CN106381301A - Application of tobacco FLS gene to regulate and control metabolism of plant flavonoid - Google Patents
Application of tobacco FLS gene to regulate and control metabolism of plant flavonoid Download PDFInfo
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Abstract
The invention discloses an application of a tobacco FLS gene to regulate and control metabolism of plant flavonoid, and application of an NtFLS1 synthetase gene and an NtFLS2 synthetase gene, whose nucleotide sequences are shown in sequence tables of SEQ ID NO:1 and SEQ ID NO:2, to regulate and control metabolism of plant flavonoid. When the function of the tobacco FLS gene is studied at an early stage, a strain, the FLS gene expression level of which is obviously inhibited, is found in a tobacco plant for transferring an FLS gene overexpression vector. In the invention, the phenomenon that metabolism of flavonoid is affected due to inhibition of expression of the tobacco FLS gene, and the foundation is laid for further studying metabolic pathways of tobacco flavonoid.
Description
Technical field
The invention belongs to gene engineering field is and in particular to one grows tobacco FLS gene in regulation and control plant flavonoids generation
Application in thanking.
Background technology
Flavonoid is the widely distributed secondary metabolite of plant kingdom, is synthesized by phenylpropyl alcohol alkane approach.Its synthesis with
Coumaric acyl CoA and malonyl CoA is precursor, in chalcone synthase（Chalcone synthase, CHS）In the presence of formed
The chalcone of yellow, this step is first rate-limiting step of flavonoid route of synthesis.Then by enzyme, namely chalcone isomerase
（Chalconeisomerase, CHI）Catalysis chalcone generates colourless naringenin, and naringenin is through flavonol -3- hydroxylase
（Flavanone 3- hydroxylase, F3H）Catalysis, generates dihydrokaempferol class（DHK）, DHK subsequently respectively formed dihydro
Quercetin（DHQ）And dihydromyricetin（DHM）.Above three flavanone alcohols compound can be in flavonol synzyme
（Flavonol synthase, FLS）Effect is lower to enter flavonol route of synthesis, produces kaempferol, Quercetin and ampelopsin；?
Can be by flavanonol reductase（DFR）Catalysis enter anthocyanidin route of synthesis, finally produce delphinidin (Delphindin),
Cyanidin (Cyanidin) and pelargonidin (Pelargonidin).Because the substrate of FLS with DFR catalytic reaction is identical, therefore
There is competitive relation.
FLS belongs to 2- ketoglutaric acid dependency dioxygenase 2-oxoglutarate-dependentdioxygenase,
2-ODD) family member, has cloned it at present in the plants such as petunia, Rhizoma Solani tuber osi, arabidopsiss, Lisianthus, Semen sojae atricolor and has encoded base
Cause.Find that FLS gene family has 5 and 6 homologous geness respectively in Fructus Vitis viniferae and arabidopsis gene group, function has differentiation.As
Arabidopsiss FLS1（AtFLS1）Flavanonol can not only be catalyzed and change into flavonol moreover it is possible to be catalyzed Fructus Citri grandiss with F3H collective effect
Pi Su oxidation generates dihydrokaempferol.Research shows to can result in using Antisense RNA Technique suppression FLS gene expression in petunia
Anthocyanidin synthesis strengthens and produces pink flowers.In pale reddish brown Lisianthus, suppression FLS gene expression leads to pattern to redden, and greatly
Amount accumulation flavanonol, flavone alcohol content is less than test limit.
Content of the invention
It is an object of the invention to provide one grow tobacco FLS gene regulation and control plant flavonoids metabolism in application
The object of the present invention is achieved like this, nucleotide sequence such as sequence table SEQ ID NO：1 and SEQ ID NO：Shown in 2
The application in regulation and control plant flavonoids metabolism of NtFLS1 synthase gene and NtFLS2 synthase gene.
During early-stage Study Nicotiana tabacum L. FLS gene function of the present invention, send out in the tobacco plant of conversion FLS gene overexpression carrier
The strain that an existing FLS gene expression dose is significantly inhibited.Present invention demonstrates that suppression FLS gene expression to flavonoid path
Other structures gene such as NtCHS, NtCHI, the expression of NtF3H, NtANS no affects.Suppression FLS gene expression makes Nicotiana tabacum L. two
Hydrogen quercetin content improves 12 times.Suppression FLS gene expression does not result in Quercetin, kaempferol, dihydrokaempferol content become
Change.Suppression FLS gene expression can remarkably promote metabolism and flow to anthocyanidin route of synthesis, so that the middle anthocyanidin content of Nicotiana tabacum L. is improved
2.4 again.
Brief description
Fig. 1 is NtFLS1 and NtFLS2 Gene Expression Profile Analysis schematic diagram of the present invention；
Fig. 2 is flavonoid path expression of structural gene horizontal analysiss schematic diagram；
Fig. 3 is flavonoid substances content schematic diagram in transgene tobacco；
Fig. 4 is transgene tobacco floral organ phenotype and Study on Content Analysis of Anthocyanidin schematic diagram.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but never in any form to the present invention in addition
Limit, based on present invention teach that any conversion of being made or replacement, belong to protection scope of the present invention.
