CN108251438A - One participates in gene and its application that Peach fruits reference state linalool is formed - Google Patents

One participates in gene and its application that Peach fruits reference state linalool is formed Download PDF

Info

Publication number
CN108251438A
CN108251438A CN201810164164.6A CN201810164164A CN108251438A CN 108251438 A CN108251438 A CN 108251438A CN 201810164164 A CN201810164164 A CN 201810164164A CN 108251438 A CN108251438 A CN 108251438A
Authority
CN
China
Prior art keywords
ppugt85a2
linalool
seq
reference state
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810164164.6A
Other languages
Chinese (zh)
Other versions
CN108251438B (en
Inventor
张波
吴伯萍
陈昆松
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN201810164164.6A priority Critical patent/CN108251438B/en
Publication of CN108251438A publication Critical patent/CN108251438A/en
Application granted granted Critical
Publication of CN108251438B publication Critical patent/CN108251438B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01017—Glucuronosyltransferase (2.4.1.17)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Nutrition Science (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention provides one and participates in the gene PpUGT85A2 that Peach fruits reference state linalool is formed, and PpUGT85A2 full length sequences are obtained by PCR amplification, and PpUGT85A2 clusters are obtained in UGT85 families by phylogenetic analysis.The external functional verification carried out in Escherichia coli shows that linalool can be converted into without volatile reference state form by PpUGT85A2 under conditions of UDP glucose are as saccharide donor.The overexpression PpUGT85A2 in Peach fruits and tobacco, significantly increase reference state linalool linalyl β D glucoside contents, increase the deposit of aroma substance, hydrolysis enzyme r e lease reference state linalool is used during adopting post-processing for agricultural product, and then the fragrance quality of processed food is promoted, there is important application value.

