CN101851634A

CN101851634A - Improving drought and cold tolerance of plants using the arginine decarboxylase gene PtADC

Info

Publication number: CN101851634A
Application number: CN 201010120741
Authority: CN
Inventors: 刘继红; 王静; 孙培培; 张庆福
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2010-03-10
Filing date: 2010-03-10
Publication date: 2010-10-06
Anticipated expiration: 2030-03-10
Also published as: CN101851634B

Abstract

The invention belongs to the plant genetic engineering field. The invention in particular relates to the citrus fruit tree genetic engineering technical field. Drought resistant cold resistant argininedecarboxylase gene PtADC is obtained by separation and cloning from citrus fruit tree poncirus trifoliata, the code sequence thereof is shown as 85-2340 bits of SEQ ID NO: 1. The gene is introduced into a model plant Arabidopsis or tobacco to carry out biological function verification and genetic transformation, thus obtaining a transgenic plant. The transgenic plant has obvious drought resistant cold resistant capability. The gene of the invention is hopeful to be further applied in citrus fruit trees.

Description

Utilize trifoliate orange arginine decarboxylase gene PtADC to improve the plant drought tolerance to cold

Technical field

The invention belongs to plant genetic engineering field.Be specifically related to from trifoliate orange (Poncirus trifoliata) to separate, the clone obtains trifoliate orange arginine decarboxylase gene PtADC, then this gene is imported in the model plant (Arabidopis thaliana) and carry out biological function verification, the drought resistant cold resistant capability of the transfer-gen plant of acquisition is significantly improved.

Background technology

Abiotic stress (for example arid, low temperature, saline and alkaline etc.) influences growth and development of plant, regional distribution and throughput, is the severe challenge that agriculture production faces, and is the bottleneck of many regional agricultural developments.With the arid is example, and according to statistics, arid area, the world total area accounts for more than 40% of land area, and the whole world is annual because water stress causes the summation of the loss loss that other all environmental factor caused no better than of crop production.Therefore cultivate the key that degeneration-resistant new crop varieties is the resisting abiotic adverse circumstance.

Genetic transformation is to utilize means such as biology, physics or chemistry that foreign gene is imported recipient cell to obtain the technology of transgenic animal, plant and microorganism, and transgenosis is expressed the render transgenic biology increases a goal gene control simultaneously at the original good character of reservation proterties in recipient cell.Since nineteen eighty-three, the first transgenic plant were come out, plant transgene research had obtained considerable progress.Some genetically modified crops carry out the commercialization plantation, and area constantly increases, and by 2008, the whole world has 25 countries business-like transgenic plant, and cultivated area is totally 1.25 hundred million ha.The key of cultivating transgenic line is that the clone is important and have a gene of corresponding function.

In the long-term evolution process, higher plant has formed the corresponding adverse circumstance acknowledgement mechanism of a cover, will change on physiology, biochemical level during environment stress, and what is more important is replied on transcriptional level, coerces the injury that causes to alleviate.According to the effect of gene product, plant abiotic stress stress response gene can be divided into two classes, and the coded product of a genoid is the functional protein and the osmoregulation factor; The coded product of another kind of gene is a transcription factor.The former mainly contains lea protein gene, antioxidase and polyphenoils genes involved, aquaporin, proline(Pro) synthesis associated protein, the key enzyme that synthesizes the osmoregulation material and molecular chaperones etc.

(polyamine is the biologically active substance that extensively is present in prokaryotic organism and the eukaryote PA) to polyamines, is the aliphatic nitrogenous alkali cpd of a class lower molecular weight.In the higher plant common polyamines have putrescine (putrescine, Put), spermidine (spermidine, Spd), spermine (spermine, Spm) etc.Under the physiological pH condition, polyamines has the characteristic of polycation, by covalent linkage form and groups interactions such as nucleic acid, protein and phosphatide such as ionic linkage, hydrogen bond and hydrophobic interactions, regulates its physiological function; In addition, have the aliphatics characteristic on the polyamines chemical structure, in hydrophobic environment, play the stabilizing membrane system.Therefore, polyamines is a kind of important chemical substance in the plant environment stress.

The biosynthesizing of plant polyamines has two approach, i.e. ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) approach.Be subjected to abiotic stress plant and coerce down, the polyamines accumulation increases.Studies show that in a large number the polyamines accumulation is mainly derived from the up-regulated expression and the active special enhancing of ADC of ADC gene under the plant environment stress.In addition, the external source polyamine treatment can alleviate abiotic stress and coerce the injury that causes, and improves the anti-adversity ability of tissue or cell.Above-mentioned studies show that, polyamines are played important effect in the resistance of plant.Therefore, allowing plant accumulate more polyamines may be a novel method that improves its resistance, and the synthetic key gene of the polyamines that overexpression plays an important role in adverse circumstance is replied is a valid approach.

Because the gene that the ADC gene plays an important role in adverse circumstance is replied, the cDNA of some plant ADC is cloned and is identified, as oat (Bell etc., 1990), soybean (Nam etc., 1997), pea (Perez-Amador etc., 1995), tomato (Rastogi etc., 1993), carnation (Chang etc., 2000), Arabidopis thaliana (Waston etc., 1996; Urano etc., 2003), grape (Primikirios etc., 1999), paddy rice (Chattopadhyay etc., 1997; ), peach (Liu etc., 2009).On the basis of the cDNA that clones ADC, carried out gene transformation research, the result shows that behind the commentaries on classics ADC gene, the polyamine level of transformant rises, and under some environment stress, transformant shows stronger resistance.Masgrau etc. (1997) change the cDNA of oat ADC in the tobacco over to, and transformant ADC increased activity 12-20 times, the drought resistance of plant is enhanced.Equally, Burtin etc. (1997) change the cDNA of oat ADC in the tobacco over to, and transformant ADC increased activity 10-20 doubly.The cDNA of oat ADC is changed in the paddy rice, and the putrescine amount rising 2-10 of transfer-gen plant is (Capell etc., 1998 doubly; Bassie etc., 2000; Noury etc., 2000).Roy etc. (2001) change the cDNA of oat ADC in the paddy rice over to, and the putrescine of transfer-gen plant and ADC activity are compared photograph high 200% and 300-400% respectively, and s-generation plant is contrast rising significantly of biomass under salt stress.

Though there is the more ADC gene that studies show that in some plants, to clone, in trifoliate orange, do not see the report of this gene clone and functional verification as yet, more do not see and use the bibliographical information that trifoliate orange PtADC gene-transformed plant improves the resisting abiotic adverse circumstance.

Summary of the invention

The objective of the invention is to clone trifoliate orange (Poncirus trifoliata) arginine decarboxylase gene PtADC, obtain drought-resistant and low temperature resistant transfer-gen plant of coercing by genetic transformation, for the breeding of citrus resisting abiotic adverse circumstance molecular designing provides genetic resources,, water-saving agriculture green for implementing from now on provides thinking, helps reducing agriculture production cost.

