CN108251438B

CN108251438B - Gene participating in formation of peach fruit combined linalool and application thereof

Info

Publication number: CN108251438B
Application number: CN201810164164.6A
Authority: CN
Inventors: 张波; 吴伯萍; 陈昆松
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-02-27
Filing date: 2018-02-27
Publication date: 2020-04-14
Anticipated expiration: 2038-02-27
Also published as: CN108251438A

Abstract

The invention provides a gene PpGT 85A2 participating in formation of peach fruit combined-state linalool, a PpGT 85A2 full-length sequence is obtained through PCR amplification, PpGT 85A2 obtained through evolutionary tree analysis is clustered in a UGT85 family, in-vitro functional verification carried out in escherichia coli shows that PpGT 85A2 can convert linalool into a combined-state form without volatility under the condition that UDP-glucose is used as a sugar donor, PpGT 85A2 is excessively expressed in peach fruits and tobacco, the content of combined-state linalyl- β -D-glucoside is remarkably increased, the storage of aroma substances is increased, the combined-state linalool is released by using hydrolytic enzyme in the postharvest processing process of agricultural products, the aroma quality of processed food is further improved, and the application value is important.

Description

Gene participating in formation of peach fruit combined linalool and application thereof

Technical Field

The invention belongs to the field of plant molecular biotechnology and genetic engineering, and relates to a gene participating in formation of combined linalool of peach fruits and application thereof.

Background

Volatile compounds are usually present in plants in both free and glycosidically bound forms. The volatile compounds in free form can be released freely from the plant. The combined-state aroma substance has no volatility and no aroma per se. Research shows that the combined aroma substances are rich in fruits and are important storage forms of the aroma substances. These combined aroma substances can be released into free aroma substances through hydrolysis during the ripening or postharvest processing process, and the aroma of fruits or foods is obviously increased. Linalool has a floral taste and can be present in a bound form. Therefore, the identification of the genes formed by the combined linalool and other substances has important potential application value for improving the reserve amount of the aromatic substance linalool by using a genetic engineering method and finally improving the aromatic quality of peach fruits and other agricultural products.

Studies have shown that UDP-glucosyltransferase (UGT) is responsible for catalyzing the biosynthesis of bound species. The genome sequencing result shows that the peach contains at least 160 UGT gene family members, and in addition, the lack of a transgenic system causes great technical challenges in identifying and cloning to obtain the UGT participating in the synthesis of the binding-state linalool.

Disclosure of Invention

The invention aims to provide a gene PpGUT 85A2 which is involved in formation of the cinnamool in the peach fruit combination state, and the nucleotide sequence of the gene is shown as SEQ: NO. 1.

The primers of the full-length PpGUT 85A2 sequence obtained by cloning are SEQ: NO.2 and SEQ: NO.3, and the primers for carrying out gene expression analysis are SEQ: NO.6 and SEQ: NO. 7.

The gene has the following characteristic functions:

1. the nucleotide sequence of the gene is shown in SEQ NO. 1.

2. Gene expression characteristics: as the peach fruits mature, the PpGUT 85A2 expression is continuously increased, and the gene expression is in positive correlation with the accumulation of the binding linalool in the fruits.

3. The gene in vitro functional characteristics are as follows: the pET6xHN-N-PpUGT85A2 recombinant vector is expressed in Escherichia coli, and the activity is detected in vitro through purifying protein, so that the protein coded by PpUGT85A2 can convert free linalool into a binding state form without volatility.

4. The over-expression of PpGUT 85A2 in peach fruits obviously increases the accumulation of the content of the linalool in a binding state. The tobacco over-expresses PpUGT85A2, and the accumulation of the content of the linalool in a binding state is obviously increased.

The invention also aims to provide application of the gene PpGUT 85A2 in genetic engineering, particularly adopts sequences of SEQ: NO.10 and SEQ: NO.11 to construct a transgenic vector, and over-expresses PpGUT 85A2 in peach fruits and tobacco to remarkably increase the content of the linalool in a binding state and improve the flavor quality of processed food.