Application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene of the present invention is nucleotide sequence such as sequence
Table SEQ ID NO：1 and SEQ ID NO：NtFLS1 synthase gene shown in 2 and NtFLS2 synthase gene are in regulation and control plant
Application in flavonoid metabolism.
Application in regulation and control plant flavonoids metabolism for the described Nicotiana tabacum L. FLS gene is it is characterised in that described plant is
Nicotiana tabacum L..
The aminoacid sequence such as SEQ ID of NtFLS1 synthase gene coding：Shown in No.3；NtFLS2 synthase gene is compiled
The aminoacid sequence such as SEQ ID of code：Shown in No.4.
The cloning process of described NtFLS1 synthase gene comprises the following steps：
The total serum IgE of A, extraction tobacco leaf and flower, reverse transcription obtains cDNA first chain；
B, pcr amplification reaction is carried out as template using cDNA first chain obtain genes of interest NtFLS1；
C, genes of interest NtFLS1 fragment is connected to pTOPO carrier it is sequenced.
In step B, the primer of pcr amplification reaction is：
NtFLS1_BamHI：GGATCCATGAAAACCCTAACAATGG
NtFLS1_XhoI：CTCGAGTCACTGAGGAAGCTTGTTAAG.
In step B, the condition of pcr amplification reaction is：98 DEG C of denaturations 30s；98 DEG C of degeneration 10s, 55 DEG C of annealing 30s, 72 DEG C
Extend 2min, totally 30 circulations；Last 72 DEG C of extension 7min.
The cloning process of described NtFLS2 synthase gene comprises the following steps：
The total serum IgE of A, extraction tobacco leaf and flower, reverse transcription obtains cDNA first chain；
B, pcr amplification reaction is carried out as template using cDNA first chain obtain genes of interest NtFLS2；
C, genes of interest NtFLS2 fragment is connected to pTOPO carrier it is sequenced.
In step B, the primer of pcr amplification reaction is：
NtFLS2_BamHI:GGATCCATGAAAACAGCTGAAGCTCA；
NtFLS2_XhoI: CTCGAGTCACTGAGGAAGCTTGTTAA.
In step B, the condition of pcr amplification reaction is：98 DEG C of denaturations 30s；98 DEG C of degeneration 10s, 56 DEG C of annealing 30s, 72 DEG C
Extend 2min, totally 30 circulations；Last 72 DEG C of extension 7min.
Application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene of the present invention is nucleotide sequence such as sequence
Table SEQ ID NO：1 and SEQ ID NO：NtFLS1 synthase gene shown in 2 and NtFLS2 synthase gene regulation and control plant
The method of Flavone metabolism comprises the steps：
A, structure RNAi carrier；
B, Agrobacterium-mediated Transformation：
C, RNAi carrier import Nicotiana tabacum L.；
D, culture transfer-gen plant.
To be embodied as case, the present invention will be further described below：
Embodiment 1
1st, materials and methods
1.1 vegetable material
Nicotiana tabacum（Nicotiana tabacumYunyan87）Blade, flower etc. organize liquid nitrogen flash freezer, and -80 DEG C preserve for dividing
Analysis target gene tissue expression characteristic and gene cloning.Transgenic tobacco leaf liquid nitrogen flash freezer is used for real time fluorescent quantitative and detects phase
Correlation gene expression, transgene tobacco Mature Leaf is used for flavonoid substances content detection.
1.2 method
1.2.1NtFLSGene overexpression vector construction and Nicotiana tabacum L. genetic transformation
NtFLS1Gene overexpression vector construction and Nicotiana tabacum L. genetic transformation
Using TRIzol（Invitrogen）Extract tobacco leaf and the total serum IgE of flower, reverse transcription obtains cDNA first chain
（PrimeScriptTMRT reagent Kit with gDNA Eraser, Takara）As template amplification genes of interestNtFLS1, primer sequence is NtFLS1_BamHI：GGATCCATGAAAACCCTAACAATGG, NtFLS1_XhoI:
CTCGAGTCACTGAGGAAGCTTGTTAAG.PCR adopts high-fidelity DNA polymerase（Phusion High-Fidelity DNA
Polymerase, NEB）, PCR reaction condition is 98 DEG C of denaturations 30s；98 DEG C of degeneration 10s, 55 DEG C of annealing 30s, 72 DEG C of extensions
2min, totally 30 circulations；Last 72 DEG C of extension 7min.
Genes of interest fragment is connected to pTOPO（Invitrogen）Carrier is sequenced, and usesBamHI andXhoI double digestion
pTOPO-NtFLS1And pENTRTM2B, is separately recovered and obtains purpose fragmentNtFLS1With carrier segments pENTRTM2B, using T4
DNA Ligase obtains entry vector pENTR after connectingTM2B-NtFLS1, with pK2GW7（Purpose carrier）Reacted by LR
（Invitrogen）Obtain plant expression vector pK2-NtFLS1, using agrobacterium mediation converted tobacco leaf disc, screening obtains and turns
Genetic tobacco.