The present invention is achieved in that

The present invention mainly is that the clone obtains arginine decarboxylase gene PtADC from trifoliate orange (Poncirus trifoliata), its nucleotide sequence and encoding sequence (aminoacid sequence) shown in sequence table SEQ ID:1 (this gene pairs answer protein sequence shown in sequence table SEQ ID NO:3-4); With this gene overexpression in the model plant Arabidopis thaliana, obtain transfer-gen plant by genetic transformation then, functional verification shows that this transfer-gen plant has tangible drought resisting and tolerance to cold.

In the embodiments of the invention part, we have set forth separation, functional verification and the application of trifoliate orange arginine decarboxylase gene PtADC.

Description of drawings

Sequence table SEQ ID NO:1 is the nucleotide sequence (being the cDNA sequence, total length 2493bp) of the trifoliate orange arginine decarboxylase gene PtADC that clones of the present invention.

The 85-2340 position of sequence table SEQ ID NO:1 is the encoding sequence (aminoacid sequence) of trifoliate orange arginine decarboxylase gene PtADC.

Sequence table SEQ ID NO:2 is the protein sequence of trifoliate orange arginine decarboxylase gene PtADC correspondence.

Sequence table SEQ ID NO:3-4 is the primer sequence of amplification trifoliate orange arginine decarboxylase gene PtADC cDNA sequence.

Fig. 1 is a techniqueflow chart of the present invention.

Fig. 2 is that the plant eukaryotic expression vector pMV-PtADC of one of them embodiment of the present invention makes up flow process.

Fig. 3 is a PtADC gene PCR amplification of the present invention.

Fig. 4 is that the enzyme of the expression vector pMV-PtADC that makes up of the present invention is cut detected result (1:pMV-PtADC clone enzyme is cut among the figure; M:1kb DNAmarker).

Fig. 5 is that the present invention transforms and obtains T ₀For transgenic arabidopsis strain system, the T of results ₀For seed through MS+ kantlex (Km) plate screen resistance T ₁For transgenosis heterozygosis strain system (green positive transformed plant among the figure).

Fig. 6 is part T among one of them embodiment of the present invention ₁For the transgenic arabidopsis strain is that the heterozygosis strain is PCR qualification result (M:DNA molecular weight marker 100bp DNA marker among the figure; A:NPTII gene specific pcr amplification, B:35S+ADCR gene specific pcr amplification; P: plasmid; PK: not transgenosis contrast; 1-8,10-14,19,21: transgenic line);

Fig. 7 is that single copy inserts T among one of them embodiment of the present invention ₃Bind fruit for the transgenic arabidopsis strain of isozygotying.

The single copy of Fig. 8 the present invention part inserts T ₃Changeing the strain of PtADC gene for isozygotying is that expression analysis is (among the figure: adc1-1: negative control; 21 (3) a, 5 (2) a5 (2) a, 19 (4) d, 14 (6) f: change PtADC gene strain system).

The strain of Fig. 9 embodiment of the invention transfer PtADC gene is adc1-1-OE (5 (2) a strains system among Fig. 8) and contrast polyamine content analysis (adc1-1: negative control; Adc1-1-OE: transgenic line; Adc1-1 and the adc1-1-OE 6weeks that in soil, grows, polyamine content is measured triplicate, and numerical value is mean value ± standard error." * " expression is through student t test, and adc1-1-OE polyamine content significantly (0.01＜P＜0.05) is higher than adc1-1).

Figure 10 is that the strain of embodiment transfer PtADC gene is adc1-1-OE (5 (2) a strains system is OE among the figure among Fig. 8) and non-transgenic plant (adc1-1) outside drawing of anti-penetrating power the (A sprouting situation behind 14d on the MS substratum when seed germination among the present invention; B is containing the sprouting situation behind the 14d on the MS substratum of 400mM N.F,USP MANNITOL).

Figure 11 be among the present invention embodiment to change the strain of PtADC gene be adc1-1-OE (5 (2) a strains system among Fig. 8, be OE among the figure) and non-transgenic plant (adc1-1) 2d, 4d, 6d, 8d, 10d, 12d, the anti-penetrating power statistics of 14d (A, MS substratum when seed germination; B, MS+400mM N.F,USP MANNITOL substratum).

Figure 12 be among the present invention embodiment change the strain of PtADC gene be percentage of water loss behind the room temperature dehydration behind adc1-1-OE (among Fig. 85 (2) a strains system) and the non-transgenic plant strain growth 10d (A, n=30) and relative conductivity (B).

Figure 13 be among the present invention embodiment change the strain of PtADC gene be the performance in soil, controlling the water arid behind the 3w and handle of adc1-1-OE (among Fig. 85 (2) a strains system) and non-transgenic plant strain growth (before A:adc1-1 and the OE control water, the phenotype of control water 15d, 18d; B: adc1-1 behind the control water 18d and the relative conductivity of OE; C: adc1-1 behind the control water 18d and the survival rate of OE).

Figure 14 be among the present invention embodiment to change the strain of PtADC gene be adc1-1-OE (among Fig. 85 (2) a strains system, the OE among the figure) and non-transgenic plant (adc1-1 among the figure) generative phase control after the water arid is handled phenotype relatively.

Figure 15 be among the present invention embodiment to change the strain of PtADC gene be that adc1-1-OE (among Fig. 85 (2) a strains system, the OE among the figure) and non-transgenic plant (adc1-1 among the figure) grew in the soil behind the 3w 0 ℃ of subzero treatment 1 day and rewarming phenotype (A), relative conductivity (B), chlorophyll content (C) and survival rate (D) after 15 days.

Embodiment

Below in conjunction with specific embodiment the present invention is described in detail.According to following description and these embodiment, those skilled in the art can determine essential characteristic of the present invention, and under the situation that does not depart from spirit and scope of the invention, can make various changes and modification, so that its suitable various uses and condition to the present invention.

The separating clone of embodiment 1:PtADC gene

With arginine decarboxylase is keyword search citrus est database (www.harvest.ucr.edu, HarvEST:Citrus ver.0.51), obtain 26 close sequences, above-mentioned 26 close sequences of primary election are formed a complete sequence with CAP3 (common software), with this sequence is template Primer Premier 5.0 software design primers, with RT-PCR method commonly used (with reference to J. Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi work, Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions) expand the total length that sequence.Coerce down Fructus Aurantii blade extracting RNA and reverse transcription under 4 ℃ of low temperature, the first chain cDNA of gained is used for the extension increasing sequence total length.The RNA method for extracting with reference to the specification sheets of Trizol test kit (available from Invitrogen company, operate according to the process specifications that this test kit provides), with DNaseI (the Amplification Grade of the extractive total RNA sample of 3 μ g through 1U, available from Invitrogen company) after room temperature (25 ℃) handles 15min, add 1 μ L EDTA (25mM), in 65 ℃ of incubation 10min.The synthetic MBI counter-rotating test kit specification sheets of pressing of the first chain cDNA is operated (test kit article No.: K1621 is available from Fermentas company).The forward primer of amplification PtADC is 5 '-CCCCCTCGTTTTTTCTTTTTCTT-3 '; Reverse primer is 5 '-TGTTCAACTGCTTCCATCTTTTG-3 '.Comprise 100ng cDNA in the reaction system of 20 μ L, 1 * damping fluid, 2mM MgCl ₂, 0.2mM dNTP, (Taq and Pfu, Fermentas Lithuania) add 0.4 μ M primer to the 0.5U polysaccharase.PCR is reflected at ABI 9700 (Applied Biosystem) and upward finishes by following setting: 94 ℃, and 3min, 94 ℃ of sex change 30s, 51 ℃ of annealing 30s, 72 ℃ are extended 180s, 30 circulations; 72 ℃ were extended 7min after circulation was finished.The single PCR band product (seeing accompanying drawing 3) that produces behind 1.5% agarose gel electrophoresis, is used E.Z.N.