Description

One participates in gene and its application that Peach fruits reference state linalool is formed
Technical field
The invention belongs to plant molecular biotechnology and genetic engineering fields, are related to a participation Peach fruits reference state virtue camphor tree The gene and its application that alcohol is formed.
Background technology
Volatile compound is usually existed in plant in the form of two kinds of free state and glucosides reference state.Free state form Volatile compound can freely be discharged out of plant.Bound state aroma substance does not have volatility, itself does not have fragrance. It is the important deposit form of aroma substance research shows that bound state aroma substance rich content in fruit.These reference states Aroma substance in maturation or is adopted Free aroma substance can be released to by hydrolysis during post-processing, dramatically increases The fragrance of fruit or food.Linalool has fragrance of a flower taste, can exist in the form of reference state.Therefore, differentiate reference state virtue camphor tree The gene that the substances such as alcohol are formed, it is final to improve for improving the storage level of aroma substance linalool using gene engineering method The fragrant quality of Peach fruits and other agricultural product has important potential using value.
Research shows that UDP-glucose based transferase (UGT) is responsible for the biosynthesis of catalyzed combination state substance.Genome is surveyed Sequence is the results show that how peach, along with transformation system is lacked, differentiates and clone at least containing 160 UGT gene family members The UGT for participating in the synthesis of reference state linalool is obtained, there are larger technological challenges.
Invention content
The gene PpUGT85A2 for participating in Peach fruits reference state linalool the object of the present invention is to provide one and being formed, the base The nucleotide sequence of cause such as SEQ:Shown in NO.1.
The primer that clone obtains this overall length PpUGT85A2 sequences is SEQ:NO.2 and SEQ:NO.3 carries out gene expression The primer of analysis is SEQ:NO.6 and SEQ:NO.7.
The gene expression characteristics function is as follows：
1. the nucleotide sequence of gene such as SEQ:Shown in NO.1.
2. allelic expression：With Peach fruits maturation, PpUGT85A2 expression continues to increase, in gene expression and fruit The accumulation correlation of reference state linalool.
3. the outer functional character of genosome：In expression in escherichia coli pET6xHN-N-PpUGT85A2 recombinant vectors, by pure Change albumen, vitro detection activity, it was demonstrated that free state linalool can be converted into without volatilization by the albumen of PpUGT85A2 codings The reference state form of property.
4. the overexpression PpUGT85A2 in Peach fruits significantly increases the accumulation of reference state linalool content.In tobacco Overexpression PpUGT85A2 significantly increases the accumulation of reference state linalool content.
It is a further object to provide applications of the gene PpUGT85A2 in genetic engineering, especially adopt Use SEQ:NO.10 and SEQ:NO.11 sequence construct transgene carriers, overexpression PpUGT85A2 is shown in Peach fruits and tobacco Writing increases reference state linalool content, promotes the fragrance quality of processed food.The present invention passes through in Peach fruits overexpression, it was demonstrated that The albumen of PpUGT85A2 codings can promote accumulation of the reference state linalool in Peach fruits tissue；Utilize technique for gene engineering The transgenic tobacco plant of overexpression PpUGT85A2 is obtained, the volatile materials in Peach fruits, overexpression can be changed PpUGT85A2 significantly promotes the accumulation of reference state linalool linalyl- β-D-glucoside.
Using SEQ:NO.4 and SEQ:The recombinant protein PpUGT85A2 of NO.5 sequence constructs can be with catalyzed combination state linalool Synthesis.Using SEQ:NO.10 and SEQ:NO.11 sequence constructs recombinant vector, can show in Peach fruits overexpression PpUGT85A2 Writing increases reference state linalool content.
The present invention provides a new encoding gene PpUGT85A2 from peach UGT family members, and confirm that its is heavy Histone is active.Using the technique for gene engineering overexpression gene, significantly improve in Peach fruits and transgene tobacco For increasing the deposit of aroma substance, post-processing is adopted for agricultural product for reference state linalool linalyl- β-D-glucoside contents In the process using hydrolysis enzyme r e lease reference state linalool, and then the fragrance quality of processed food is promoted, valency is applied with important Value.
Description of the drawings
Fig. 1：Reference state linalool linalyl- β-D-glucoside contents gradually increase in Peach fruits maturation.
Fig. 2：The lasting enhancing of PpUGT85A2 expression in Peach fruits maturation.
Fig. 3：Recombinant protein PpUGT85A2 catalysis free state linalool synthesis reference state linalool linalyl- β-D- glucoside。
Fig. 4：Overexpression PpUGT85A2 significantly improves the reference state linalool linalyl- β-D- in transgene tobacco Glucoside contents.
Fig. 5：Overexpression PpUGT85A2 significantly improves the reference state linalool linalyl- β-D- in Peach fruits Glucoside contents.
Specific embodiment
The present invention is further elaborated with reference to specific embodiments and the drawings, but embodiment does not limit the guarantor of the present invention Protect range.
Embodiment 1：PpUGT85A2 gene clonings
(1) experimental method
There is the AdGT4 amino acid sequences of UDP-glucose based transferase as reference sequences in Kiwi berry, apply Blastp algorithms search the homologous sequence of peach in peach genome database Peach Genome V2.0, choose matching degree highest Sequence, design primer pair SEQ:NO.2 and SEQ:NO.3 using peach cDNA as template, carries out PCR amplification and obtains PpUGT85A2 sequences Arrange SEQ:NO.1, and carry out sequence verification.PCR reaction systems are 50 μ l, and ingredient is respectively：0.5 μ l Taq enzymes (Roche), 5 μ l Buffer solution (10 ×), 4 μ l dNTP (2.5mM), upstream and downstream primer (10 μM, Invitrogen) each 2 μ l, 4 μ l cDNA, 32.5 μ l H2O.RT-qPCR response procedures are 95 DEG C of reaction 5min；95 DEG C of reaction 30s, 58 DEG C of reaction 30s, 72 DEG C of extension 2min, 37 are followed Ring；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.
