tttctaattg ctacaaacaa cagactaatc tgtttttaca tatttaatgg 1200

ttcagatgtc atcaaagatt atctgtccaa atatttatat tataagatac attttaatat 1260

ttaattatga tttagtagag cggccgatcc gttttgatat gtttagaact aaaaattgaa 1320

atatactaaa atcaaaattg gcaaacattt gaacgagtct catttatatg tcctctgacc 1380

atattattta atgggcctat cacatttccc gagcccagat aacgtctttt cagttctcct 1440

atccttttgg tcagcttgtg catggttgtt acttgttaat gatggccaac atgtttttga 1500

tcgtgcatta cacttaggta tgagaatctc aaacatacat gattatcatc aattactcac 1560

ttttaagata acgtttcttt atttcatatt caaattttta ataccccaaa cattttctta 1620

taaagcccca tttggtgaaa tcgttttcct catcacaatt taaatacaaa gaacacaaca 1680

aacgaactag cttaaggcaa tatacaagcc aagcgctcag caatggtaga tctcaacatg 1740

tcttgctcgg ccaccgatct cgccgtcctg ttgggtccta atgccacggc ggcggccaac 1800

tacatatgtg gccagctagg cgacgtcaac aacaaattca tcgacaccgc tttcgctata 1860

gacaacactt acctcctctt ctccgcctac cttgtcttct ctatgcagct tggcttcgct 1920

atgctctgtg ccggttccgt gagagccaag aatactatga acatcatgct taccaacgtc 1980

cttgacgctg cagccggtgg tctcttctat tatctgtttg gctacgcctt tgcctttgga 2040

tctccgtcca atggtttcat cggtaaacac tactttggtc tcaaagacat ccccacggcc 2100

tctgctgact actccaactt tctctaccaa tgggcctttg caatcgctgc ggctggaatc 2160

acaagtggct cgatcgctga acggacacag ttcgtggctt acctaatcta ttcctctttc 2220

ttaaccgggt ttgtttaccc ggtcgtctct cactggttct ggtcagttga tggatgggcc 2280

agcccgttcc gtaccgatgg agatttgctt ttcagcaccg gagcgataga tttcgctggg 2340

tccggtgttg ttcatatggt cggaggtatc gctggactct ggggtgcgct catcgaaggt 2400

ccacgacttg gccggttcga taacggaggc cgtgccatcg ctcttcgtgg ccactcggcg 2460

tcacttgttg tccttggaac attcctcctc tggtttggat ggtacggatt taaccccggt 2520

tccttcaaca agatcctagt cacgtacgag acaggcacat acaacggcca gtggagcgcg 2580

gtcggacgga cagctgtcac aacaacgtta gctggctgca ccgcggcgct gacaacccta 2640

tttgggaaac gtctactctc gggacattgg aacgtcactg atgtatgcaa cggcctcctc 2700

ggagggtttg cagccataac tggtggctgc tctgtcgttg agccatgggc tgcgatcatc 2760

tgcgggttcg tggcggccct agtcctcctc ggatgcaaca agctcgctga gaagctcaaa 2820

tacgacgacc ctcttgaggc agcacaacta cacggtggtt gcggtgcgtg gggactaata 2880

ttcacggctc tcttcgctca agaaaagtac ttgaaccaga tttacggcaa caaacccgga 2940

aggccacacg gtttgtttat gggcggtgga ggaaaactac ttggagctca gctgattcag 3000

atcattgtga tcacgggttg ggtaagtgcg accatgggga cacttttctt catcctcaag 3060

aaaatgaaat tgttgcggat atcgtccgag gatgagatgg ccggtatgga tatgaccagg 3120

cacggtggtt ttgcttatat gtactttgat gatgatgagt ctcacaaagc cattcagctt 3180

aggagagttg agccacgatc tccttctcct tctggtgcta atactacacc tactccggtt 3240

tgattt 3246

Claims

1. high affinity ammonium transport factor fuse gene AtPRP3-AtAMT1; 1, it is characterized in that this fusion gene is by the promoter sequence of Arabidopis thaliana root system different expression gene AtPRP3 and Arabidopis thaliana high affinity ammonium transport factor gene A tAMT1; 1 encoding sequence constructs and forms, and the fusion gene full sequence is as follows:

tagaagagtt gtaacgcaag ctgtggttgt gggtgacgat cttccttatt tcgatattgg ctatccattc aagtttaggt

tgttgctttg ttctactatt aatatagctc ggccttgtga ttatctagtt acgtttattg ctgtgtcttt tgttcatgca

tattatttca ttgctttata atgatacata atatcatact ttggtaatta cttagggatg tctttttgaa attattaatc

agtcatagaa catatatatt catccaactc taatagatgg tatatgatat gaatgtttac cataccacac aacatacgat

ggaaaaatga tgagttgatg acaggtcata tattattatg tatcttggca tgccatatgt tttgggctca caattatatt

tcaattctta accaaattgg gcttcttaaa cattcaccag gcccattgac ataatcattt cgattcagtt ttcagaaggg

tttttgactc taggatccct ttctctaaag ctcattgcca ttaacgttaa acaaaaacac tagaaatcaa caaaaactat

ttacctaaac caaaacaatc acagtttata cagagtacaa aatagtgtca taaaacccaa gtaaattaga gaattgatta

gggaatatat atgtaagcag agttactaat aaaacacctt ctgggataaa agagaagaag gtattatcag cgaaactcgc

agctttagtt taggcctcac atgaatcaga taagtacttc atcaaactta gctgggatta agaaaggccc ttccatctct

ctaatttctc ttctctattt gaaaacaact tttaaactat gaatcaaact gcgagcctta aacattctct aagcatatga

gaaaacaaag acgaagaacc ctaattctaa gatccaacat aagaaaataa acaaaaaccg aagagaaaaa tatatcgccg

aatatatata acaaatctcc gatcattagt caatcgccgc ttgagcttgg atttgaggca aagacaaata tataggattt

gcttttttca tcattagggt ttccagtttt caatgttttg ggcttatatt atcgggcctt taggtttaat tggcccatta

gttgaaacta tacacggatg gttttcttac tttctaattg ctacaaacaa cagactaatc tgtttttaca tatttaatgg

ttcagatgtc atcaaagatt atctgtccaa atatttatat tataagatac attttaatat ttaattatga tttagtagag

cggccgatcc gttttgatat gtttagaact aaaaattgaa atatactaaa atcaaaattg gcaaacattt gaacgagtct

catttatatg tcctctgacc atattattta atgggcctat cacatttccc gagcccagat aacgtctttt cagttctcct

atccttttgg tcagcttgtg catggttgtt acttgttaat gatggccaac atgtttttga tcgtgcatta cacttaggta

tgagaatctc aaacatacat gattatcatc aattactcac ttttaagata acgtttcttt atttcatatt caaattttta

ataccccaaa cattttctta taaagcccca tttggtgaaa tcgttttcct catcacaatt taaatacaaa gaacacaaca

aacgaactag cttaaggcaa tatacaagcc aagcgctcag caatggtaga tctcaacatg tcttgctcgg ccaccgatct

cgccgtcctg ttgggtccta atgccacggc ggcggccaac tacatatgtg gccagctagg cgacgtcaac aacaaattca

tcgacaccgc tttcgctata gacaacactt acctcctctt ctccgcctac cttgtcttct ctatgcagct tggcttcgct

atgctctgtg ccggttccgt gagagccaag aatactatga acatcatgct taccaacgtc cttgacgctg cagccggtgg

tctcttctat tatctgtttg gctacgcctt tgcctttgga tctccgtcca atggtttcat cggtaaacac tactttggtc

tcaaagacat ccccacggcc tctgctgact actccaactt tctctaccaa tgggcctttg caatcgctgc ggctggaatc

acaagtggct cgatcgctga acggacacag ttcgtggctt acctaatcta ttcctctttc ttaaccgggt ttgtttaccc

ggtcgtctct cactggttct ggtcagttga tggatgggcc agcccgttcc gtaccgatgg agatttgctt ttcagcaccg