Gel reclaims test kit (available from Omega company, the U.S.) and reclaims special band, and operation steps is with reference to the working instructions of this test kit.The dna solution and the commercial carrier that reclaim purifying are that pGEM-T easy carrier (available from Promega company) carries out ligation, and operation is undertaken by the specification sheets of Promega company, and the mole ratio of inserting fragment and carrier in the ligation system is 3-10: 1.The ligation cumulative volume is 5 μ L, comprising 2 * damping fluid (this test kit carries) of 2.5 μ L, and the PCR product of 1.5 μ L purifying, 0.5 μ LT-easy carrier (this test kit carries), 0.5 μ L T4 ligase enzyme (this test kit carries).16 ℃ of connections of spending the night.Get 5 μ l and connect product, adopt the thermal shock method (with reference to J. Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi work, Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions) transformed into escherichia coli DH5 α, screening positive clone in the LB solid plate that contains 50mg/L ammonia benzyl mycin, 5 cloning and sequencings of picking (examining order is finished by Shanghai associating genome company), sequencing result shows that this gene order total length is 2493bp, by order-checking, comparison is defined as the PtADC gene, sign indicating number (ORF) is read in the opening that it comprises 2256bp, 751 amino acid (referring to sequence table SEQ ID NO:1) of encoding, iso-electric point is 5.14, the molecular weight of prediction is 80.53kD.Amino acid and grape (X96791), tobacco (AF127240, AF127241), apple (AB181854) amino acids coding of the PtADC genes encoding that PtADC derives have 71%, 70%, 69% homology respectively.The multisequencing comparison result shows, the present invention clone's PtADC gene comprises two conservative amino acid structure territories: ADC family 2 pyridoxal phosphate binding sites and ADC family 2 marks, 2 sequences.IPSORT and SignalP analysis revealed amino acids coding PtADC N-end are comprising a chloroplast(id) positioning sequence.

Embodiment 2, plant conversion carrier make up

According to pMV carrier (the plant binary conversion carrier pBI121 of excision gus gene, this carrier is so kind as to give by professor Ye Zhibiao of gardening forestry institute of Hua Zhong Agriculture University, see shown in Figure 2) multiple clone site and the coding region sequence of PtADC gene, according to the principle of general design primer with Primer Premier5.0 software design go out the to increase forward and the reverse primer of the whole coding region of PtADC gene.Used forward and reverse primer 5 ' end is added with Xho I and Kpn I restriction enzyme site (the restriction enzyme site sequence underlines expression, and is as follows) respectively, and adds 3 protection bases respectively in the restriction enzyme site recognition sequence outside, is beneficial to carrying out smoothly of endonuclease reaction.The right dna sequence dna of described primer is as follows:

Forward primer: 5 '-CCG CTCGAGCCCCCTCGTTTTTTCTTTTTCTT-3 '

Reverse primer: 5 '-CG GGGTACCTGTTCAACTGCTTCCATCTTTTG-3 '

Clone's with the PtADC gene is that template is carried out pcr amplification.The annealing temperature of pcr amplification is 61 ℃.PCR reaction system and amplification program are with embodiment 1.The double digestion system: the reaction cumulative volume is 20 μ L, wherein contains the purified product 10 μ L of PCR, 10 * damping fluid (MBI, 100mmol/L Tris-HCl, pH8.3,500mmol/L KCl; 0.1% gelatin (gelation) and 15mmol/L MgCl ₂) 2 μ L, each 1 μ L of Kpn I and XhoI, distilled water 6 μ L).Cutting the back purifying that spends the night at 37 ℃ of enzymes reclaims.The double digestion system of pMV carrier: the reaction cumulative volume is 20 μ L, wherein contains through plasmid and extracts the pMV carrier DNA 8 μ L that obtain, 10 * damping fluid (MBI), 2 μ L, each 1 μ L of Kpn I and Xho I, distilled water 8 μ L.37 ℃ of enzymes are cut the back purifying that spends the night and are reclaimed.The mole ratio of inserting fragment and carrier in the ligation system is 3: 1, and the reaction cumulative volume is 10 μ L, wherein contains 10 * damping fluid (MBI), 1 μ L, T4DNA ligase enzyme 1 μ L, the double digestion of PtADC gene reclaims product 4 μ L, and the double digestion of pMV carrier reclaims product 2 μ L, distilled water 2 μ L.At 16 ℃ of reaction 14-16h.Connect product transformed into escherichia coli bacterial strain DH5 α, screening positive clone in containing the LB solid plate of 50mg/L kantlex, the extracting plasmid carries out that enzyme is cut and PCR identifies, order-checking determines not have the reading frame sudden change, acquisition contains inserts the segmental recombinant clone of purpose, and we are with this carrier called after pMV-PtADC.The application freeze-thaw method (with reference to J. Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi work, and Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions) described carrier pMV-PtADC is imported among the Agrobacterium EHA105.The structure flow process of plant eukaryotic expression vector pMV-PtADC is seen shown in Figure 2.PMV-PtADC after structure is finished through the KpnI/XhoI double digestion, obtains to be about 2 dna fragmentation (see figure 4)s of 2300bp and nearly 14000bp.

Embodiment 3, plant genetic transform

Use freeze-thaw method (with reference to J. Sa nurse Brooker, Deng work, Huang Peitang etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions) the pMV-PtADC carrier is imported among the Agrobacterium EHA105, stable and the Arabidopis thaliana T-DNA that isozygoty of transformation traits inserts (Japanese RIKEN among the AtADC1 mutant adc1-1, Shinozaki seminar is so kind as to give), positive plant obtains the transgenic line that isozygotys by the selfing three generations behind the genetic transformation.

Particularly, present embodiment utilizes Agrobacterium tumefaciens mediated Arabidopis thaliana genetic transformation step as follows:

1, the cultivation of Arabidopis thaliana

Get 4 ℃ of Arabidopis thaliana seeds behind the vernalization 2d spill and be sowed in the fully wetting matrix (according to the weight part meter: nutrition soil: vermiculite=1: 1), cultivate in the greenhouse (22 ℃, 16h illumination; 16 ℃, the 8h dark).Grow to bolting and bloom, 1cm cuts off the inflorescence of bolting for the first time under the tongue, grows to be used for transforming after more spending more preface.

2, the preparation of Agrobacterium bacterium liquid

The Agrobacterium of getting correct, the streak culture mono-clonal inoculation of detection is inoculated in 10 milliliters of LB liquid nutrient mediums and (contains kantlex (Km) 50mg/l, Rifampin (Rif) 50mg/l, pH7.0) in, 28 ℃, 250rpm are cultivated 30h, get bacterium liquid 1.5mL and be by volume and be transferred to 150mL LB (Km50mg/L at 1: 100, Rif:50mg/L) in, 28 ℃, 250rpm overnight incubation are to OD ₆₀₀=0.8; 4 ℃, in the centrifugal 10min of 3000g, collected thalline is resuspended in 5% sucrose solution, adds 0.02%Silwet L-77 (a kind of tensio-active agent is available from U.S. GE company) before transforming.