(2) experimental result
Through sequence verification, the PpUGT85A2 sequences SEQ to match with Peach fruits genome database is obtained:NO.1.
Embodiment 2：Peach fruits PpUGT85A2 is expressed and reference state linalool linalyl- β-D-glucoside contain measurer There is positive correlation
(1) experimental method
1. Peach fruits material
Peach fruits harvest in stone phase (71DAB), the quick expanding stage of fruit (94DAB) and maturity period (108DAB).It is ripe Fruit is placed in 20 DEG C of storage 3d to complete ripeness.Meanwhile the ripe Peach fruits of harvesting are respectively adopted ethylene (Ethylene) processing and accelerate Fruit maturation, 1- methyl cyclopropenes (1-MCP) delaying ripening, air-treatment are control (Control).Pulp organization freezes through liquid nitrogen It is for use that -80 DEG C of refrigerators are deposited in after depositing.3 biology of setting repeat, and each repeat 5 fruits.
2.RNA is extracted and cDNA synthesis
Peach fruits total serum IgE is extracted using CTAB methods, genome is removed with TURBO DNase Kit (Ambion) kit After DNA pollution, 1.0 μ g RNA is taken to be operated by iScript cDNA Synthesis Kit (Bio-Rad) reagents specification and are synthesized cDNA。
3. gene expression
With peach PpTEF2 (SEQ:NO.12 it is) reference gene, primer SEQ:NO.13 and SEQ:NO.14, PpUGT85A2 Primer is SEQ:NO.6 and SEQ:NO.7.Real-time quantitative PCR (qPCR) reaction system includes 10 μ l Ssofast EvaGreen Supermix (Bio-Rad), upstream and downstream primer (10 μM) each 1 μ l, 2 μ l cDNA, 6 μ l H2O.Response procedures are 95 DEG C of reactions 3min；95 DEG C of reaction 10s, 60 DEG C of reaction 30s, 45 recycle.Instrument is Bio-Rad CFX96 real-time fluorescence quantitative PCRs Instrument, every time detection all include with H2O makees the negative control of reaction template.
The RNA of different stage of ripeness Peach fruits be material, carry out transcript profile sequencing, analysis Peach fruits UGT gene families into The expression pattern of member.
4. Peach fruits bound state aroma content of material is analyzed
The Meat Sample 50g after grinding is taken, is dissolved in 100mL ddH2(the NH of O and 34g4)2SO2, ultrasonic 5min is excessively fixed Property filter paper after supernatant centrifugation (12,000g, 4 DEG C), LC-18SPE pillars (6mL CNW, Dusseldorf, Germany) 6mL first Alcohol is with after the activation of 6mL distilled water, crossing the above-mentioned gained supernatants of 25mL, soluble saccharic acid being washed away, then use 25mL with isometric distilled water Dichloromethane washes away Free aroma, is finally eluted bound state aroma with 25mL methanol.The eluate of collection, is gone with nitrogen evaporator Except 45 DEG C of rotary evaporated to dryness after most of methanol, 200 μ L (0.2M pH=5) citrate-phosphate sodium buffering is added in residue Liquid dissolves, then is repeatedly eluted with dichloromethane, takes supernatant in 4mL ml headspace bottles, 400 μ L enzymes of addition (AR2000, 2mg), after 37 DEG C of water enzyme digestion 48h, 10 μ L internal standards (0.07 μ g μ l are added in-1Sec-n-octyl alcohol) vortex mixing, in HS-SPME methods It is extracted.After constant temperature balance 30min, inhaled using 65 μm of PDMS/DVB of extracting head, Stableflex (pink) (SUPELCO) Attached 30min.Capillary column model DB-WAX (30m*0.25mm*0.25mm), more highly polar, pillar highest tolerable temperature is 250℃.Column temperature rise program：40 DEG C of holdings 2min, 3 DEG C/min are warming up to 100 DEG C, and 5 DEG C/min is warming up to 245 DEG C, injection port Temperature is 240 DEG C, and 250 DEG C of detector temperature, carrier gas is helium, flow velocity 1ml/min, Splitless injecting samples, constant flow rate pattern.
(2) experimental result
Real-time quantitative PCR enhances (attached drawing 1) the results show that PpUGT85A2 expression is lasting in Peach fruits maturation, adjoint There is continuing to increase (attached drawing 2) for reference state linalool linalyl- β-D-glucoside contents.
Embodiment 3：Escherichia coli heterogenous expression PpUGT85A2 recombinant protein catalyzed combination state linalool linalyl- β-D- Glucoside is synthesized
(1) experimental method
1. construction of recombinant vector and Escherichia coli conversion
With reference to primer pair SEQ:NO.4 and SEQ:NO.5 is expanded using round pcr and is obtained PpUGT85A2, PCR system and anti- Program is answered with embodiment 1.PCR product and destination carrier (pET6xHN-N, Clonetch, Takara) use restriction enzyme It is connected after SalI and HindIII digestions, carries out sequence verification, plasmid converts E. coli competent BL21 (DE3) with heat shock method PLysS (Promega), the picking positive bacterium colony PCR detection verification.
2. induced expression
Picking individual colonies add in 20mL and contain in the LB of ammonia benzyl antibiotic, and 37 DEG C are incubated overnight.Bacterium solution after being incubated overnight According to 1:50 ratio is transferred to the LB that 1L contains ammonia benzyl antibiotic, cultivates to OD600=0.5-0.6.The IPTG of 1mM is added in 16 DEG C induction overnight is to 0D600=1.8-2.0.4000g, 15min centrifugation are precipitated, and are suspended again with about 60mL 1*PBS solution, Gone to after -80 DEG C of ultra low temperature freezers freeze 30 DEG C of water-baths melt it is broken.
3. protein purification
Broken bacterium solution is centrifuged (4 DEG C, 10000rpm, 30min), obtained supernatant withHV degermings After (0.45 μm, diameter 33mm, Millipore USA) filtering of film, HisTALON is usedTM(Clontech, Takara) weight Power column purification obtains crude protein.With desalting column PD-10, ((GE Healthcare UK) carries out desalination to crude protein liquid, and by albumen Tris-HCI buffer solutions (100mM Tris, 2mM DTT, PH 7.5) are replaced, add in 10% glycerine by protein storage ultralow Temperature refrigerator.
4. albumen active determination in vitro