NtFLS2Gene overexpression vector construction and Nicotiana tabacum L. genetic transformation
Using TRIzol（Invitrogen）Extract tobacco leaf and the total serum IgE of flower, reverse transcription obtains cDNA first chain
（PrimeScriptTMRT reagent Kit with gDNA Eraser, Takara）As template amplification genes of interestNtFLS2, primer sequence is NtFLS2_BamHI:GGATCCATGAAAACAGCTGAAGCTCA, NtFLS2_XhoI:
CTCGAGTCACTGAGGAAGCTTGTTAA.PCR adopts high-fidelity DNA polymerase（Phusion High-Fidelity DNA
Polymerase, NEB）, PCR reaction condition is 98 DEG C of denaturations 30s；98 DEG C of degeneration 10s, 56 DEG C of annealing 30s, 72 DEG C of extensions
2min, totally 30 circulations；Last 72 DEG C of extension 7min.
Genes of interest fragment is connected to pTOPO（Invitrogen）Carrier is sequenced, and usesBamHI andXhoI double digestion
pTOPO-NtFLS2And pENTRTM2B, is separately recovered and obtains purpose fragmentNtFLS2With carrier segments pENTRTM2B, using T4
DNA Ligase obtains entry vector pENTR after connectingTM2B-NtFLS2, with pK2GW7（Purpose carrier）Reacted by LR
（Invitrogen）Obtain plant expression vector pK2-NtFLS2, using agrobacterium mediation converted tobacco leaf disc, screening obtains and turns
Genetic tobacco.
1.2.2 transgene tobacco gene expression dose is analyzed
Extract transgenic tobacco leaf total serum IgE, reverse transcription obtains cDNA first chain（PrimeScriptTMRT reagent Kit
With gDNA Eraser, Takara）.Using SYBR Green method（SYBR Green l Master Mix, Roche）To mesh
Mark gene and flavonoid path other structures gene carry out fluorescence real-time quantitative PCR（qRT-PCR）Analysis（LightCycler
480, Roche）, quantitative primer is as follows：
The primer that the analysis of table 1 quantitative fluorescent PCR uses
1.2.3 flavonoid substances content analysis
Grind into powder in the blade liquid nitrogen of transgenic and the fixing leaf position of comparison Nicotiana tabacum L. period of maturation collection, after lyophilization, weighs
20 mg dry samples add the methanol trituration of 3 ml 75%, ultrasonic Treatment 30 min, and 10000 rpm are centrifuged 10 min, take on 1ml
Transfer to clearly 10 ml pipes, then, plus 1 ml water and 2 ml chloroforms remove chlorophyll.Rotation concussion mixture 1 min, 10 000
Rpm is centrifuged 10 min, collects upper solution and is used for analyzing.
LC-MS is utilized to measure Flavonoid Content referring generally to the method that Li etc. delivers.Using Waters ACQUITY UPLC
system（Waters）Flavonoid in conjunction with AB Sciex Triple Quad 5500 spectrometer analysis tobacco leaf and in spending contains
Amount, chromatographic column use Waters BEH C18 column (150 × 2.1 mm i.d., 1.7 um particle sizes,
Waters Corporation), column temperature is set to 30 DEG C, and sample size is 1 μ l, and solvent orange 2 A and B are water and acetonitrile respectively, first
The ammonium acetate of sour (0.1%, v/v) and 0.2 mmol/L is separately added into solvent orange 2 A and B to promote chromatographic isolation, and eluent gradient sets
For 10% solvent B 1min, 10-90% solvent B 8min, 90-100% solvent B 2min.Total run time is 13min, wherein wraps
Include 2min equilibration time.Liquid chromatography eluant directly introduces ESI interface with flow velocity 0.2 ml/min, in conjunction with positive and negative ionization pattern
Analysis flavonoid.The impact energy of 20,30,40, and 50 V is used for MS2 and crushes.Gas curtain 40 psi；Collision gas 6 psi；
Ion spray voltage ± 4000 V；700 DEG C of temperature；Ion source gas 1,60 psi；Ion source gas 2,50 psi.In broken reality
Test middle helium as collision gas.
1.2.4 Study on Content Analysis of Anthocyanidin
The method that method Primary Reference Mehrtens etc. delivers, slightly modified.Collection flower grinds after being placed in 60 DEG C of dryings 48 hours
Clay into power, weigh 0.2 gram of powder and add 1 ml acid methanol（Containing 1%HCL, v/v）In shaken at room temperature 18 hours, 16800g was centrifuged
After 15 minutes, transferase 45 00 l supernatant, in centrifuge tube, adds 500 l ultra-pure waters and 300 l chloroforms to remove chlorophyll.