3, transform

Fresh Arabidopis thaliana plant is inverted, makes its bud and immerse penetrating fluid (5% sucrose solution: 5g sucrose+100mLddH down ₂O) in, keep 3s and stirring gently at least, the globule of penetrating fluid is overlying on the described inflorescence.Arabidopis thaliana plant after transforming is placed vertically, build preservative film, the insulation of watering (22 ℃) is in low light intensity (50 μ mol m ^-2s ^-1) descend growth 24h to be placed on normal illumination (100 μ mol m ^-2s ^-1) grow under the condition, the collection seed bears pods up to blooming.

4, positive transgenic arabidopsis is tentatively definite

To change 4 ℃ of vernalization 2d of adc1-1 overexpression adc1-1-OE seed, 0.1g in the centrifuge tube of 1.5mL, add 1mL 1.5% clorox surface sterilization 8min, fully vibration, wash 5 times with the sterilization distilled water after discarding clorox, be sowed at 0.1% agar on the culture dish of the MS substratum with 50mg/l kantlex (Km), the resistance seedling is the energy normal growth on resistant panel, be green, non-resistance seedling then yellow is die.Treat to move in the soil when green seedling grows to four true leaves, cover the preservative film water conservation, striping behind the 4d during to 8 true leaves of seedling, gets final product the clip blade, extracts its blade genomic dna, carries out the Molecular Identification of positive plant.Agrobacterium is infected the seed that plant tied the present age and is designated as T ₀For seed, the plant that this seed grows through resistance screening is T ₁For plant.Use T ₁Blade be PCR and detect, positive plant is carried out mark and is gathered in the crops seed and is designated as T ₁For seed, the plant that the MS substratum screening of this seed through containing 50mg/l kantlex (Km) grows is T ₂For plant, the seed of being tied is T ₂For seed.Use a part of T ₂The planting seed in generation is on resistant panel, if T ₃For plant all is green resistance seedling, proves that then this individual plant is the pure lines transfer-gen plant.

The molecule of embodiment 4, transformed plant and physiology are identified

1, Arabidopsis leaf total DNA extraction

Get an amount of Arabidopsis leaf and put into the 1.5mL centrifuge tube, add liquid nitrogen, after fully grinding; DNA extraction CTAB damping fluid (the 100mM Tris-HCl that adds 65 ℃ of preheatings of 700 μ L, pH 8.0), 1.5M NaCl, 50mM EDTA, pH 8.0) solution adds 1% polyvinylpyrrolidone, 2% cetyltriethylammonium bromide, 65 ℃ of water-baths fully dissolve standby, after preceding 65 ℃ of water-bath preheatings, add the 1-4% mercaptoethanol, mixing), 65 ℃ of temperature are bathed 60-90min, take out every 15min and put upside down mixing up and down gently; 10000g, centrifugal 10min; Get supernatant, add 600 μ L chloroforms, put upside down mixing and leave standstill 3min; 10000g, centrifugal 15min; Get supernatant 450 μ L, add 900 μ L precooling dehydrated alcohols, 420 μ L5M NaCl, behind the mixing, freezing 30min, 10000g, centrifugal 10min; After abandoning supernatant, with the ethanol of 1mL 75% concentration, wash 3 times after, add an amount of ddH ₂The O dissolving.

2, positive transgenic arabidopsis PCR detects

Carry out pcr amplification with NPTII primer and gene inner primer (35S+PtADC), described primer sequence and response procedures see Table 1-1,1-2,1-3.

Table 1-1 primer sequence information

Table 1-2PCR response procedures

Table 1-3PCR reaction system

10 * PCR damping fluid composition: 100mmol/L Tris-HCl, pH8.3,500mmol/L KCl; 0.1%gelation (gelatin) and 15mmol/L MgCl ₂

3, the overexpression analysis of transgenic arabidopsis

Extract and receive adc1-1 and the T that plants, plants simultaneously simultaneously ₃Generation single copy overexpression strain is blade RNA, removes residual DNA, and reverse transcription method is described with embodiment 1, with PtADC gene specific primer (forward primer: 5 '-GGTCGAAACCGGAGCTGTTG-3 '; Reverse primer: GSP2,5 '-GCCCCCGATGTCAATCACTT-3 ') carry out RT-PCR, response procedures is 94 ℃, 3min, 94 ℃ of sex change 30s, 61 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; 72 ℃ were extended 5min after circulation was finished.Be internal reference (forward primer: R 5 '-CGTGGATCACAGCAATACAGAGCC-3 ' with Tubulin; Reverse primer: 5 '-CCTCCTGCACTTCCACTTCGTCTTC-3 '.

Utilize flower-dipping method (Clough and Bent, Floral dip:a simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana.Plant J, 1998,16:735-743) transform T ₀For transgenic arabidopsis strain system, the T of results ₀For seed through the screening of MS minimum medium+50mg/l kantlex (Km) plate and PCR identify resistance T ₁For transgenosis heterozygosis strain system (seeing accompanying drawing 5, Fig. 6).Individual plant is received T ₁In generation, plant, and through MS minimum medium+50mg/l kantlex (Km) plate screening, gets T again ₂For strain system, if Km resistance (green seedling): non-Km resistance (albefaction seedling) is that 3: 1 strain is that single copy inserts strain system, with the T of tool Km resistance ₂Receive kind for individual plant, get single-strain seed that single copy inserts strain system through MS minimum medium+50mg/l kantlex (Km) plate screening, that be entirely Km resistance (green seedling) is T ₃For homozygous lines (seeing accompanying drawing 7).Choose T ₃Carry out the overexpression analysis for homozygous lines, find in the strain of choosing to be that the ADC expression amount is the highest among 5 (2) a, called after adc1-1-OE (seeing accompanying drawing 8) chooses this strain system and does further research.

4, the polyamine content analysis of transgenic arabidopsis

Adc1-1 with 6 ages in week, OE puts in order strain material liquid nitrogen grinding, get the sample powder about 0.1g, the perchloric acid that adds 1mL 5%, ice bath 30min behind the mixing, 4 ℃ of centrifugal 15min of 10000g, get supernatant to another centrifuge tube, precipitation adds the perchloric acid mixing of 1mL 5% again, ice bath 30min collects supernatant liquor twice after the centrifugal 15min of 10000g gets supernatant, behind the mixing, get 200 μ L, add 200 μ L saturated sodium carbonates, the dansyl chloride (10mg/mL) of 5 μ L hexane diamines (100 μ M) and 400 μ L shakes up back 60 ℃ of dark reaction 1h, the proline(Pro) that adds 100 μ L100mg/mL, after 60 ℃ of dark reaction 30min, add the toluene of 400 μ L, the centrifugal 10min of 10000g room temperature behind the mixing.Inhale 400 μ L supernatants, dry in the vacuum-drying instrument, add the 1mL dissolve with methanol, get 20 μ L sample on the high performance liquid chromatograph behind the organic membrane filtration of 0.22 μ m.The fluoroscopic examination wavelength: excitation wavelength is 365nm, and emission wavelength is 510nm.Adopt gradient elution, elution program sees Table 1-2.Flow velocity 1mL/min, 30 ℃ of column temperatures.Polyamine content adopts the relative calibration method in the tissue, organizes the ratio=place peak area ratio of amount with the internal reference hexane diamines amount of polyamines, and unit is nmol g ^-1FW.The result shows that overexpression plant putrescine content has improved 2 times, detects through t, and there were significant differences with contrast for transfer-gen plant, and spermidine, spermine content constant substantially (seeing accompanying drawing 9).