Reaction carries out in Tris-HCl buffer solutions (100mM Tris, pH7.5,2.0mM DTT), 200 μ l reaction systems 1 μ g purifying proteins of middle addition, 1mM UDP-glucose and 1mM substrates.After being incubated 16 hours at 30 DEG C, by adding in 10 μ l 24% (v/v) TCA terminates reaction, is extracted with ethyl acetate, is then evaporated to dryness.By glycoside compounds dissolving in methyl alcohol, lead to Cross LC-MS analysis products.The use of instrument is Agilent 1290Infinity LC System (Agilent Technologies, USA), chromatographic column for SunFire C18 analytical columns (5 μm, 4.6 × 250mm；Waters, USA), detect wave A length of 200-400nm.Chromatographic condition is using 100% pure water as solvent A and 100% acetonitrile as solvent B, the flow velocity of 1mL/min It is detected.Mass spectrum by be equipped with ESI sources Agilent6460 triple quadrupole mass spectrometers (Agilent Technologies, USA), analyzed with negative ion mode.Scanning range is 100-1000m/z, and nebulizer pressure is set as 45psi.Linalyl-β- The Detection wavelength of D-glucoside is 210nm, and m/z values are 315 [M-H]-。
(2) experimental result
The recombinant protein PpUGT85A2 induced in Escherichia coli, can be using linalool as substrate, and UDP-glucose is sugar Donor, catalyzed combination state linalool synthesis (attached drawing 3).
Embodiment 4：Transgene tobacco overexpression PpUGT85A2 promotes reference state linalool linalyl- β-D- Glucoside is accumulated
(1) experimental method
1. vector construction
With reference to primer pair SEQ:NO.10 and SEQ:NO.11, using round pcr expand obtain PpUGT85A2, PCR system and Response procedures are the same as embodiment 1.PCR product and destination carrier (0029 62-SK of pGreen) with restriction enzyme Sac I and It after Bam HI digestions, connects respectively, converts Escherichia coli, picking positive bacteria falls behind sequence verification, sequence is confirmed correct matter Grain utilizes electric shocking method conversion Agrobacterium strains GV3101::pSoup.
2. transfer-gen plant is identified
After technique for gene engineering conversion Nicotiana tabacum obtains transformed plant, need further through PCR and qPCR means It is verified.Tobacco leaf DNA is extracted using CTAB methods, template is done using the DNA of gained, with reference to primer pair SEQ:NO.10 and SEQ:NO.11 carries out transfer-gen plant identification with PCR.
The plant leaf of the PCR that learnt from else's experience identifications extracts tobacco total serum IgE using TRIzol (Invitrogen), synthesizes cDNA Process is as described in Example 1.In RT-qPCR detections, PpUGT85A2 primer pairs are SEQ:NO8 and SEQ NO.9, with Nicotiana tabacum EF1- α genes (SEQ:NO.15) as reference gene, primer SEQ:NO.16 and SEQ:NO.17.RT-qPCR reaction systems and Response procedures are the same as embodiment 1.
3. bound state aroma content of material tests and analyzes
The tobacco leaf being fully ground detects for bound state aroma substance, and extraction and detection process are the same as embodiment 2.
1g tobacco leafs is taken to be dissolved in 30mL ddH2O and 10g (NH4)2SO2, add octanol (0.07 μ g μ l-1) conduct Internal standard is tied with using headspace solid-phase microextraction (HS-SPME) with reference to gas chromatography mass spectrometry (GC-MS) detection architecture after vortex mixing State fragrance is closed to measure.Splitter is DB-Wax capillary chromatographic columns (0.25mm, 30m, 0.25 μm, J＆W Scientific).Temperature It spending program and is warming up to 100 DEG C for 40 DEG C of holdings 2min, 3 DEG C/min, 5 DEG C/min is warming up to 245 DEG C, and injector temperature is 240 DEG C, 250 DEG C of detector temperature, carrier gas are helium, flow velocity 1ml/min, Splitless injecting samples, constant flow rate pattern.
(2) experimental result
Compared with wild tobacco plant, the transgenic tobacco plant reference state linalool of overexpression PpUGT85A2 Linalyl- β-D-glucoside contents dramatically increase (attached drawing 4).
Embodiment 5：Overexpression PpUGT85A2 promotes reference state linalool linalyl- β-D- in Peach fruits Glucoside is accumulated
(1) experimental method
1. vector construction
The structure of carrier and carrier, method for transformation are shown in embodiment 4.By the Agrobacterium coated plate after conversion, 2 are cultivated at 28 DEG C Monoclonal is selected after it and is transferred to the LB culture solutions containing kanamycins (50mg/L) Yu gentamicin (25mg/L), culture is extremely OD600To centrifuge (4000g, 5min) after 0.8-1.0.Isometric penetrating fluid (10mM MES, the 10mM MgCI of precipitation2, 150mM acetosyringones, PH 5.6) resuspension, and in the static induction 2h of room temperature.
2. real-time quantitative PCR (qPCR) analyzes gene expression
With peach PpTEF2 (SEQ:NO.12 it is) reference gene, primer SEQ:NO.13 and SEQ:NO.14, PpUGT85A2 Primer is SEQ:NO.8 and SEQ:NO.9.QPCR reaction systems are shown in embodiment 1.
3. Peach fruits infect
The Peach fruits in veraison are chosen, after cleaning and sterilizing, epidermis is removed in superclean bench, chooses middle part pulp It is cut into the slice that thickness is about 1cm.Each fruit chooses the equatorial plane of non-suture line position and its face of contraposition, cuts 2, and Slice is placed in preculture 24-30h on MS solid mediums.The penetrating fluid of resuspension pours into vacuum infiltration after 2h is induced In device, and the sarcocarp section on culture medium is put into, carries out vacuumizing infiltration, vacuum pressure is -70Kpa.Work as slice surface After no longer there is bubble, start slowly to deflate and restore pressure, process takes around 15-20min.Fruit slice nothing after infiltration Bacterium water sterilization 2 times is placed into after drying on new MS culture mediums, is cultivated 2-3 days.Wherein, the every pulp cut, half are used to ooze Bacterium solution thoroughly containing recombinant vector PpUGT85A2-pGreen0029 62-SK, half are used for permeating containing only sky as negative control The bacterium solution of load.With liquid nitrogen cryopreservation in -80 DEG C of refrigerators after sample collection.The measure of bound state aroma substance is the same as the fruit in embodiment 1 Real bound state aroma substance-measuring method.
(2) experimental result
The overexpression PpUGT85A2 in Peach fruits significantly promotes reference state linalool linalyl- β-D- The increase (attached drawing 5) of glucoside contents.
Sequence table
<110>Zhejiang University
<120>One participates in gene and its application that Peach fruits reference state linalool is formed
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1452
<212> DNA
<213>Flowering peach (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>Flowering peach (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 (5)