After 8600g is centrifuged 5 minutes, shift supernatant（Water-methanol phase）To new centrifuge tube, with spectrophotometric determination 530 nm and 657
Light absorption value at nm.Calculate anthocyanidin content using following equation：Anthocyanidin content=（A530-0.25*A657）/ M, wherein M be for
Extract the sample quality of anthocyanidin（Gram）.
2nd, result and analysis
2.1 Nicotiana tabacum L.FLSGene Expression Profile Analysis
Being searched in NCBI in discovery tobacco gene group by Blast has twoFLSGene, respectivelyFLS1（Accession number：
DQ435530）WithFLS（Accession number：AB289451）, for avoiding and other plantsFLSGene is obscured, and names respectively in this research
ForNtFLS1WithNtFLS2.The expression pattern of two genes is similar, expression highest in blade, secondly has certain in spending
Horizontal expression,NtFLS1WithNtFLS2Gene be to spend respectively in the expression in blade in 3.3 and 7.0 times (Fig. 1).Additionally,
The two all has low expression level in stem, but expression is extremely low in root.
2.2 transfer-gen plant flavonoid expression of structural gene horizontal analysiss
According to expression pattern analysis result, reverse transcription after the total serum IgE mixing of tobacco leaf and flower is obtained cDNA template,
Amplification obtainsNtFLS2Gene.Using the over-express vector of Gateway this gene of technique construction, the expression of wherein target gene is subject to
Drive to CaMV 35S promoter composing type.T0 shows most of plant for the qRT-PCR analysis result of transfer-gen plantNtFLS2Gene expression dose significantly improves compared with the control, but finds the target gene expression of strain KD36 and to photograph
Ratio is remarkably decreased.Collect T1 and carry out screening for seed and in resistance culture base and obtain resistance seedling, transplant to greenhouse plantation.
Period of maturation collection KD36 and empty vector control（VC）The blade of each 10-15 strain carries out gene expression dose analysis.In KD36 strainNtFLS2Gene expression dose declines about 62% compared with the control, and illustrating that T0 declines for plant target gene expression can be by
Stable heredity（Fig. 2）.Additionally,NtFLS1Gene expression dose decline about 88%, this because ofNtFLS1WithNtFLS2In core
The flat concordance of sour water is higher, reaches 98.5%（Fig. 2）.To the other structures gene of transfer-gen plant flavonoid route of synthesis
Expression carries out qRT-PCR analysis, and result showsNtCHS、NtCHI,NtF3H,NtANSDeng gene expression dose and to photograph
Than no significant changes（Fig. 2）.
2.3 transfer-gen plant metabolic analysis
FLS is catalyzed the reaction that flavanonol generates flavonol, and in Nicotiana tabacum L., flavanone alcohols compound mainly includes dihydro mountain
How phenol and dihydroquercetin, flavonoid drugs are mainly kaempferol and Quercetin.Metabolic analysis result shows, KD36 strain
Middle dihydrokaempferol, kaempferol, quercetin content and empty vector control strain no significant difference, dihydroquercetin content reaches 6.26
μ g/g, is to compare about 12 times（Fig. 3）.Additionally, KD36 strain pattern is redder compared with the control, anthocyanidin testing result show KD36
In strain, anthocyanidin content significantly improves, and is 2.4 times of comparison, and this shows to regulate and controlFLSGene expression has larger to anthocyanidin synthesis
Impact（Fig. 4）.
3rd, conclusion
In plant genetic engineering research, when the overexpression gene importing is consistent with endogenous gene obtain homology very high when, part turn
In gene plant, the expression of exogenous gene and endogenous gene is suppressed, i.e. co-suppression phenomenon.The frequency that co-suppression phenomenon occurs
Related to the promoter intensity being used, all have been reported that in many transgenic researches.Oryza sativa L. UDPG pyrophosphatase gene
（Ugp1）In the middle part of over-express vector transformed plant, co-suppression phenomenon in plant division system, and endogenous Ugp1 gene expression is totally constrained,
Lead to plant temperature sensitive male sterility.TurnNtFLS2The expression of most of strain target gene in gene overexpression carrier plant
Level is significantly increased than comparison（Data is not delivered）, but also obtain oneNtFLS2The strain that gene expression dose is suppressed
System, this plantNtFLS1The expression of gene is also suppressed, and this should be the result of co-suppression phenomenon.We have discovered that
This phenomenon can be from the stable heredity of T0 generation to T1 for plant.