The evaluation of resistance of embodiment 5, transfer-gen plant

Be sheerly seed treatment with step 4 among the embodiment 3 with crowd Arabidopis thaliana adc1-1 that receives and OE, be sowed on MS minimum medium (pH5.8), the MS minimum medium+400mM N.F,USP MANNITOL infiltration substratum (pH5.8), each is to get 30 seeds for every ware, handle and repeat 3 times, identical experimental result repeats 3 times at least.On common MS substratum, the seed germination situation does not have difference between adc1-1 and the OE, on the infiltration substratum, these seed germinations all are suppressed, but OE is subjected to the inhibition program to be lower than adc1-1, shows that OE seed osmotic adjustment ability is strong, can restrain oneself osmotic stress (accompanying drawing 10,11) preferably.

With crowd Arabidopis thaliana adc1-1 that receives and commentaries on classics adc1-1 overexpression pure lines adc1-1-OE seed, be sowed at after the sterilising treatment (pH5.8) on the MS minimum medium, getting each transgenic arabidopsis strain is that 30 strains of the big seedling of 10d place the 50min that dewaters naturally on the culture dish, measured fresh weight in per 10 minutes, calculate percentage of water loss, and measure the specific conductivity of last time point (50 minutes), be contrast with (when just having opened the culture dish lid) of the material before dewatering.The result shows that the OE dehydration is slow than adc1-1, and the OE specific conductivity is than adc1-1 low (seeing accompanying drawing 12) behind the dehydration 50min.

Big adc1-1, adc1-1-OE controlled the test of water drought stress with 3 ages in week, is to cut off the water under the 50% normal growth condition to cultivate 18d in relative humidity.The result shows that behind the 15d that stops to water, the OE blade is full, and glossiness is strong, adc1-1 has the wilting phenomenon to occur, and behind the control water 18d, OE lotus leaf seat lower blade is partly wilted, and adc1-1 begins death at this moment, and the OE specific conductivity is low than adc1-1, and survival rate is higher than adc1-1 (seeing accompanying drawing 13).Control the water drought stress during bolting, OE survives situation and obviously is better than adc1-1 (seeing accompanying drawing 14).

With 16h light (50 μ mol m ^-2s ^-1Each 17 of the potted plant transgenic arabidopsis seedlings in)/8h half-light cycle, 23 ± 1 ℃ of 4 weeks of growing down are in 16h light (50 μ mol m ^-2s ^-1)/8h dark situation is handled 24h for following 0 ℃, and the result shows that the OE blade is full after the subzero treatment, and vitality is strong, the adc1-1 yellow leaf, and a little less than the vitality, behind the rewarming 15d, OE is continued growth still, has bolting to take place, and adc1-1 is dead.After low temperature (0 ℃) was handled, the specific conductivity of OE was lower than adc1-1, and chlorophyll content is higher than adc1-1, and survival rate is higher than adc1-1, shows that the OE low temperature tolerance ability is higher than adc1-1 (seeing accompanying drawing 15).

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉utilize trifoliate orange arginine decarboxylase gene PtADC to improve the plant drought tolerance to cold

<130>

<141>2010-03-03

<160>4

<170>PatentIn?version?3.1

<210>1

<211>2493

<212>DNA

＜213〉trifoliate orange (Poncirus trifoliata)

<220>

<221>gene

<222>(1)..(2493)

<223>

<220>

<221>CDS

<222>(85)..(2340)

<223>

<400>1

ccccctcgtt?ttttcttttt?cttttttttc?ttctttaatt?ctttaaaaaa?aggccgcact 60

tttactcgtc?tccgcggaaa?gagg?atg?ccg?gcc?ctc?ggg?tgt?tgc?gta?gac 111

Met?Pro?Ala?Leu?Gly?Cys?Cys?Val?Asp

1 5

gct?gcc?gta?gcg?cct?cct?gcc?tac?gcc?aac?tcc?cca?ctc?ggc?tcc?ctc 159

Ala?Ala?Val?Ala?Pro?Pro?Ala?Tyr?Ala?Asn?Ser?Pro?Leu?Gly?Ser?Leu

10 15 20 25

ccc?gcg?ccg?ccg?ccg?ctg?ccg?ctt?tct?ttt?aac?tcc?ggc?aca?tca?cca 207

Pro?Ala?Pro?Pro?Pro?Leu?Pro?Leu?Ser?Phe?Asn?Ser?Gly?Thr?Ser?Pro

30 35 40

ccc?aca?cct?atg?tcc?cct?acc?tcc?gcc?tcg?gct?ggt?tct?gtt?gcc?gcc 255

Pro?Thr?Pro?Met?Ser?Pro?Thr?Ser?Ala?Ser?Ala?Gly?Ser?Val?Ala?Ala

45 50 55

gac?gtg?gac?gct?tca?cat?tgg?tcg?ccg?tct?cac?tcg?gca?tcg?ttg?tat 303

Asp?Val?Asp?Ala?Ser?His?Trp?Ser?Pro?Ser?His?Ser?Ala?Ser?Leu?Tyr

60 65 70

aaa?atc?gac?tcc?tgg?ggt?gcg?ccc?tac?ttc?gcc?gtc?aac?cca?tcc?ggc 351

Lys?Ile?Asp?Ser?Trp?Gly?Ala?Pro?Tyr?Phe?Ala?Val?Asn?Pro?Ser?Gly

75 80 85

aac?gtc?tcc?gtc?cgt?ccg?tac?ggc?cac?gcc?acg?ctg?gcc?cac?cag?gag 399

Asn?Val?Ser?Val?Arg?Pro?Tyr?Gly?His?Ala?Thr?Leu?Ala?His?Gln?Glu

90 95 100 105

att?gac?ctg?ctc?aag?att?gtt?aag?aag?gtt?acg?gat?ccg?aaa?tca?gtc 447

Ile?Asp?Leu?Leu?Lys?Ile?Val?Lys?Lys?Val?Thr?Asp?Pro?Lys?Ser?Val

110 115 120

ggt?ggg?ctc?ggg?ttg?cag?ctc?cct?ctc?atc?gtc?cgg?ctg?ccg?gac?gtg 495

Gly?Gly?Leu?Gly?Leu?Gln?Leu?Pro?Leu?Ile?Val?Arg?Leu?Pro?Asp?Val

125 130 135

ctc?agg?gac?cgg?ctt?gag?tcc?ctt?cag?tcg?gcc?ttt?gaa?ttc?gct?atc 543

Leu?Arg?Asp?Arg?Leu?Glu?Ser?Leu?Gln?Ser?Ala?Phe?Glu?Phe?Ala?Ile

140 145 150

cag?acg?caa?tgc?tac?gag?gcc?cat?tat?cag?ggg?gtg?ttc?ccc?gtg?aaa 591

Gln?Thr?Gln?Cys?Tyr?Glu?Ala?His?Tyr?Gln?Gly?Val?Phe?Pro?Val?Lys

155 160 165

tgt?aac?cag?gac?cgg?ttc?gtt?gtg?gag?gat?att?gtg?aaa?ttc?ggg?tcg 639

Cys?Asn?Gln?Asp?Arg?Phe?Val?Val?Glu?Asp?Ile?Val?Lys?Phe?Gly?Ser

170 175 180 185

cag?ttc?cgg?ttc?ggg?ttg?gaa?gcc?ggg?tcg?aaa?ccg?gag?ctg?ttg?ttg 687

Gln?Phe?Arg?Phe?Gly?Leu?Glu?Ala?Gly?Ser?Lys?Pro?Glu?Leu?Leu?Leu

190 195 200

gct?atg?agt?tgc?ttg?tgc?aaa?gga?agc?cct?gaa?gct?ttg?ctc?gtt?tgt 735

Ala?Met?Ser?Cys?Leu?Cys?Lys?Gly?Ser?Pro?Glu?Ala?Leu?Leu?Val?Cys

205 210 215

aat?gga?ttc?aaa?gat?gct?gaa?tac?atc?acc?ctc?gct?ttg?ctg?gcg?agg 783

Asn?Gly?Phe?Lys?Asp?Ala?Glu?Tyr?Ile?Thr?Leu?Ala?Leu?Leu?Ala?Arg

220 225 230

aag?cta?gct?ttg?aac?gca?gtg?att?gtg?cta?gag?caa?gaa?gag?gag?gtc 831

Lys?Leu?Ala?Leu?Asn?Ala?Val?Ile?Val?Leu?Glu?Gln?Glu?Glu?Glu?Val

235 240 245

gat?ttg?gtt?att?gag?ata?agc?aag?aag?ctg?aat?gtc?cga?ccc?gtg?att 879

Asp?Leu?Val?Ile?Glu?Ile?Ser?Lys?Lys?Leu?Asn?Val?Arg?Pro?Val?Ile

250 255 260 265

ggc?gct?cgg?gcc?aag?ttg?aga?acc?aaa?cat?tcg?ggt?cat?ttc?ggg?gcg 927

Gly?Ala?Arg?Ala?Lys?Leu?Arg?Thr?Lys?His?Ser?Gly?His?Phe?Gly?Ala

270 275 280

acc?tcc?ggg?gag?aaa?ggc?aaa?ttt?ggg?tta?aca?acc?tgc?cag?att?ctg 975

Thr?Ser?Gly?Glu?Lys?Gly?Lys?Phe?Gly?Leu?Thr?Thr?Cys?Gln?Ile?Leu

285 290 295

cgg?gtt?gtg?aag?aaa?ctt?gag?ctg?gct?gaa?atg?ctt?gat?tgc?ttc?cag 1023

Arg?Val?Val?Lys?Lys?Leu?Glu?Leu?Ala?Glu?Met?Leu?Asp?Cys?Phe?Gln

300 305 310

ctg?ttg?cat?ttt?cat?atc?gga?tcc?cag?atc?cca?tca?acg?gct?ttg?ctt 1071

Leu?Leu?His?Phe?His?Ile?Gly?Ser?Gln?Ile?Pro?Ser?Thr?Ala?Leu?Leu

315 320 325

acc?gat?ggt?gtt?ggt?gaa?gct?gct?cag?att?tat?tgc?gaa?tta?gtc?cgt 1119

Thr?Asp?Gly?Val?Gly?Glu?Ala?Ala?Gln?Ile?Tyr?Cys?Glu?Leu?Val?Arg

330 335 340 345

ctt?ggt?gct?aat?atg?caa?gtg?att?gac?atc?ggg?ggc?ggg?ttg?ggt?atc 1167

Leu?Gly?Ala?Asn?Met?Gln?Val?Ile?Asp?Ile?Gly?Gly?Gly?Leu?Gly?Ile

350 355 360

gat?tat?gac?gga?tcc?aaa?tcg?gct?gat?tcg?gat?ctc?tca?gtc?gct?tat 1215

Asp?Tyr?Asp?Gly?Ser?Lys?Ser?Ala?Asp?Ser?Asp?Leu?Ser?Val?Ala?Tyr

365 370 375

acc?ctc?gaa?gaa?tat?gcc?tct?gcc?gtt?gtt?caa?gca?att?cgc?tat?gtt 1263