gagcgataga tttcgctggg tccggtgttg ttcatatggt cggaggtatc gctggactct ggggtgcgct catcgaaggt

ccacgacttg gccggttcga taacggaggc cgtgccatcg ctcttcgtgg ccactcggcg tcacttgttg tccttggaac

attcctcctc tggtttggat ggtacggatt taaccccggt tccttcaaca agatcctagt cacgtacgag acaggcacat

acaacggcca gtggagcgcg gtcggacgga cagctgtcac aacaacgtta gctggctgca ccgcggcgct gacaacccta

tttgggaaac gtctactctc gggacattgg aacgtcactg atgtatgcaa cggcctcctc ggagggtttg cagccataac

tggtggctgc tctgtcgttg agccatgggc tgcgatcatc tgcgggttcg tggcggccct agtcctcctc ggatgcaaca

agctcgctga gaagctcaaa tacgacgacc ctcttgaggc agcacaacta cacggtggtt gcggtgcgtg gggactaata

ttcacggctc tcttcgctca agaaaagtac ttgaaccaga tttacggcaa caaacccgga aggccacacg gtttgtttat

gggcggtgga ggaaaactac ttggagctca gctgattcag atcattgtga tcacgggttg ggtaagtgcg accatgggga

cacttttctt catcctcaag aaaatgaaat tgttgcggat atcgtccgag gatgagatgg ccggtatgga tatgaccagg

cacggtggtt ttgcttatat gtactttgat gatgatgagt ctcacaaagc cattcagctt aggagagttg agccacgatc

tccttctcct tctggtgcta atactacacc tactccggtt tgattt。

2. the described high affinity ammonium transport factor fuse gene AtPRP3-AtAMT1 of claim 1; 1 preparation method is characterized in that, the pcr amplification primer of the promotor of designed Arabidopis thaliana root system different expression gene AtPRP3 is as follows, and wherein downstream primer is introduced the BglII point of contact:

Upstream primer: 5 '-TAGAAGAGTTGTAACGCAAGCTGTG-3 '

Downstream primer: 5 '- AGATCTACCATTGCTGAGCGCTTGGC-3 '.

3. the described high affinity ammonium transport factor fuse gene AtPRP3-AtAMT1 of claim 1; 1 preparation method is characterized in that, designed Arabidopis thaliana high affinity ammonium transport factor AtAMT1; The pcr amplification primer of 1 gene is as follows, and upstream primer is introduced the BglII point of contact, and downstream primer is introduced the BstEII point of contact:

Upstream primer: 5 '- AGATCTCAACATGTCTTGCTCGGCCA-3 '

Downstream primer: 5 ' GGTCACCAAATCAAACCGGAGTAGGT-3 '.

4. the described high affinity ammonium transport factor fuse gene AtPRP3-AtAMT1 of claim 1; 1 preparation method is characterized in that, AtPRP3-AtAMT1; The structure of 1 fusion gene, the concrete operations step is as follows:

(4) with EcoRI/BstEII double digestion pCAMBIA1301 plasmid, reclaim big carrier segments;

(5) mix above-mentioned AtPRP3 gene promoter fragment, AtAMT1 gene fragment fragment and pCAMBIA1301 carrier segments, under ligase enzyme catalysis, carry out ligation, finish the AtPRP3-AtAMT1 on the pCAMBIA1301 carrier; The structure of 1 fusion gene.

5. the described high affinity ammonium transport factor fuse gene AtPRP3-AtAMT1 of claim 1; 1 application is characterized in that, utilizes AtPRP3-AtAMT1; 1 fusion gene transforms mini-tomato and Arabidopis thaliana, obtains transgenosis mini-tomato and Arabidopis thaliana that low nitrogen resisting is coerced ability.

6. the described high affinity ammonium transport factor fuse gene AtPRP3-AtAMT1 of claim 1; 1 application is characterized in that, uses AtPRP3-AtAMT1; 1 fusion gene transforms any plant and obtains to have the transgenosis new variety that low nitrogen resisting is coerced ability.