QRT-PCR analysis result showsNtFLS1WithNtFLS2The expression pattern of gene is consistent, expresses in leaf scape root
Level reduces successively, illustrates that the two there may be functional redundancy, and flavonol synthase Main Function site is in leaf tissue.This two
After individual gene expression dose is suppressed, dihydroquercetin content improves to about 12 times of comparison, and this explanation flavonol synzyme is urged
The reaction that the dihydroquercetin changed changes into Quercetin is suppressed, and leads to substrate content to rise；But product Quercetin contains
There are not significant changes in amount, suppression FLS gene expression in the research with Mahajan etc. leads to Nicotiana tabacum L. Quercetin to contain compared with the control
The result that amount declines 25-93% is inconsistent, and this may be relevant with factors such as Reducing sugar and cultivation steps, concrete reason
Need to be analyzed further.Dihydrokaempferol, kaempferol content and empty vector control strain no significant difference, this explanation in KD36 strain
Flavonol synthase activity is suppressed the metabolic fluxes not affecting Rhizoma Kaempferiae phenol metabolism branch approach, and reason may have at 2 points：One is
In tobacco gene group exceptNtFLS1WithNtFLS2Also there is other gene code flavonol synzyme, single-minded catalysis outside gene
Dihydrokaempferol is converted into kaempferol, and NtFLS1 and NtFLS2 is only responsible for the conversion of dihydroquercetin；Two is that kaempferol divides
The approach of propping up metaboilic level in the tobacco-containing material that Nicotiana tabacum L. or this institute use is relatively low and unrelated with flavonol synthase activity,
Reaction substrate content is relatively low to be its restriction factor.Additionally, in addition to target gene expression is remarkably decreased, flavonoid way
The expression of remaining structural gene of footpath compared with the control not occur significant changes, this with except dihydroquercetin content notable
Rise outer, the no significant changes phase confirmation of remaining metabolic intermediate content, illustrate that flavonoid metabolism network regulatory mechanism is complicated.
Desk study of the present invention Nicotiana tabacum L. flavonol synthase gene function, result shows to suppress this gene expression except energy
Enough significantly improve outside dihydroquercetin content, less on the approach impact of associated class Flavone metabolism branch, but metabolism can be remarkably promoted
Flow to anthocyanidin route of synthesis, promote the understanding to tobacco Flavone metabolism network.
SEQUENCE LISTING
<110>Yunnan Academy of Tobacco Agricultural Science
<120>One grow tobacco FLS gene regulation and control plant flavonoids metabolism in application
<130> 2016
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 1016
<212> DNA
<213>NtFLS1 nucleotide
<400> 1
atgaaaaccc taacaatgga agttgcaaga gtacaagcaa tagcgtcaat aacaaaatgc 60
atggacacaa ttccatcaga atacatacgg tcagagaacg agcagcccgc atccacaacg 120
ttgcatggtg tggttcttca agttccagta atcgacatag acgatacaaa tgtagtgaaa 180
ctcatatcgg atgctagcaa agagtggggg atctttcaag tgataaatca tggaattcca 240
gatgaggtta ttgcgaattt gcaaaaagta ggaaaagagt tctttgaggt tgtaccacaa 300
gaggagaaag aagtgattgc aaagactcca gggtcgcaga atattgaagg gtatggtact 360
tctttgcaga aggaacttga agggaaaaga ggttgggttg atcatttgtt ccataagatt 420
tggcctcctt ctgccatcaa ttatcgttat tggcctaaaa gccctccttc ctacagggaa 480
gcaaatgagg agtacgcaaa gaggctgcga gaagttgcgg agaagatctt taagagctta 540
tcacttgggc ttgggttaga agcccatgaa atgatggagg cagcaggtgg tgaagaaata 600
gtttacttgt tgaagatcaa ttactaccca ccatgcccaa ggcccgattt ggcacttgga 660
gttgtggccc atacagacat gtcccatata accattcttg tcccaaatga agtccaaggc 720