Thr?Leu?Glu?Glu?Tyr?Ala?Ser?Ala?Val?Val?Gln?Ala?Ile?Arg?Tyr?Val

380 385 390

tgt?gac?cgg?aag?aat?gtt?aag?cat?cct?gtg?ctt?tgc?agc?gag?agc?ggt 1311

Cys?Asp?Arg?Lys?Asn?Val?Lys?His?Pro?Val?Leu?Cys?Ser?Glu?Ser?Gly

395 400 405

cgt?gct?att?gtg?tct?cat?cat?tcc?att?ttg?ata?ttt?gaa?gct?gtt?tcg 1359

Arg?Ala?Ile?Val?Ser?His?His?Ser?Ile?Leu?Ile?Phe?Glu?Ala?Val?Ser

410 415 420 425

gct?agt?gtc?tcc?cgt?gcc?gcc?ccg?gtt?gct?atg?agt?cct?ctt?ggt?ttg 1407

Ala?Ser?Val?Ser?Arg?Ala?Ala?Pro?Val?Ala?Met?Ser?Pro?Leu?Gly?Leu

430 435 440

cag?tat?ttg?gta?gaa?ggg?ttg?act?gag?gat?gct?cgt?tcg?gat?tat?aca 1455

Gln?Tyr?Leu?Val?Glu?Gly?Leu?Thr?Glu?Asp?Ala?Arg?Ser?Asp?Tyr?Thr

445 450 455

aag?atg?act?act?gct?gct?ctg?aga?ggt?gag?ttt?gag?acc?tgt?ttg?ttc 1503

Lys?Met?Thr?Thr?Ala?Ala?Leu?Arg?Gly?Glu?Phe?Glu?Thr?Cys?Leu?Phe

460 465 470

tat?gct?gat?caa?ttg?aaa?caa?aga?tgc?att?gaa?cag?ttt?aag?gat?ggg 1551

Tyr?Ala?Asp?Gln?Leu?Lys?Gln?Arg?Cys?Ile?Glu?Gln?Phe?Lys?Asp?Gly

475 480 485

act?ttg?ggt?att?gaa?cag?tta?gct?act?gtt?gat?ggg?ttg?tgt?gat?ttt 1599

Thr?Leu?Gly?Ile?Glu?Gln?Leu?Ala?Thr?Val?Asp?Gly?Leu?Cys?Asp?Phe

490 495 500 505

gta?gct?aag?gaa?ata?ggt?gca?tct?gat?cct?gtt?cgt?act?tat?cat?gtg 1647

Val?Ala?Lys?Glu?Ile?Gly?Ala?Ser?Asp?Pro?Val?Arg?Thr?Tyr?His?Val

510 515 520

aat?cta?tct?att?ttc?act?tca?att?ccg?gat?tat?tgg?ggc?att?ggt?cag 1695

Asn?Leu?Ser?Ile?Phe?Thr?Ser?Ile?Pro?Asp?Tyr?Trp?Gly?Ile?Gly?Gln

525 530 535

ttg?ttt?cct?att?gtt?cca?att?cat?cat?ttg?gat?gag?agg?cct?gga?gtg 1743

Leu?Phe?Pro?Ile?Val?Pro?Ile?His?His?Leu?Asp?Glu?Arg?Pro?Gly?Val

540 545 550

agg?ggg?att?tta?tcg?gat?ttg?act?tgt?gat?agt?gat?ggt?aag?atc?gat 1791

Arg?Gly?Ile?Leu?Ser?Asp?Leu?Thr?Cys?Asp?Ser?Asp?Gly?Lys?Ile?Asp

555 560 565

aag?ttc?att?ggt?ggt?ggt?acg?agc?ttg?cct?tta?cat?gaa?atg?gtt?ggt 1839

Lys?Phe?Ile?Gly?Gly?Gly?Thr?Ser?Leu?Pro?Leu?His?Glu?Met?Val?Gly

570 575 580 585

ggt?ggt?ggt?ggt?gag?cgt?ggg?cct?tat?tat?ttg?ggg?atg?ttc?ttg?ggc 1887

Gly?Gly?Gly?Gly?Glu?Arg?Gly?Pro?Tyr?Tyr?Leu?Gly?Met?Phe?Leu?Gly

590 595 600

ggg?gct?tat?gag?gag?gcc?ctc?ggt?gga?gtc?cac?aac?ttg?ttt?ggt?ggt 1935

Gly?Ala?Tyr?Glu?Glu?Ala?Leu?Gly?Gly?Val?His?Asn?Leu?Phe?Gly?Gly

605 610 615

cca?agc?gtg?gta?cgc?gtc?ttg?cag?agt?gat?ggt?ccg?cac?agc?ttc?gct 1983

Pro?Ser?Val?Val?Arg?Val?Leu?Gln?Ser?Asp?Gly?Pro?His?Ser?Phe?Ala

620 625 630

gtg?act?cgg?gcc?atg?cct?ggg?ccg?tct?tgt?ggg?gat?gtc?ctc?cgg?gtg 2031

Val?Thr?Arg?Ala?Met?Pro?Gly?Pro?Ser?Cys?Gly?Asp?Val?Leu?Arg?Val

635 640 645

atg?cag?cac?gag?ccc?gag?ctc?atg?ttt?gag?acc?ctc?aaa?cac?cgt?gct 2079

Met?Gln?His?Glu?Pro?Glu?Leu?Met?Phe?Glu?Thr?Leu?Lys?His?Arg?Ala

650 655 660 665

gag?gaa?tgt?tgt?gga?cag?gag?cat?ggt?agt?aat?ggt?ggg?aat?ggt?gat 2127

Glu?Glu?Cys?Cys?Gly?Gln?Glu?His?Gly?Ser?Asn?Gly?Gly?Asn?Gly?Asp

670 675 680

act?gat?gat?tac?ggc?atg?gct?aat?aat?tct?gct?tta?gct?agc?agc?ctt 2175

Thr?Asp?Asp?Tyr?Gly?Met?Ala?Asn?Asn?Ser?Ala?Leu?Ala?Ser?Ser?Leu

685 690 695

gct?cag?tac?ttt?cac?agc?atg?cca?tat?ctt?gtg?gtg?cca?tcc?tct?tgt 2223

Ala?Gln?Tyr?Phe?His?Ser?Met?Pro?Tyr?Leu?Val?Val?Pro?Ser?Ser?Cys

700 705 710

tct?ttg?act?gct?atc?aat?aat?ggt?ggt?ggg?tta?tac?tat?tgc?aac?ggg 2271

Ser?Leu?Thr?Ala?Ile?Asn?Asn?Gly?Gly?Gly?Leu?Tyr?Tyr?Cys?Asn?Gly

715 720 725

gag?gat?tac?gat?gct?gtt?gtt?gat?tct?tct?cca?aat?gag?gat?gag?cag 2319

Glu?Asp?Tyr?Asp?Ala?Val?Val?Asp?Ser?Ser?Pro?Asn?Glu?Asp?Glu?Gln

730 735 740 745

tgg?tca?tac?tgc?tat?gct?tag?atgtgtattc?ttagtatcac?ctgtcaccct 2370

Trp?Ser?Tyr?Cys?Tyr?Ala

750

atcgaaatgc?ttcatctaat?ccttaagctt?gtcttgttgc?aagtttttat?tcgccgtaat 2430

tttaatttca?ttgtctattt?taatttttag?attttcattg?caaaagatgg?aagcagttga 2490

aca 2493

<210>2

<211>751

<212>PRT

＜213〉trifoliate orange (Poncirus trifoliata)