1. one participates in the gene PpUGT85A2 that Peach fruits reference state linalool is formed, which is characterized in that its nucleotide sequence is such as SEQ：Shown in NO.1.
2. one according to claim 1 participates in the gene PpUGT85A2 that Peach fruits reference state linalool is formed, feature It is, the primer that clone obtains this overall length PpUGT85A2 sequences is SEQ:NO.2 and SEQ:NO.3 carries out gene expression analysis Primer be SEQ:NO.6 and SEQ:NO.7.
3. the one described in claim 1 gene PpUGT85A2 for participating in the formation of Peach fruits reference state linalool is in genetic engineering In application.
4. application according to claim 3, which is characterized in that the application is using SEQ:NO.10 and SEQ:NO.11 sequences Row structure transgene carrier, overexpression PpUGT85A2 dramatically increases reference state linalool content in Peach fruits and tobacco, carries Rise the fragrance quality of food.
5. application according to claim 4, which is characterized in that using SEQ:NO.4 and SEQ:The recombination of NO.5 sequence constructs Albumen PpUGT85A2 catalyzed combination states linalool synthesizes.
CN201810164164.6A 2018-02-27 2018-02-27 Gene participating in formation of peach fruit combined linalool and application thereof Active CN108251438B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810164164.6A CN108251438B (en) 2018-02-27 2018-02-27 Gene participating in formation of peach fruit combined linalool and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810164164.6A CN108251438B (en) 2018-02-27 2018-02-27 Gene participating in formation of peach fruit combined linalool and application thereof