ctccaagtgt tcaaggatga ccattggtat gatgtcaagt acataccaaa tgccctaatt 780
atccacattg gtgaccaagt tgagattctt agcaatggga aatacaagag tgtgtaccat 840
aggacaacag tgacaaagga caagacaaga atgtcatggc cagttttctt ggagccacca 900
tcagagcatg aagttgggcc aattcctaag ctggttaatg aggccaatcc accaaaattc 960
aagaccaaga agtacaagga ttatgtctat tgtaagctta acaagcttcc tcagtg 1016
<210> 2
<211> 1041
<212> DNA
<213>NtFLS2 nucleotide
<400> 2
atgaaaacag ctgaagctca gagtgcaaca accctaacaa tggaggttgc aagagtacag 60
gcaatagcgt caataacaaa atgcatggac acaataccat cagaatatat taggtcagag 120
aacgagcagc cagcgtccac aacgttgcat ggtgtgctac ttcaagttcc agtaattgac 180
atagacgata aaaatgtagt gaaactcata tcggatgcta gcaaagaatg ggggatattt 240
caagtgataa atcatggaat tccagatgag gtaattgcga atttgcaaaa agtagggaag 300
gaattctttg aggttgtacc acaagaggag aaagaggtga ttgcaaaaac tccagggtct 360
cagaatattg aagggtatgg tacttctttg cagaaagaac ttgaagggaa aaggggttgg 420
gttgatcatt tgttccataa gatttggcct ccttctgcca tcaattatcg ttattggcct 480
aaaaatcctc cctcctacag ggaagcaaat gaggaatacg caaagaggct gcgagaagtt 540
gtggagaaaa tctttaagag cttatcactt gggcttgggt taggagccca tgaaatgatg 600
gaggcagcag gtggtgaaga tattgtttac ttgttgaaaa tcaattatta cccaccatgc 660
ccaaggcctg atttggcact tggtgttgtg gcccatacag acatgtccca tataaccatt 720
cttgtcccaa atgaagtcca aggcctccaa gtgttcaagg atggccattg gtatgatgtc 780
aagtacatac ctaatgccct aattgtccac attggtgacc aagttgagat tcttagcaat 840
gggaaataca agagtgtgta ccataggaca acagtgacaa aggacaagac aagaatgtca 900
tggccagtat tcttggagcc accatcagag catgaagtag ggccaatttc taagctggtt 960
aatgaggcca atccacccaa attcaagacc aagaagtaca aggattatgt ttattgtaag 1020
cttaacaagc ttcctcagtg a 1041
<210> 3
<211> 338
<212> PRT
<213>NtFLS1 aminoacid
<400> 3
Met Lys Thr Leu Thr Met Glu Val Ala Arg Val Gln Ala Ile Ala Ser
1 5 10 15
Ile Thr Lys Cys Met Asp Thr Ile Pro Ser Glu Tyr Ile Arg Ser Glu
20 25 30
Asn Glu Gln Pro Ala Ser Thr Thr Leu His Gly Val Val Leu Gln Val
35 40 45
Pro Val Ile Asp Ile Asp Asp Thr Asn Val Val Lys Leu Ile Ser Asp
50 55 60
Ala Ser Lys Glu Trp Gly Ile Phe Gln Val Ile Asn His Gly Ile Pro
65 70 75 80
Asp Glu Val Ile Ala Asn Leu Gln Lys Val Gly Lys Glu Phe Phe Glu
85 90 95
Val Val Pro Gln Glu Glu Lys Glu Val Ile Ala Lys Thr Pro Gly Ser
100 105 110
Gln Asn Ile Glu Gly Tyr Gly Thr Ser Leu Gln Lys Glu Leu Glu Gly
115 120 125
Lys Arg Gly Trp Val Asp His Leu Phe His Lys Ile Trp Pro Pro Ser
130 135 140
Ala Ile Asn Tyr Arg Tyr Trp Pro Lys Ser Pro Pro Ser Tyr Arg Glu
145 150 155 160
Ala Asn Glu Glu Tyr Ala Lys Arg Leu Arg Glu Val Ala Glu Lys Ile
165 170 175
Phe Lys Ser Leu Ser Leu Gly Leu Gly Leu Glu Ala His Glu Met Met
180 185 190
Glu Ala Ala Gly Gly Glu Glu Ile Val Tyr Leu Leu Lys Ile Asn Tyr
195 200 205
Tyr Pro Pro Cys Pro Arg Pro Asp Leu Ala Leu Gly Val Val Ala His
210 215 220
Thr Asp Met Ser His Ile Thr Ile Leu Val Pro Asn Glu Val Gln Gly
225 230 235 240
Leu Gln Val Phe Lys Asp Asp His Trp Tyr Asp Val Lys Tyr Ile Pro
245 250 255
Asn Ala Leu Ile Ile His Ile Gly Asp Gln Val Glu Ile Leu Ser Asn
260 265 270
Gly Lys Tyr Lys Ser Val Tyr His Arg Thr Thr Val Thr Lys Asp Lys
275 280 285
Thr Arg Met Ser Trp Pro Val Phe Leu Glu Pro Pro Ser Glu His Glu
290 295 300
Val Gly Pro Ile Pro Lys Leu Val Asn Glu Ala Asn Pro Pro Lys Phe
305 310 315 320
Lys Thr Lys Lys Tyr Lys Asp Tyr Val Tyr Cys Lys Leu Asn Lys Leu
325 330 335
Pro Gln
<210> 4
<211> 346
<212> PRT
<213>NtFLS2 aminoacid
<400> 4
Met Lys Thr Ala Glu Ala Gln Ser Ala Thr Thr Leu Thr Met Glu Val
1 5 10 15
Ala Arg Val Gln Ala Ile Ala Ser Ile Thr Lys Cys Met Asp Thr Ile
20 25 30
Pro Ser Glu Tyr Ile Arg Ser Glu Asn Glu Gln Pro Ala Ser Thr Thr
35 40 45
Leu His Gly Val Leu Leu Gln Val Pro Val Ile Asp Ile Asp Asp Lys
50 55 60
Asn Val Val Lys Leu Ile Ser Asp Ala Ser Lys Glu Trp Gly Ile Phe
65 70 75 80
Gln Val Ile Asn His Gly Ile Pro Asp Glu Val Ile Ala Asn Leu Gln
85 90 95
Lys Val Gly Lys Glu Phe Phe Glu Val Val Pro Gln Glu Glu Lys Glu
100 105 110
Val Ile Ala Lys Thr Pro Gly Ser Gln Asn Ile Glu Gly Tyr Gly Thr
115 120 125
Ser Leu Gln Lys Glu Leu Glu Gly Lys Arg Gly Trp Val Asp His Leu
130 135 140
Phe His Lys Ile Trp Pro Pro Ser Ala Ile Asn Tyr Arg Tyr Trp Pro
145 150 155 160
Lys Asn Pro Pro Ser Tyr Arg Glu Ala Asn Glu Glu Tyr Ala Lys Arg
165 170 175
Leu Arg Glu Val Val Glu Lys Ile Phe Lys Ser Leu Ser Leu Gly Leu
180 185 190
Gly Leu Gly Ala His Glu Met Met Glu Ala Ala Gly Gly Glu Asp Ile
195 200 205
Val Tyr Leu Leu Lys Ile Asn Tyr Tyr Pro Pro Cys Pro Arg Pro Asp
210 215 220
Leu Ala Leu Gly Val Val Ala His Thr Asp Met Ser His Ile Thr Ile
225 230 235 240
Leu Val Pro Asn Glu Val Gln Gly Leu Gln Val Phe Lys Asp Gly His
245 250 255
Trp Tyr Asp Val Lys Tyr Ile Pro Asn Ala Leu Ile Val His Ile Gly
260 265 270
Asp Gln Val Glu Ile Leu Ser Asn Gly Lys Tyr Lys Ser Val Tyr His
275 280 285
Arg Thr Thr Val Thr Lys Asp Lys Thr Arg Met Ser Trp Pro Val Phe
290 295 300
Leu Glu Pro Pro Ser Glu His Glu Val Gly Pro Ile Ser Lys Leu Val
305 310 315 320
Asn Glu Ala Asn Pro Pro Lys Phe Lys Thr Lys Lys Tyr Lys Asp Tyr
325 330 335
Val Tyr Cys Lys Leu Asn Lys Leu Pro Gln
340 345
<210> 5
<211> 25
<212> DNA
<213> NtFLS1_BamHI
<400> 5
ggatccatga aaaccctaac aatgg 25
<210> 6
<211> 27
<212> DNA
<213> NtFLS1_XhoI
<400> 6
ctcgagtcac tgaggaagct tgttaag 27
<210> 7
<211> 26
<212> DNA
<213> NtFLS2_BamHI
<400> 7
ggatccatga aaacagctga agctca 26
<210> 8
<211> 26
<212> DNA
<213> NtFLS2_XhoI
<400> 8
ctcgagtcac tgaggaagct tgttaa 26
<210> 9
<211> 19
<212> DNA
<213> NtCHS-F
<400> 9
ggctccgtcc atcggtcaa 19
<210> 10
<211> 18
<212> DNA
<213> NtCHS-R
<400> 10
tcattgggtc cacgaaac 18
<210> 11
<211> 18
<212> DNA
<213> NtCHI-F
<400> 11
tctcgccgct aaatggaa 18
<210> 12
<211> 18
<212> DNA
<213> NtCHI-R
<400> 12
tacccgtcaa aggcaaga 18
<210> 13
<211> 18
<212> DNA
<213> NtF3H-F
<400> 13
atgcccaact tatctcac 18
<210> 14
<211> 18
<212> DNA
<213> NtF3H-R
<400> 14
ccacttcacc ctgtagat 18
<210> 15
<211> 18
<212> DNA
<213> NtFLS1-F
<400> 15
aagactccag ggtcgcag 18
<210> 16
<211> 18
<212> DNA
<213> NtFLS1-R
<400> 16
ctgtaggaag gagggctt 18
<210> 17
<211> 18
<212> DNA
<213> NtFLS2-F
<400> 17
aaaactccag ggtctcag 18
<210> 18
<211> 18
<212> DNA
<213> NtFLS2-R
<400> 18
ctgtaggagg gaggattt 18
<210> 19
<211> 20
<212> DNA
<213> NtANS1-F
<400> 19
gctatcccta aagagtatgt 20
<210> 20
<211> 18
<212> DNA
<213> NtANS1-R
<400> 20
tctttgtggc atttctcg 18
Claims (10)
1. one grow tobacco application in regulation and control plant flavonoids metabolism for the FLS gene it is characterised in that nucleotide sequence such as sequence
Table SEQ ID NO：1 and SEQ ID NO：NtFLS1 synthase gene shown in 2 and NtFLS2 synthase gene are in regulation and control plant
Application in flavonoid metabolism.
2. application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene according to claim 1 is it is characterised in that institute
The plant stated is Nicotiana tabacum L..
3. Nicotiana tabacum L. FLS gene according to claim 1 regulation and control plant flavonoids metabolism in application it is characterised in that
The aminoacid sequence such as SEQ ID of NtFLS1 synthase gene coding：Shown in No.3；The amino of NtFLS2 synthase gene coding
Acid sequence such as SEQ ID：Shown in No.4.
4. application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene according to claim 1 or 3, its feature exists
Cloning process in described NtFLS1 synthase gene comprises the following steps：
The total serum IgE of A, extraction tobacco leaf and flower, reverse transcription obtains cDNA first chain；
B, pcr amplification reaction is carried out as template using cDNA first chain obtain genes of interest NtFLS1；
C, genes of interest NtFLS1 fragment is connected to pTOPO carrier it is sequenced.
5. application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene according to claim 4 is it is characterised in that B
In step, the primer of pcr amplification reaction is：
NtFLS1_BamHI：GGATCCATGAAAACCCTAACAATGG
NtFLS1_XhoI：CTCGAGTCACTGAGGAAGCTTGTTAAG.
6. application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene according to claim 4 is it is characterised in that B
In step, the condition of pcr amplification reaction is：98 DEG C of denaturations 30s；98 DEG C of degeneration 10s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min,
Totally 30 circulations；Last 72 DEG C of extension 7min.
7. application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene according to claim 1 or 3, its feature exists
Cloning process in described NtFLS2 synthase gene comprises the following steps：
The total serum IgE of A, extraction tobacco leaf and flower, reverse transcription obtains cDNA first chain；
B, pcr amplification reaction is carried out as template using cDNA first chain obtain genes of interest NtFLS2；
C, genes of interest NtFLS2 fragment is connected to pTOPO carrier it is sequenced.
8. application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene according to claim 7 is it is characterised in that B
In step, the primer of pcr amplification reaction is：
NtFLS2_BamHI:GGATCCATGAAAACAGCTGAAGCTCA；
NtFLS2_XhoI: CTCGAGTCACTGAGGAAGCTTGTTAA.
9. application in regulation and control plant flavonoids metabolism for the Nicotiana tabacum L. FLS gene according to claim 7 is it is characterised in that B
In step, the condition of pcr amplification reaction is：98 DEG C of denaturations 30s；98 DEG C of degeneration 10s, 56 DEG C of annealing 30s, 72 DEG C of extension 2min,
Totally 30 circulations；Last 72 DEG C of extension 7min.
10. Nicotiana tabacum L. FLS gene according to claim 1 regulation and control plant flavonoids metabolism in application it is characterised in that
Nucleotide sequence such as sequence table SEQ ID NO：1 and SEQ ID NO：NtFLS1 synthase gene shown in 2 and NtFLS2 synthesis
The method that enzyme gene regulates and controls plant flavonoids metabolism comprises the steps：
A, structure RNAi carrier；
B, Agrobacterium-mediated Transformation：
C, RNAi carrier import Nicotiana tabacum L.；
D, culture transfer-gen plant.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110029117A (en) * | 2019-04-04 | 2019-07-19 | 中国热带农业科学院南亚热带作物研究所 | Flavonol synthase gene SmFLS and its application and flavonols synzyme |
| CN110938615A (en) * | 2018-09-21 | 2020-03-31 | 中国科学院上海生命科学研究院 | Oxalate metabolism related enzyme and application thereof in oxalate degradation |
| CN113430180A (en) * | 2021-07-14 | 2021-09-24 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato flavonol synthetase IbFLS1 and coding gene and application thereof |
| CN113755460A (en) * | 2021-09-10 | 2021-12-07 | 浙江华睿生物技术有限公司 | Flavone reductase for preparing dihydroquercetin |
| CN113826502A (en) * | 2021-09-10 | 2021-12-24 | 中南林业科技大学 | Method for increasing flavonoid content in camellia oleifera seeds |
| CN115197951A (en) * | 2022-05-10 | 2022-10-18 | 浙江农林大学 | Tea tree flavonol synthesis candidate gene CsNAC086 and application thereof |
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| CN110938615A (en) * | 2018-09-21 | 2020-03-31 | 中国科学院上海生命科学研究院 | Oxalate metabolism related enzyme and application thereof in oxalate degradation |
| CN110029117A (en) * | 2019-04-04 | 2019-07-19 | 中国热带农业科学院南亚热带作物研究所 | Flavonol synthase gene SmFLS and its application and flavonols synzyme |
| CN110029117B (en) * | 2019-04-04 | 2022-11-01 | 怀化学院 | Flavonol synthetase gene SmFLS and application thereof, and flavonol synthetase |
| CN113430180A (en) * | 2021-07-14 | 2021-09-24 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato flavonol synthetase IbFLS1 and coding gene and application thereof |
| CN113755460A (en) * | 2021-09-10 | 2021-12-07 | 浙江华睿生物技术有限公司 | Flavone reductase for preparing dihydroquercetin |
| CN113826502A (en) * | 2021-09-10 | 2021-12-24 | 中南林业科技大学 | Method for increasing flavonoid content in camellia oleifera seeds |
| CN113826502B (en) * | 2021-09-10 | 2023-10-20 | 中南林业科技大学 | Method for improving flavonoid content in camellia oleifera seeds |
| CN115197951A (en) * | 2022-05-10 | 2022-10-18 | 浙江农林大学 | Tea tree flavonol synthesis candidate gene CsNAC086 and application thereof |
| CN115197951B (en) * | 2022-05-10 | 2023-06-02 | 浙江农林大学 | Tea tree flavonol synthesis candidate gene CsNAC086 and application thereof |
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