<400>2

Met?Pro?Ala?Leu?Gly?Cys?Cys?Val?Asp?Ala?Ala?Val?Ala?Pro?Pro?Ala

1 5 10 15

Tyr?Ala?Asn?Ser?Pro?Leu?Gly?Ser?Leu?Pro?Ala?Pro?Pro?Pro?Leu?Pro

20 25 30

Leu?Ser?Phe?Asn?Ser?Gly?Thr?Ser?Pro?Pro?Thr?Pro?Met?Ser?Pro?Thr

35 40 45

Ser?Ala?Ser?Ala?Gly?Ser?Val?Ala?Ala?Asp?Val?Asp?Ala?Ser?His?Trp

50 55 60

Ser?Pro?Ser?His?Ser?Ala?Ser?Leu?Tyr?Lys?Ile?Asp?Ser?Trp?Gly?Ala

65 70 75 80

Pro?Tyr?Phe?Ala?Val?Asn?Pro?Ser?Gly?Asn?Val?Ser?Val?Arg?Pro?Tyr

85 90 95

Gly?His?Ala?Thr?Leu?Ala?His?Gln?Glu?Ile?Asp?Leu?Leu?Lys?Ile?Val

100 105 110

Lys?Lys?Val?Thr?Asp?Pro?Lys?Ser?Val?Gly?Gly?Leu?Gly?Leu?Gln?Leu

115 120 125

Pro?Leu?Ile?Val?Arg?Leu?Pro?Asp?Val?Leu?Arg?Asp?Arg?Leu?Glu?Ser

130 135 140

Leu?Gln?Ser?Ala?Phe?Glu?Phe?Ala?Ile?Gln?Thr?Gln?Cys?Tyr?Glu?Ala

145 150 155 160

His?Tyr?Gln?Gly?Val?Phe?Pro?Val?Lys?Cys?Asn?Gln?Asp?Arg?Phe?Val

165 170 175

Val?Glu?Asp?Ile?Val?Lys?Phe?Gly?Ser?Gln?Phe?Arg?Phe?Gly?Leu?Glu

180 185 190

Ala?Gly?Ser?Lys?Pro?Glu?Leu?Leu?Leu?Ala?Met?Ser?Cys?Leu?Cys?Lys

195 200 205

Gly?Ser?Pro?Glu?Ala?Leu?Leu?Val?Cys?Asn?Gly?Phe?Lys?Asp?Ala?Glu

210 215 220

Tyr?Ile?Thr?Leu?Ala?Leu?Leu?Ala?Arg?Lys?Leu?Ala?Leu?Asn?Ala?Val

225 230 235 240

Ile?Val?Leu?Glu?Gln?Glu?Glu?Glu?Val?Asp?Leu?Val?Ile?Glu?Ile?Ser

245 250 255

Lys?Lys?Leu?Asn?Val?Arg?Pro?Val?Ile?Gly?Ala?Arg?Ala?Lys?Leu?Arg

260 265 270

Thr?Lys?His?Ser?Gly?His?Phe?Gly?Ala?Thr?Ser?Gly?Glu?Lys?Gly?Lys

275 280 285

Phe?Gly?Leu?Thr?Thr?Cys?Gln?Ile?Leu?Arg?Val?Val?Lys?Lys?Leu?Glu

290 295 300

Leu?Ala?Glu?Met?Leu?Asp?Cys?Phe?Gln?Leu?Leu?His?Phe?His?Ile?Gly

305 310 315 320

Ser?Gln?Ile?Pro?Ser?Thr?Ala?Leu?Leu?Thr?Asp?Gly?Val?Gly?Glu?Ala

325 330 335

Ala?Gln?Ile?Tyr?Cys?Glu?Leu?Val?Arg?Leu?Gly?Ala?Asn?Met?Gln?Val

340 345 350

Ile?Asp?Ile?Gly?Gly?Gly?Leu?Gly?Ile?Asp?Tyr?Asp?Gly?Ser?Lys?Ser

355 360 365

Ala?Asp?Ser?Asp?Leu?Ser?Val?Ala?Tyr?Thr?Leu?Glu?Glu?Tyr?Ala?Ser

370 375 380

Ala?Val?Val?Gln?Ala?Ile?Arg?Tyr?Val?Cys?Asp?Arg?Lys?Asn?Val?Lys

385 390 395 400

His?Pro?Val?Leu?Cys?Ser?Glu?Ser?Gly?Arg?Ala?Ile?Val?Ser?His?His

405 410 415

Ser?Ile?Leu?Ile?Phe?Glu?Ala?Val?Ser?Ala?Ser?Val?Ser?Arg?Ala?Ala

420 425 430

Pro?Val?Ala?Met?Ser?Pro?Leu?Gly?Leu?Gln?Tyr?Leu?Val?Glu?Gly?Leu

435 440 445

Thr?Glu?Asp?Ala?Arg?Ser?Asp?Tyr?Thr?Lys?Met?Thr?Thr?Ala?Ala?Leu

450 455 460

Arg?Gly?Glu?Phe?Glu?Thr?Cys?Leu?Phe?Tyr?Ala?Asp?Gln?Leu?Lys?Gln

465 470 475 480

Arg?Cys?Ile?Glu?Gln?Phe?Lys?Asp?Gly?Thr?Leu?Gly?Ile?Glu?Gln?Leu

485 490 495

Ala?Thr?Val?Asp?Gly?Leu?Cys?Asp?Phe?Val?Ala?Lys?Glu?Ile?Gly?Ala

500 505 510

Ser?Asp?Pro?Val?Arg?Thr?Tyr?His?Val?Asn?Leu?Ser?Ile?Phe?Thr?Ser

515 520 525

Ile?Pro?Asp?Tyr?Trp?GlyIle?Gly?Gln?Leu?Phe?Pro?Ile?Val?Pro?Ile

530 535 540

His?His?Leu?Asp?Glu?Arg?Pro?Gly?Val?Arg?Gly?Ile?Leu?Ser?Asp?Leu

545 550 555 560

Thr?Cys?Asp?Ser?Asp?Gly?Lys?Ile?Asp?Lys?Phe?Ile?Gly?Gly?Gly?Thr

565 570 575

Ser?Leu?Pro?Leu?His?Glu?Met?Val?Gly?Gly?Gly?Gly?Gly?Glu?Arg?Gly

580 585 590

Pro?Tyr?Tyr?Leu?Gly?Met?Phe?Leu?Gly?Gly?Ala?Tyr?Glu?Glu?Ala?Leu

595 600 605

Gly?Gly?Val?His?Asn?Leu?Phe?Gly?Gly?Pro?Ser?Val?Val?Arg?Val?Leu

610 615 620

Gln?Ser?Asp?Gly?Pro?His?Ser?Phe?Ala?Val?Thr?Arg?Ala?Met?Pro?Gly

625 630 635 640

Pro?Ser?Cys?Gly?Asp?Val?Leu?Arg?Val?Met?Gln?His?Glu?Pro?Glu?Leu

645 650 655

Met?Phe?Glu?Thr?Leu?Lys?His?Arg?Ala?Glu?Glu?Cys?Cys?Gly?Gln?Glu

660 665 670

His?Gly?Ser?Asn?Gly?Gly?Asn?Gly?Asp?Thr?Asp?Asp?Tyr?Gly?Met?Ala

675 680 685

Asn?Asn?Ser?Ala?Leu?Ala?Ser?Ser?Leu?Ala?Gln?Tyr?Phe?His?Ser?Met

690 695 700

Pro?Tyr?Leu?Val?Val?Pro?Ser?Ser?Cys?Ser?Leu?Thr?Ala?Ile?Asn?Asn

705 710 715 720

Gly?Gly?Gly?Leu?Tyr?Tyr?Cys?Asn?Gly?Glu?Asp?Tyr?Asp?Ala?Val?Val

725 730 735

Asp?Ser?Ser?Pro?Asn?Glu?Asp?Glu?Gln?Trp?Ser?Tyr?Cys?Tyr?Ala

740 745 750

<210>3

<211>23

<212>DNA

＜213〉trifoliate orange (Poncirus trifoliata)

<220>

<221>primer_bind

<222>(1)..(23)

<223>

<400>3

ccccctcgtt?ttttcttttt?ctt 23

<210>4

<211>23

<212>DNA

＜213〉trifoliate orange (Poncirus trifoliata)

<220>

<221>primer_bind

<222>(1)..(23)

<223>

<400>4

tgttcaactg?cttccatctt?ttg 23

Claims

1. An isolated and cloned PtADC gene with drought and cold resistance functions derived from Poncirus trifoliata, its cDNA sequence is as shown in the sequence table SEQ ID NO: 1.

2. An isolated and cloned PtADC gene with drought and cold resistance functions derived from Poncirus trifoliata, its coding sequence is shown in the 85th-2340th position of SEQ ID NO: 1 in the sequence table.

3. the primer pair of gene described in amplification claim 1, its dna sequence is as follows:

Forward primer: 5'-CCCCCTCGTTTTTTTCTTTTTCTT-3';

Reverse primer: 5'-TGTTCAACTGCTTCCATCTTTTG-3'.

4. The application of the gene according to claim 1 in improving the drought resistance and cold tolerance of plants.

5. The application of the gene according to claim 2 in improving the drought resistance and cold tolerance of plants.