Publications (2)

Publication Number Publication Date
CN108251438A true CN108251438A (en) 2018-07-06
CN108251438B CN108251438B (en) 2020-04-14

Family

ID=62745749

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810164164.6A Active CN108251438B (en) 2018-02-27 2018-02-27 Gene participating in formation of peach fruit combined linalool and application thereof

Country Status (1)

Country Link
CN (1) CN108251438B (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305856A (en) * 2019-06-27 2019-10-08 华中农业大学 A kind of application of cytochrome P 450 enzymes
CN110836942A (en) * 2019-11-29 2020-02-25 浙江省农业科学院 Method for analyzing smelly components in fruit and vegetable products
CN111172176A (en) * 2020-03-12 2020-05-19 浙江大学 Transcription factor PpMADS2 involved in synthesis and regulation of linalool from peach trees and application thereof
CN111217899A (en) * 2020-03-12 2020-06-02 浙江大学 Transcription factor PpbHLH1 involved in synthesis and regulation of linalool from peach trees and application thereof
CN111307973A (en) * 2020-03-09 2020-06-19 西北农林科技大学 Method for releasing combined-state aroma substances of kiwi fruit juice
CN111500606A (en) * 2020-05-06 2020-08-07 浙江大学 Gene participating in biosynthesis of linalool from peach trees and application of gene