The recombinant protein PpGUT 85A2 constructed by adopting the sequences of SEQ No.4 and SEQ No.5 can catalyze the synthesis of the combined linalool. The recombinant vector is constructed by adopting the sequences of SEQ: NO.10 and SEQ: NO.11, and the PpGT 85A2 is over-expressed in peach fruits, so that the content of the bonding-state linalool can be obviously increased.

The invention provides a novel coding gene PpGUT 85A2 derived from peach UGT family members, and confirms that the recombinant protein of the gene PpGUT 85A2 has activity, the gene is over-expressed by using a genetic engineering technology, the content of the combined linalool linyl- β -D-glucoside in peach fruits and transgenic tobacco is obviously improved, and the combined linalool is released by using hydrolytic enzyme in the postharvest processing process of agricultural products for increasing the storage of aroma substances, so that the aroma quality of processed food is improved, and the gene has important application value.

Drawings

FIG. 1 shows that the content of bonded linalool linalyl- β -D-glucoside gradually increases during the ripening process of peach fruits.

FIG. 2: PpGUT 85A2 expression is continuously enhanced in the process of peach fruit ripening.

FIG. 3 shows that the recombinant protein PpUGT85A2 catalyzes free linalool to synthesize bound linalool linyl- β -D-glucoside.

FIG. 4 shows that the over-expression PpUGT85A2 obviously improves the content of the combined linalyl- β -D-glucoside in the transgenic tobacco.

FIG. 5 shows that the over-expression PpUGT85A2 obviously improves the content of the combined linalyl- β -D-glucoside in the peach fruits.

Detailed Description

The invention will be further elucidated with reference to the following specific examples and the accompanying drawings, without limiting the scope of the invention.

Example 1: PpGUT 85A2 gene clone

(I) Experimental method

The method comprises the steps of taking an AdGT4 amino acid sequence with UDP-glucosyltransferase function in kiwi fruits as a reference sequence, searching a homologous sequence of the Peach in a Peach Genome database Peach Genome V2.0 by applying a blastp algorithm, selecting a sequence with the highest matching degree, designing a primer pair SEQ: NO.2 and SEQ: NO.3, carrying out PCR amplification by taking Peach cDNA as a template to obtain a PpGUT 85A2 sequence SEQ: NO.1, and carrying out sequencing verification. The PCR reaction system is 50 ul, and the components are respectively: mu.l Taq enzyme (Roche), 5. mu.l buffer (10X), 4. mu.l dNTP (2.5mM), 2. mu.l each of upstream and downstream primers (10. mu.M, Invitrogen), 4. mu.l cDNA, 32.5. mu.l H₂And O. The RT-qPCR reaction program is reaction at 95 ℃ for 5 min; reacting at 95 ℃ for 30s, reacting at 58 ℃ for 30s, extending at 72 ℃ for 2min, and performing 37 cycles; finally, the extension is carried out for 10min at 72 ℃ and the product is stored at 4 ℃.

(II) results of the experiment

Through sequencing verification, PpUGT85A2 sequence SEQ NO.1 matched with peach fruit genome database is obtained.

Example 2 expression of peach fruit PpGUT 85A2 and content of linalyl- β -D-glucoside in bound form have positive correlation

(I) Experimental method

1. Peach fruit material

The peach fruits are harvested in a hard kernel period (71DAB), a quick fruit expansion period (94DAB) and a maturation period (108 DAB). Storing the mature fruit at 20 deg.C for 3d until the mature fruit is completely ripe. Meanwhile, the collected mature peach fruits are respectively treated by Ethylene (Ethylene) to accelerate fruit ripening, 1-methylcyclopropene (1-MCP) delays ripening, and air treatment is used as Control. Freezing the pulp tissue with liquid nitrogen, and storing in a refrigerator at-80 deg.C for use. 3 biological replicates were set, 5 fruits per replicate.

RNA extraction and cDNA Synthesis

Total RNA of peach fruits is extracted by a CTAB method, genomic DNA pollution is removed by a TURBO DNase Kit (Ambion) Kit, and 1.0 mu g of RNA is taken to synthesize cDNA according to the operation of an iScript cDNA Synthesis Kit (Bio-Rad) reagent instruction.

3. Gene expression

Peach PpTEF2(SEQ: NO.12) is used as an internal reference gene, primers are SEQ: NO.13 and SEQ: NO.14, and PpGT 85A2 primers are SEQ: NO.6 and SEQ: NO. 7. The real-time quantitative PCR (qPCR) reaction system comprises 10 mu l of Ssofast EvaGreenSupermix (Bio-Rad), 1 mu l of cDNA (2 mu l) and 6 mu l H of upstream and downstream primers (10 mu M) respectively₂And O. The reaction program is reaction at 95 ℃ for 3 min; the reaction is carried out at 95 ℃ for 10s, at 60 ℃ for 30s and for 45 cycles. The used instrument is a Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument, and each detection comprises H₂O is used as a negative control of the reaction template.

RNA of peach fruits at different maturation stages is taken as a material, transcriptome sequencing is carried out, and the expression mode of UGT gene family members of the peach fruits is analyzed.

4. Peach fruit combined state aroma substance content analysis

Taking 50g of the ground pulp sample, dissolving in 100mL ddH₂O and 34g of (NH)₄)₂SO₂Sonication for 5min, centrifugation (12,000g, 4 ℃) of the supernatant after qualitative filter paper, LC-18SPE cartridge (6mL CNW, Dusseldoff, Germany) activated with 6mL methanol and 6mL distilled water, passing 25mL of the above obtained supernatant, washing off soluble sugar acids with equal volume of distilled water, washing off free aroma with 25mL dichloromethane, and finally eluting the bound aroma with 25mL methanol. The collected eluate was evaporated to dryness at 45 ℃ after removing most of methanol with a nitrogen blower, dissolved in 200. mu.L (0.2M pH 5) of citric acid-sodium phosphate buffer, eluted with dichloromethane several times, the supernatant was taken out in a 4mL headspace bottle, 400. mu.L of enzyme (AR2000, 2mg) was added thereto, and after 48 hours of hydrolysis in 37 ℃ water bath, 10. mu.L of an internal standard (0.07. mu.g. mu.l) was added^-12-octanol) and extracted by HS-SPME method. After 30min of equilibration at constant temperature, an extraction head of 65 μm PDMS/DVB, stableflex (ping) (SUPELCO) was used for adsorption for 30 min. The capillary column model was DB-WAX (30m 0.25mm), more polar, and the maximum temperature tolerance of the column was 250 ℃. Column box temperature program: keeping the temperature at 40 ℃ for 2min, heating to 100 ℃ at 3 ℃/min, heating to 245 ℃ at 5 ℃/min, and injecting the sampleThe temperature of the detector is 240 ℃, the temperature of the detector is 250 ℃, the carrier gas is helium, the flow rate is 1ml/min, the split-flow sample injection is not carried out, and the flow rate is constant.

(II) results of the experiment

The real-time quantitative PCR result shows that PpUGT85A2 expression is continuously enhanced in the process of peach fruit ripening (figure 1), and the PpUGT85A2 expression is accompanied with continuous increase of the content of the linalyl- β -D-glucoside in the binding state (figure 2).

Example 3 Escherichia coli heterologous expression PpGUT 85A2 recombinant protein catalysis combination state linalool linyl- β -D-glucoside Synthesis

(I) Experimental method

1. Recombinant vector construction and E.coli transformation

The primer pair SEQ: NO.4 and SEQ: NO.5 are combined, PpUGT85A2 is obtained by utilizing PCR technology amplification, and the PCR system and the reaction procedure are the same as the example 1. The PCR product and the target vector (pET6xHN-N, Clonetch, Takara) are cut by restriction enzymes SalI and HindIII and then are connected for sequencing verification, the plasmid is transformed into escherichia coli competence BL21(DE3) pLysS (Promega) by a heat shock method, and positive colonies are picked for PCR detection verification.

2. Inducible expression

Single colonies were picked, added to 20mL of LB containing ampicillin, and cultured overnight at 37 ℃. The overnight-cultured bacterial solution was transferred to 1L of LB containing ampicillin at a ratio of 1:50 and cultured to OD₆₀₀0.5-0.6. Induction to 0D overnight at 16 ℃ with the addition of 1mM IPTG₆₀₀1.8-2.0. And centrifuging at 4000g for 15min to obtain precipitate, re-suspending with about 60mL of 1 × PBS solution, freezing at-80 deg.C and ultra-low temperature, and transferring to 30 deg.C water bath for thawing and crushing.

3. Protein purification

Centrifuging the crushed bacteria solution (4 deg.C, 10000rpm,30min), collecting supernatant

After filtration through HV sterile membranes (0.45 μm, diameter 33mm, Millipore USA), HisTALON was used^TM(Clontech, Takara) gravity column purification gave crude protein. Desalting the crude protein solution with desalting column PD-10((GE Healthcare UK), and replacing the protein with Tris-HCI buffer (100mM Tris,2mM DTT, pH 7.5), 10% glycerol was added to store the protein in the ultra-low temperature refrigerator.

4. In vitro protein activity assay

The reaction was carried out in Tris-HCl buffer (100mM Tris, pH7.5, 2.0mM DTT), 1. mu.g of purified protein, 1mM UDP-glucose and 1mM substrate were added to 200. mu.l of the reaction System, after incubation at 30 ℃ for 16 hours, the reaction was terminated by adding 10. mu.l 24% (v/v) TCA, extraction with ethyl acetate and evaporation to dryness, the glycoside compound was dissolved in methanol, and the product was analyzed by LC-MS, using an apparatus of Agilent 1290Infinity LC System (Agilent Technologies, USA), a column of SunAire C63 18 (5. mu.m, 4.6X 250 mM; Waters, USA) at a detection wavelength of 200 psi 400nm, chromatographic conditions with 100% pure water as solvent A and 100% acetonitrile as solvent B, 1mL/min, a mass spectrum was detected by an Agilent6460 quadrupole mass spectrometer (Agilent Technologies, USA) equipped with a source at a scanning pressure of 1000. negative ion, 45-1000 nm, and a scanning pressure of 1000. mu.210. mu.5. mu.m/300. mu.]^-。

(II) results of the experiment

The recombinant protein PpGUT 85A2 induced in Escherichia coli can catalyze the synthesis of the combined linalool by taking linalool as a substrate and UDP-glucose as a sugar donor (shown in figure 3).

Example 4 overexpression of PpGUT 85A2 in transgenic tobacco promotes accumulation of linalyl- β -D-glucoside in bound form

(I) Experimental method

1. Vector construction

The primer pair SEQ: NO.10 and SEQ: NO.11 were combined, PpUGT85A2 was obtained by PCR amplification, and the PCR system and reaction procedure were the same as in example 1. The PCR product and the target vector (pGreen 002962-SK) are cut by restriction enzymes Sac I and Bam HI, then are respectively connected, transformed into escherichia coli, verified by sequencing after positive bacteria are selected, and plasmids with the sequences confirmed to be correct are transformed into agrobacterium strain GV3101:: pSoup by an electric shock method.

2. Identification of transgenic plants

After the common tobacco is transformed by the genetic engineering technology to obtain a transformed plant, the verification needs to be further carried out by means of PCR and qPCR. The DNA of the tobacco leaf is extracted by a CTAB method, the obtained DNA is used as a template, and a PCR is used for identifying the transgenic plant by combining a primer pair SEQ: NO.10 and SEQ: NO. 11.

The method comprises the steps of taking plant leaves identified by PCR, extracting total RNA of tobacco by utilizing TRIzol (Invitrogen), synthesizing cDNA (complementary deoxyribonucleic acid) as described in example 1, wherein in RT-qPCR detection, a PpUGT85A2 primer pair is SEQ: NO8 and SEQ NO.9, a common tobacco EF1- α gene (SEQ: NO.15) is used as an internal reference gene, and primers are SEQ: NO.16 and SEQ: NO. 17. RT-qPCR reaction system and reaction program are the same as those in example 1.

3. Detection and analysis of content of bound aroma substances

The fully ground tobacco leaves were used for the detection, extraction and detection of the bound aroma in the same manner as in example 2.

1g of tobacco lamina was dissolved in 30mL ddH2O and 10g (NH)₄)₂SO₂Then octanol (0.07. mu.g. mu.l) was added^-1) As an internal standard, vortex mixing is carried out, and then combined aroma determination is carried out by using a headspace solid phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) detection system. The separation column is DB-Wax capillary chromatography column (0.25mm, 30m, 0.25 μm, J)&W Scientific). The temperature program is that the temperature is kept at 40 ℃ for 2min, the temperature is increased to 100 ℃ at 3 ℃/min, the temperature is increased to 245 ℃ at 5 ℃/min, the temperature of a sample inlet is 240 ℃, the temperature of a detector is 250 ℃, the carrier gas is helium, the flow rate is 1ml/min, the split-flow sample injection is not carried out, and the flow rate is constant.

(II) results of the experiment

Compared with wild tobacco plants, the content of linalyl- β -D-glucoside in the transgenic tobacco plants excessively expressing PpUGT85A2 is obviously increased (figure 4).

Example 5 overexpression of PpGUT 85A2 in peach fruit promotes accumulation of linalyl- β -D-glucoside in bound form

(I) Experimental method

1. Vector construction

The vector and the construction and transformation method of the vector are shown in example 4. The transformed Agrobacterium is plated, cultured at 28 ℃ for 2 days, monocloned and selectedTransferring into LB culture medium containing kanamycin (50mg/L) and gentamicin (25mg/L), and culturing to OD₆₀₀After a time of 0.8-1.0, centrifugation (4000g,5 min). Equal volume of permeate (10mM MES,10mM MgCI) was used for precipitation₂150mM acetosyringone, pH 5.6) and induced at room temperature for 2 h.

2. Real-time quantitative PCR (qPCR) analysis of gene expression

Peach PpTEF2(SEQ: NO.12) is used as an internal reference gene, primers are SEQ: NO.13 and SEQ: NO.14, and PpGT 85A2 primers are SEQ: NO.8 and SEQ: NO. 9. The qPCR reaction system is shown in example 1.

3. Infection of peach fruit

Selecting peach fruits in the color conversion period, cleaning and sterilizing, removing the epidermis on a superclean workbench, selecting middle pulp, and cutting into slices with the thickness of about 1 cm. Selecting equatorial plane and contraposition plane of each fruit except suture line, cutting 2 pieces, and pre-culturing the cut pieces on MS solid culture medium for 24-30 h. Inducing the resuspended penetrant for 2 hr, pouring into vacuum infiltration apparatus, placing the pulp slice on the culture medium, and vacuum infiltrating at-70 Kpa. When no air bubbles appear on the surface of the slice, the pressure is restored by starting slow deflation, and the process takes about 15-20 min. Sterilizing the penetrated fruit slices with sterile water for 2 times, air drying, placing on a new MS culture medium, and culturing for 2-3 days. Wherein, half of each piece of pulp is used for permeating bacterial liquid containing the recombinant vector PpUGT85A2-pGreen 002962-SK, and half is used as a negative control for permeating bacterial liquid only containing no load. After the sample was collected, it was frozen in a freezer at-80 ℃ with liquid nitrogen. Measurement of bound aroma substances the same procedure as for the measurement of bound aroma substances of fruits as in example 1 was applied.

(II) results of the experiment

The over-expression of PpGUT 85A2 in peach fruits significantly promoted the increase of the content of linalyl- β -D-glucoside in the bound form (figure 5).

Sequence listing

<110> Zhejiang university

<120> a gene participating in formation of peach fruit combined linalool and application thereof

<160>17

<170>SIPOSequenceListing 1.0

<210>1

<211>1452

<212>DNA

<213> Biluo (Prunus persica)

<400>1

atgagtccag ttgcctccaa agagaagcca catgcagttt ttgtaccctt cccagctcag 60

ggtcacataa accctatgct gcaattagcc aagctcctca actacaaagg cttccacata 120

acctttgtga acacagagtt caaccacaag cgcatgcttg aatcccaagg ttcccacgct 180

ctcgacggcc tcccttcgtt tcgattcgaa accattcccg acggcctccc tccggctgat 240

gccgatgcca ggcgcaactt gcctttggtg tgcgattcca ctagtaaaac ctgcttggca 300

cccttcgagg cgcttttgac caagctcaac tcttcacccg attcccctcc tgtgacttgt 360

attgttgctg atggtgtcac cagcttcacc cttgatgcag cagagcattt cgggatccca 420

gaagtgcttt tctggacaac tagtgcttgt ggcttgatgg gctacgttca gtattaccgt 480

ctcattgaga agggcctcac tccttttaaa gatgccaagg attttgcaaa tgggtatttg 540

gatacagaga ttgattggat cccaggcatg aaggatgtca gattgaagga catgcctagc 600

tttattagaa ccacagaccc aaacgacatc atgctgcatt atatggtgtc tgaaacagag 660

cgatccaaaa aggcttctgc tattattttg aacacatttg atgccttgga gcaagaagtt 720

gtggatgccc tttccacttt gctacctcct atttactcca ttggacccct tcagctacca 780

tacagcgaga ttccatcaga atacaatgat ttgaaggcga tcggatcgaa cctgtgggca 840

gagaatacag agtgccttaa ctggctggac accaaagagc ccaactctgt ggtttatgtc 900

aactttggaa gcaccacagt catgacaaat gagcagctgg ttgagttttc ttggggactt 960

gcaaatagca agaagccatt tttgtggatc atcaggcctg gccttgttgc tggagaaacc 1020

gctgtggtgc cgcctgagtt tttggaggag accaaagaga ggggtatgtt ggcaagttgg 1080

tgccctcaag aacaggttct gctccactca gccattggag ggttcttgac tcacagtggc 1140

tggaactcta ccctcgaggc cttgtgtggc ggagtgcctc tcatctgctg gcctttcttt 1200

gcagagcagc aaacaaatgt taggtacagc tgcacacagt ggggcatagg cattgagata 1260

gacggggaag ttaaaagaga ttacattgac ggtcttgtga ggacattgat ggatggagaa 1320

gagggcaaaa agatgaggaa gaaagccctt gaatggaaga agttggcaga ggacgccact 1380

gccccaaaag ggtcgtctta cttggctctg gaaaatgtgg ttagcaaagt gcttctttcc 1440

ccaagagatt ag 1452

<210>2

<211>22

<212>DNA

<213> Artificial sequence (Unknown)

<400>2

atgagtccag ttgcctccaa ag 22

<210>3

<211>22

<212>DNA

<213> Artificial sequence (Unknown)

<400>3

ctaatctctt ggggaaagaa gc 22

<210>4

<211>37

<212>DNA

<213> Artificial sequence (Unknown)

<400>4

aaggcctctg tcgacatgag tccagttgcc tccaaag 37

<210>5

<211>37

<212>DNA

<213> Artificial sequence (Unknown)

<400>5

agaattcgca agcttctaat ctcttgggga aagaagc 37

<210>6

<211>21

<212>DNA

<213> Artificial sequence (Unknown)

<400>6

agcaaagtgc ttctttcccc a 21

<210>7

<211>20

<212>DNA

<213> Artificial sequence (Unknown)

<400>7

aaaagcctcg gcaaacggta 20

<210>8

<211>20

<212>DNA

<213> Artificial sequence (Unknown)

<400>8

cactcagcca ttggagggtt 20

<210>9

<211>20

<212>DNA

<213> Artificial sequence (Unknown)

<400>9

cagcagatga gaggcactcc 20

<210>10

<211>37

<212>DNA

<213> Artificial sequence (Unknown)

<400>10

gcccaagctg agctcatgag tccagttgcc tccaaag 37

<210>11

<211>37

<212>DNA

<213> Artificial sequence (Unknown)

<400>11

gactctagag gatccatctc ttggggaaag aagcact 37

<210>14

<211>2532

<212>DNA

<213> Biluo (Prunus persica)

<400>14

atggtgaagt tcacagctga ggagctccgt aggattatgg actacaaaca caacattcgt 60

aacatgtctg ttattgcgca tgttgatcac gggaagtcaa cccttaccga ctcccttgtt 120

gctgctgctg gtatcattgc acaagaagtt gctggtgatg tccgcatgac agatacccgt 180

gcagatgagg cagagcgtgg tatcacaatc aaatctactg gtatctctct ctactatgag 240

atgactgatg aagctttgaa gagctacaag ggggagagaa acggaaatga gtacctcatc 300

aatctcattg attcccctgg gcacgttgac ttttcatctg aagtcacagc tgcccttcgc 360

attactgatg gtgcacttgt ggtggttgat tgcattgagg gtgtttgtgt ccaaacagag 420

actgtgcttc gtcaagcctt gggagaaagg atcaggcctg ttttgactgt taacaagatg 480

gacaggtgct tccttgagct ccaggtcgat ggagaggagg cttaccaaac attccagagg 540

gttattgaga atgctaatgt tattatggct acatacgaag accctcttct tggtgatgtc 600

caggtctatc cagagaaagg aacagttgcc ttttctgctg gtttgcacgg atgggctttt 660

actctgacca actttgccaa gatgtatgca tccaagtttg gagttgatga gtcaaagatg 720

atggaaaggc tctggggtga gaactacttt gacccagcta ccaagaaatg gaccagcaag 780

aacactggtt ctgctacctg caagcgtggt ttcgttcagt tctgttatga acccatcaag 840

cagattatca acacctgcat gaatgatcag aaggagaagt tgtggcccat gttgacaaag 900

cttggtgtga cgatgaagag tgatgaaaag gagctgatgg ggaaggggtt gatgaagcgt 960

gtcatgcaga cctggctacc agccagcagt gccctattgg aaatgatgat ctttcacctt 1020

ccctctcctt caactgccca gagataccgt gttgagaact tgtacgaggg tccccttgat 1080

gatcaatatg caaatgctat caggaactgc gatcctgaag ggcctcttat gctctatgta 1140

tctaagatga ttcccgcatc tgacaagggt cgattctttg cctttggtcg tgtctttgct 1200

ggtaaagtcc agacaggttt gaaggttaga atcatgggtc caaattatgt tcctggagag 1260

aagaaggatt tgtatgttaa gaacgtgcag aggactgtta tttggatggg aaagaaacaa 1320

gaaactgttg aggatgttcc ttgtggtaac actgttgcct tggtcggtct tgatcagttt 1380

atcaccaaga atgctacgtt gacaaatgag aaggaagcgg atgctcaccc cattcgtgct 1440

atgaagttct ctgtttcacc tgttgtgcgt gttgctgttc aatgcaaggt ggcttctgac 1500

cttcccaaac tggttgaagg tctcaaacgt ctggccaagt ctgatcctat ggttgtctgt 1560

tccattgagg aatccggtga gcacattatt gctggtgctg gtgaacttca tcttgagatt 1620

tgtttgaagg atctacaaga tgatttcatg ggtggagctg agattataaa atctgacccc 1680

gttgtgtctt tccgtgagac tgtcctggag aagtctagtc gtactgtgat gagcaagtca 1740

cccaacaagc ataaccgtct gtatatggaa gctcgaccct tggaggaagg tcttcctgag 1800

gccattgatg atggccgtat tggcccaaga gatgatccca aaattcgttc caagatattg 1860

gctgaagagt ttggttggga caaggatctt gctaagaaaa tctggtgttt tggccctgag 1920

accaccggtc ctaacatggt ggtggatatg tgtaagggag ttcagtacct gaatgaaatt 1980

aaggactctg ttgttgctgg tttccagtgg gcttcaaagg aaggtgcatt ggcagaagaa 2040

aacatgaggg gtatttgctt tgaagtctgt gatgtggttc ttcatgctga tgccatccac 2100

agaggaggtg gtcaggtcat tcccactgct aggagggtca tctatgcttc ccagctcact 2160

gccaagccaa ggctccttga acctgtatat cttgttgaaa tccaagctcc agagcaggct 2220

cttggtggta tctacagtgt tcttaatcag aaacgtgggc acgtgtttga ggaaatgcag 2280

aggcctggta caccactcta caatatcaag gcatacctcc ccgtcattga atcttttggg 2340

ttctctggtc aactgagggc ttcgacttca gggcaggcct tcccacaatg tgtctttgat 2400

cattgggaga tgatgtcgtc tgatccattg gaagctggat cccaggcttc acagcttgtt 2460

acagatatcc gtaagaggaa gggtttgaag gagcaaatga ccccactatc cgagtttgag 2520

gacaaactct ga 2532

<210>15

<211>22

<212>DNA

<213> Artificial sequence (Unknown)

<400>15

ggtgtgacga tgaagagtga tg 22

<210>16

<211>22

<212>DNA

<213> Artificial sequence (Unknown)

<400>16

tgaaggagag ggaaggtgaa ag 22

<210>17

<211>1344

<212>DNA

<213> tobacco (Nicotiana tabacum)

<400>17

atgggtaaag agaaggttca catcaacatt gtggtcattg gccacgtcga ctctggtaag 60

tcgactacca ctggtcactt gatctacctt ggtggtattg acaagcgtgt cattgagagg 120

tttgagaaag aagctgctga gatgcacaag cggtcattca agtatgcctg ggtgcttgac 180

aaactaaagg ctgagcgtga ccggggtatc actattgata tcgccttgtg gaagtttgag 240

accaccaagt actactgcac tgtgattgat cgtcctggac acagggattt catcaagaat 300

atgattactg gtacctcccg tgcctgtcgt gtcctgattg ttgcctccac cactggcttt 360

gcacaagctg gtaatctcaa ggatggacag acccgtgaaa gactgcttat tgactccacc 420

actggtaaca cccttggtgt cacccaaatg atttgctgct gcaacaagat ggatgctacc 480

acccccaagt attccaaggc taggtacgat gaaatcgtga aggaggtttc ttcctacctc 540

aagaaggtag gatacaaccc tgacaagatc ccctttgtcc ccatctctgg tttggaaggt 600

gacaacatgc tcgaaagatc aacaaacctt gactggtaca agggcccaac acttcttgat 660

gctcttgacc agattaatga gcccaagagg cccacagaca agcctctcag gctcccactt 720

caggatgttt acaagattgg tggaattggt actgtccctg ttggtcgtgt ggaaactggt 780

gtcctcaagc ctggtatggt tgttactttt ggtcccactg gtctgaccac tgaagttgga 840

tctgttgaga tgcaccacga agctcttcag gaggcacttc ctggtgacaa tgttggattt 900

aatgtcaaga atgttgcggt taaggatctc aagcgtgggt ttgttgcttc caactccgga 960

tgtcccagca atgggtgctt cagctttacc tcccaagcta tcatcatgaa ccattcagga 1020

cagattggca atggatatgc tccagttctg gactgccaca cctcccatgc tgtcagtgca 1080

gaaattttga ccaatattgc caggcgttct ggtcaggaga ttgcgaaaga gcccaggttc 1140

cttttgaatg gttgtgctgg ttttgttatg atgattccca ccctacccat ggttgttgca 1200

aggatcctct ctgcgtaccc accattggag cgttttgcgt tcaggagcat gcgtcaaact 1260

gttgctgttg gtgttatcaa gaacgttgtc aagaaggacc caactggtgc tatggtcacc 1320

aaggctgctc agaagaagaa atga 1344

<210>18

<211>21

<212>DNA

<213> Artificial sequence (Unknown)

<400>18

gcccaacact tcttgatgct c 21

<210>19

<211>20

<212>DNA

<213> Artificial sequence (Unknown)

<400>19

gacaccagtt tccacacgac 20

Claims

1. Gene participating in formation of peach fruit combined linaloolPpUGT85A2The application of the linalool in catalyzing the synthesis of plant combined-state linalool is characterized in that the application is realized by constructing a transgenic vector and performing over-expression in peach fruits and tobaccoPpUGT85A2So as to remarkably increase the content of linalyl- β -D-glucoside in combined state, and the genePpUGT85A2The nucleotide sequence of (A) is shown as SEQ NO. 1.