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305856A (en) * 2019-06-27 2019-10-08 华中农业大学 A kind of application of cytochrome P 450 enzymes
CN110836942A (en) * 2019-11-29 2020-02-25 浙江省农业科学院 Method for analyzing smelly components in fruit and vegetable products
CN111307973A (en) * 2020-03-09 2020-06-19 西北农林科技大学 Method for releasing combined-state aroma substances of kiwi fruit juice
CN111172176A (en) * 2020-03-12 2020-05-19 浙江大学 Transcription factor PpMADS2 involved in synthesis and regulation of linalool from peach trees and application thereof
CN111217899A (en) * 2020-03-12 2020-06-02 浙江大学 Transcription factor PpbHLH1 involved in synthesis and regulation of linalool from peach trees and application thereof
CN111172176B (en) * 2020-03-12 2021-07-13 浙江大学 Transcription factor PpMADS2 involved in synthesis and regulation of linalool from peach trees and application thereof
CN111500606A (en) * 2020-05-06 2020-08-07 浙江大学 Gene participating in biosynthesis of linalool from peach trees and application of gene

Also Published As

Publication number Publication date
CN108251438B (en) 2020-04-14

Similar Documents

Publication Publication Date Title
CN108251438A (en) One participates in gene and its application that Peach fruits reference state linalool is formed
Kannangara et al. Characterization and expression profile of two UDP‐glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava
Mingossi et al. SacRALF1, a peptide signal from the grass sugarcane (Saccharum spp.), is potentially involved in the regulation of tissue expansion
KR20180051530A (en) Plants with reduced asparagine content
CN105861517B (en) A kind of Radix Notoginseng antibacterial peptide gene PnSN1 and its application
CN112969785A (en) Cellulose synthase-like enzyme and use thereof
EP2235051B1 (en) Glycosyltransferases, polynucleotides encoding these and methods of use
CN114555796A (en) Compositions and methods for modifying plant characteristics without modifying the genome of a plant
CN108138199B (en) Plants containing low copy number of RI gene
EA008669B1 (en) A method of increasing the total or soluble carbohydrate content or sweetness of an endogenous carbohydrate by catalysing the conversion of an endogenous sugar to an alien sugar
CN109913469B (en) Transcription factor PpNAC1 involved in synthesis and regulation of peach ester aromatic substances and application thereof
Manivannan et al. Identification of a sulfatase that detoxifies glucosinolates in the phloem-feeding insect Bemisia tabaci and prefers indolic glucosinolates
CN114032223B (en) Esculin and ash bark glycoside glycosyltransferase protein, and coding gene and application thereof
WO2019219015A1 (en) Rnai target gene that is highly lethal to aphids and use thereof
EP3479685A1 (en) Herbicide resistant transformed plant
KR101730074B1 (en) A flavonol synthase gene and a transgenic plant with the same
CN108823178B (en) Emodin glycosyltransferase protein FtUGT73BE5, and coding gene and application thereof
EP1053336A1 (en) Promoter inductible in plants, sequence incorporating same and resulting product
KR101131770B1 (en) Promoter inducible by drought stress isolated from rice and uses thereof
CN111218462B (en) Gene for coding suberect spatholobus stem chalcone synthetase and application thereof
CN114181956A (en) Wheat stripe rust resistance related metabolite, synthesis related gene and application thereof
CA2320401A1 (en) Nucleic acid comprising the sequence of a promoter inductible by stress and a gene sequence coding for a stilbene synthase
CN107337721B (en) Aphid-resistant gene PPA-7, preparation, encoded protein and application
CN111217899A (en) Transcription factor PpbHLH1 involved in synthesis and regulation of linalool from peach trees and application thereof
CN106794232A (en) The VIP3 polypeptides of modification

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant