CN105399803B

CN105399803B - Tobacco light receptor gene NtPHYB1, its coding albumen and the application in the regulation and control of tobacco leaf polyphenol

Info

Publication number: CN105399803B
Application number: CN201510636307.5A
Authority: CN
Inventors: 赵杰宏; 韩洁; 余婧
Original assignee: Guizhou Institute of Tobacco Science
Current assignee: Guizhou Institute of Tobacco Science
Priority date: 2015-09-30
Filing date: 2015-09-30
Publication date: 2018-11-13
Anticipated expiration: 2035-09-30
Also published as: CN105399803A

Abstract

It grows tobacco light receptor gene the present invention provides oneNtPHYB1, its encode albumen and tobacco leaf polyphenol regulation and control in application, including：It is substituted in the protein that amino acid sequence forms shown in SEQ ID No.1, or the amino acid sequence shown in SEQ ID No.1, lacks and ors add one or several amino acid and with same active protein, and the gene of the above-mentioned albumen of codingNtPHYB1.The gene can be used for metabolic regulation and the improvement of tobacco leaf polyphenols.

Description

Tobacco light receptor gene NtPHYB1, its encode albumen and tobacco leaf polyphenol regulation and control in Using

Technical field

The present invention relates to genetic engineering field, more particularly to a tobacco light receptor gene, its encode albumen and its Application in tobacco leaf polyphenol metabolic regulation.

Background technology

Polyphenol compound is weighing apparatus to cigarette quality, color and luster and flue gas physiological strength etc. important Measure a key factor of tobacco quality.Once it has been proposed that using the ratio of content of phenolic compounds and protein nitrogen content as sentencing The jealous foundation of disconnected tobacco fragrant.

Can influence content of the polyphenols in tobacco it has been known that there is many factors, including tobacco bred and type, it is raw Position, maturity, illumination, temperature, nutrition and modulator approach etc..Gene, cultivation management and the comprehensive of modulation technique make With the component and content for determining polyphenols in various types tobacco.The quantity of polyphenol compound and the difference of component are all It can heredity.The Polyphenols and its derivative species found in tobacco is various, more because of the difference of tobacco type and kind Aldehydes matter content variation range is in 0.4-6.0%.It is reported that the polyphenol content of high kind its offspring of polyphenol content is also high, it is more Its offspring's polyphenol content of the low kind of phenol content is also low.This illustrates that the polyphenol content in tobacco is controlled by gene, between kind The difference of polyphenol level can pass to offspring.It thus can be by breeding method come the suitable tobacco product of selection and breeding polyphenol content Kind, but traditional breeding method is time-consuming and laborious, along with the development of Protocols in Molecular Biology, is dug using genetic engineering means Pick, verification and the critical function gene using regulation and control polyphenol metabolism, it is important by being provided for the molecular regulation of tobacco leaf polyphenols Theory and technology support, to improve cigarette quality.

Invention content

A kind of adjusting tobacco leaf polyphenol content that the purpose of the present invention provides to overcome the above disadvantages, to carry The tobacco NtPHYB1 albumen of high cigarette quality.

It is a further object of the present invention to provide encode the tobacco NtPHYB1 albumenNtPHYB1Gene.

It is also another object of the present invention to provide thisNtPHYB1Gene and its coding albumen NtPHYB1 are more in adjusting tobacco leaf Application in phenol content.

The present invention tobacco NtPHYB1 albumen be：Amino acid sequence forms shown in SEQ ID No.1 protein or It is substituted in the amino acid sequence shown in SEQ ID No.1, lacks and ors add one or several amino acid and with same Active protein.

The tobacco NtPHYB1 albumen of the present inventionNtPHYB1（Full name isNicotiana tabacum Phytochrome B）Gene has nucleotide sequence shown in SEQ ID No.2.

Above-mentioned tobaccoNtPHYB1Gene is from tobacco K326（Nicotiana tabacum cv. K326）Lead in kind Cross the gene that RT-PCR is cloned into.

Above-mentioned tobaccoNtPHYB1Gene, is overexpressed and RNA interference plant expression vectors are respectively PEarleyGate100 and pB7GWIWG2 (I).

Compared with prior art, the present invention there is apparent advantageous effect, as can be known from the above technical solutions：The present invention'sNtPHYB1Similitude between the protein sequence and the amino acid sequence of arabidopsis AtPHYB albumen of gene is 75%, therefore is speculated With arabidopsisPHYBThe function of gene is similar.

It willNtPHYB1It is gene constructed to arrive over-express vector pEarleyGate100 and rna interference vector pB7GWIWG2 (I) On, and expand in bacillus coli DH 5 alpha numerous.By agriculture bacillus mediated leaf dish method for transformation, respectively by pEarleyGate100 and What pB7GWIWG2 (I) was carriedNtPHYB1Gene is transferred in Nicotiana tabacum, obtains Transformation of tobacco plant.The result shows that being overexpressedNtPHYB1With up-regulation phenylalanine lyase（NtPAL）Transcriptional level, to improve the work(of tobacco leaf polyphenol content Energy；Or it is interfered by RNANtPHYB1Polyphenol content in tobacco leaf can be reduced, to realize tobacco leaf polyphenol content Improvement.

Therefore,NtPHYB1Gene and its coding albumen can adjust tobacco leaf polyphenol content, fast and efficiently realize tobacco Quality-improving.

The present invention can also be provided containingNtPHYB1The cloning vector or all kinds of expression vectors of nucleotide sequence or its segment, Host cell containing the carrier is planted containing the conversion plant cell and transgenosis of the nucleotide sequence or its specific fragment Object.

Description of the drawings

Fig. 1 is tobacco light receptor geneNtPHYB1Encode the amino acid sequence and arabidopsis of albumenPHYBGene code egg The comparison of white amino acid sequence.

Fig. 2 is the structural schematic diagram for cloning intermediate carrier pGWCm.

Fig. 3 is the structural schematic diagram of plant over-express vector pEarleyGate100.

Fig. 4 is the structural schematic diagram of plant RNA interference carrier pB7GWIWG2 (I).

Fig. 5 is tobacco light receptor geneNtPHYB1Positioning in transgenic plant cells.

Fig. 6 is tobacco light receptor geneNtPHYB1Influence to tobacco leaf polyphenol content.

Fig. 7 is the expression analysis of Polyphenols key enzyme in transgenic tobacco leaves.

Specific implementation mode

Embodiment 1

（1）Tobacco light receptor geneNtPHYB1Clone

It is utilized respectively shown in SEQ ID No.3NtPHYB1Clone forward primer 5'- Shown in ATGGCTTCTGGAAGTAGAACAAAG-3' and SEQ ID No.4NtPHYB1Clone reverse primer 5'- CTAGCCAAGACTCTTTGAACCTCTG-3' is by the method for RT-PCR from tobacco K326（Nicotiana tabacum cv. K326）Middle amplification gene.

PCR response procedures are:95 DEG C of 5min pre-degenerations, 95 DEG C of 30S, 50 DEG C of 35S, 72 DEG C of 3min30S, 30 are followed Ring, 72 DEG C of 10min extend.

NtPHYB1The acquisition of cloning vector：The PCR product that amplification obtains directly is cloned into according to TA cloning process such as figure It is cloned on intermediate carrier pGWCm shown in 2.It will first be returned with gel reclaims kit after the Ahd I inscribe enzyme hydrolysis of pGWCm carriers Digestion products are received to obtain carrier T.Then PCR product and carrier T carry out connecting for 12 hours in 16 DEG C, connection product are converted big Enterobacteria DH5 α, and expand wherein, screening positive clone, and be sequenced.

It obtainsNtPHYB1Gene, gene order is as shown in SEQ ID No.2；The amino of protein encoded by them Acid sequence is as shown in SEQ ID No.1.

（2）Tobacco light receptor geneNtPHYB1Plant expression vector and genetic transformation

By tobacco light receptor geneNtPHYB1Cloning vector respectively with plant over-express vector as shown in Figure 3 pEarleyGate100（Purchased from Invitrogen）And plant RNA interference carrier pB7GWIWG2 (I) equal proportion as shown in Figure 4 It is reacted by LR after mixing（Two kinds of plasmid each 50ng, LR enzyme 1ml mend ddH₂O to final volume 5ml, 25 DEG C of reactions 6 are small after mixing When more than）, willNtPHYB1Structure is respectively used to be overexpressed in plant on pEarleyGate100 and pB7GWIWG2 (I) Tobacco light receptor gene is interfered with RNANtPHYB1, study its function.The genetic transforming method of tobacco is using agriculture bacillus mediated Leaf disc transformation method carries out, referring to《Molecular Cloning:A Laboratory guide》, 2005.Selection markers in plant are Bar, to obtain Tobacco light receptor geneNtPHYB1Transformed plant.

Embodiment 2

（1）Tobacco light receptor geneNtPHYB1Encode the amino acid sequence analysis of albumen

Tobacco light receptor geneNtPHYB1Protein sequence and arabidopsis PHYB albumen between similitude be 75%, and Conserved functional domains height is similar, as shown in Figure 1.Therefore speculate tobaccoNtPHYB1Gene and arabidopsisPHYBThe function class of gene Seemingly.

（2）Tobacco light receptor geneNtPHYB1Encode the positioning of albumen in transgenic plants

By tobacco light receptor geneNtPHYB1WithGFPFusion, conversion is in protoplasts of Arabidopsis thaliana broken by ultrasonic.Specific side Method is as follows：

1）Protoplast extracts：

The Arabidopsis leaf for growing surrounding or so is cut into thin strips, is put into mannitol solution；Prepare enzymolysis liquid：It will be above-mentioned 55 DEG C of heating 10min of solution, after being placed at room temperature for, are added CaCl₂10mM, beta -mercaptoethanol 5mM, BSA 0.1% (W/V), 0.45 The filtering of μM filter, is protected from light 4 DEG C and saves backup；Arabidopsis leaf slice is pulled out, is put into enzymolysis liquid（1% cellulase R- 10,0.4% macerozyme R-10,0.4M mannitol, 20mM KCl, 20mM MES, pH value 5.7）, 23 DEG C are protected from light, 40~60 rpm enzymes Solve 3h；Metallic screen filters（200 mesh）Into small beaker（It operates on ice）, filtrate is drawn in 10mL plastic centrifuge tubes, 100 × g from Heart 1min, abandons supernatant；Isometric W5 solution that precooling is added into 10mL centrifuge tubes rinses protoplast, gently overturns mixing, 100 × g centrifuges 1min, abandons supernatant, adds 1/2 volume W5 solution of precooling, and flicking makes precipitation suspend, and places on ice 30min；100 × g centrifuges 1min, is put when taking out on ice, abandons supernatant, and the MMg solution that is pre-chilled in right amount is added to rinse protoplast, and 100 × g centrifuges 1min, abandons supernatant, adds the MMg solution suspension protoplasts being pre-chilled in right amount.

2）PEG is converted

Take the Plasmid DNA of 10 μ L fusion expression vectors（10μg）In 1.5mL centrifuge tubes, it is added with the yellow pipette tips for cutting head 100 μ L protoplast extracting solutions are gently inhaled and beat mixing, add the PEG/Ca of 110 μ L²⁺Solution, mixing, 23 DEG C are protected from light warm bath 30min is converted；440 μ L W5 solution are added into centrifuge tube, 23 DEG C, 100 × g centrifuges 1min, supernatant is removed, to precipitation 100 μ L W5 solution of middle addition, mixing, 23 DEG C, 100 × g centrifuges 1min, and suction abandons supernatant, adds 100 μ L W5 solution, be repeated 1 times, 23 DEG C, 100 × g centrifuges 1min, and supernatant is abandoned in suction, and mixing will be precipitated by adding 1mL W5 solution；Suspension is poured into six orifice plates （First with 5% the abundant rinse of BSA solution）, it is protected from light, lies against 23 DEG C of 12~16h of light culture；100 × g centrifuges 1min, directly inhales Take a certain amount of protoplast pellet in laser confocal microscope（Laser Scanning Confocal Microscope） Lower observation.

GFP fluorescence signals in transformed cells indicate tobacco light receptor geneNtPHYB1Encode the positioning of albumen.Such as Fig. 5 It is shown, show tobacco light receptor geneNtPHYB1The albumen of coding is equal in the cytoplasm and nucleus of protoplasts of Arabidopsis thaliana broken by ultrasonic It is distributed.

Embodiment 3

（1）Tobacco light receptor geneNtPHYB1Influence to tobacco leaf polyphenol content

The Transformation of tobacco plant obtained from embodiment 1 and its parent（Wild type K326 controls）In phytotron（16 Hour illumination/8 hour dark photoperiod, light intensity 80mmolm^-2·s^-1）It cultivates simultaneously.Respectively in the prosperous long-term and early flowering season Take its polyphenol content of measuring blade in the middle part of plant.The results are shown in Figure 6, shows to be overexpressedNtPHYB1Transgenosis cigarette The content of polyphenols in blade of grass piece, neochlorogenic acid, chlorogenic acid and 2,4-o- coffee chinic acids is higher than wild type, andNtPHYB1RNA interference of transgene tobacco leafs in, content compared with wild type reduce.And the sample in two periods is with identical Variation tendency.

（2）The expression analysis of Polyphenols key enzyme in transgenic tobacco leaves

Transgenic tobacco leaves polyphenol is measured using real-time fluorescence quantitative PCR (quantitative real time RT-PCR) Class key enzymeNtPAL4WithNtCHSMRNA expressions.Real-time fluorescence quantitative PCR is carried out using ABI StepOne, is used SYBR Green I detect fluorescence signal, and the primer of Real-time PCR is：

As shown in SEQ ID No.5NtPAL4Real-time PCR sense primers：

5’- TTATTTCCCCTCAACCTACCCA-3’

As shown in SEQ ID No.6NtPAL4Real-time PCR downstream primers：

5’- CATCATCTCCCATCATTTTCAAGT-3’

As shown in SEQ ID No.7NtCHS Real-time PCR sense primers：

5’- CTGGCGGCACTGTTCTCC-3’

As shown in SEQ ID No.8NtCHS Real-time PCR downstream primers：

5’- AGGGCTTGTCCGACCATACTA-3’

Reaction system is：

SYBR Primix Ex Taq（2×）（TaKaRa） 7.5 μl

Sense primer（10μM） 0.3 μl

Downstream primer（10μM） 0.3 μl

ROX Reference Dye（50x） 0.3 μl

cDNA 1.0 μl

ddH₂O（Sterilize distilled water） 5.6 μl

Response parameter is two-step method：95 °C of 10S, thermal starting；95 °C of 5S, 60 °C of 1 min, 40 cycles.With gene core Sheet data analysis software Genesis is standardized and maps to the expression of gene.

The results are shown in Figure 7, shows to be overexpressed tobacco light receptor geneNtPHYB1Transgenic tobacco leaves in, Polyphenols generation Thank to enzyme key geneNtPAL4AndNtCHSExpression up-regulation, and in RNA interference of transgene tobacco leaves, the table of two genes Up to lowering.

Sequence explanation：

SEQ ID No.1 show the amino acid sequence of tobacco NtPHYB1；SEQ ID No.2 show tobacco NtPHYB1 Nucleotide sequence；SEQ ID No.3 and SEQ ID No.4 show NtPHYB1 clone's forward primers and reverse primer；SEQ ID No.5 and SEQ ID No.6 show the sense primer and downstream primer of NtPAL4 Real-time PCR； SEQ ID No.7 and SEQ ID No.8 show the sense primer and downstream primer of NtCHS Real-time PCR.

The above described is only a preferred embodiment of the present invention, being not intended to limit the present invention in any form, appoint What is simply repaiied to any made by above example according to the technical essence of the invention without departing from technical solution of the present invention content Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.

<110>Guizhou Province Tabacco Science and Technology Institute

<120>Tobacco light receptor gene NtPHYB1, its coding albumen and the application in the regulation and control of tobacco leaf polyphenol

<130>

<140>

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<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 1131

<212> PRT

<213>Tobacco K326（Nicotiana tabacum）

<400> 1

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Pro Phe Tyr Ala Ile Leu His Arg Val Asp Val Gly Ile Val Ile Asp

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<212> DNA

<213>Tobacco K326（Nicotiana tabacum）

<400> 2

atggcttctg gaagtagaac aaagcattct catcagtcag gtcaagttca agctcaatct 60

tcaggcacaa gtaatgttaa ttacaaagat tcaataagca aagccatagc acagtacaca 120

gctgatgcta ggcttcatgc tgtgtttgaa caatctggtg agtctggcaa gtcttttgat 180

tattcacagt ctgttaaaac tactacacaa tctgttgtcc ctgaacagca aattactgct 240

tatttgacta aaatccaaag agggggtcac attcagcctt ttggttgtat gatagctgta 300

gatgaggcta gttttcgtgt tattgcttat agcgaaaatg cgtgcgaaat gcttagtcta 360

actccacaat cagttccaag ccttgagcgg cctgagatcc tcactgttgg aactgatgtt 420

aggacccttt ttactccttc tagctctgtt ttgcttgaaa gagcatttgg ggcgcgcgag 480

atcactttgc tgaatcctat ttggatacat tccaagaatt ctggcaagcc attttacgca 540

attttgcata gggttgatgt cgggattgta attgatttgg agcctgctag aacagaggac 600

cctgctttat ccattgctgg cgcagtgcag tcgcaaaaac ttgcagtgag ggctatttct 660

catttgcaat cacttcctgg tggggatgtt aagcttttgt gtgatactgt ggttgagagt 720

gtgagggagt taaccgggta tgatcgggtt atggtatata aatttcatga ggatgagcat 780

ggggaggtag tggctgagag caaaagaccg gatttagagc cctatattgg tttgcattat 840

cctgctaccg acattcctca agcttcacgg tttttgttta agcagaacag ggtaagaatg 900

attgtggact gccatgccag ccctgtgcgg gttgttcagg atgaatcact gatgcagcct 960

ttatgtttag ttggttccac acttagagcc cctcatggtt gccacgcgca gtacatggca 1020

aatatggggt ctattgcgtc attaactcta gcagttatta tcaatggaaa cgatgaggaa 1080

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ctccaattga atatggagtt gcaactggca tcacagttgt ctgagaaaca tgtgttgagg 1260

acacaaacac tgttatgtga catgctcctt cgagactcac ctacggggat tgttacccag 1320

agccccagta ttatggacct tgtgaagtgc gatggcgctg ctctgtactg tcaggggaag 1380

tactatccat taggcgttac accaactgaa gctcagataa aggacattgt ggagtggtta 1440

ttgacttacc atggggactc aacaggttta agtactgaca gtttggctga tgcagggtat 1500

cctggggcag cttcgcttgg tgatgcagtt tgtggtatgg ctgttgctta tataacttct 1560

aaagatttct tgttttggtt tcgctcccat acagcgaaag agataaagtg gggtggtgca 1620

aagcatcatc ctgaagacaa ggatgacggg cagagaatgc atccacgttc ttctttcaag 1680

gcatttctgg aagttgctaa aagccggagc ttaccatggg aaaatgcaga aatggatgca 1740

attcactctc tgcagcttat tctgcgagat tcatttaagg atgccgaggc aagtaattct 1800

aaggctgttg tgcatgctca gcttggggaa atggagttgc aagggataga tgaactgagt 1860

tctgttgcca gagaaatggt tagattgata gagactgcaa ctgctcccat atttgctgtt 1920

gatgtcgaag gtcgcattaa tgggtggaat gcaaaggtcg ctgaattgac agatttatct 1980

gttgaagaag caatggggaa gtccttggtt catgatcttg tgcataaaga gtcacaggag 2040

actgctgaga agcttctctt caatgctctg agaggcgaag aagataaaaa tgtagaaata 2100

aagttaagga catttggacc cgagcaactg aagaaggctg tttttgtggt ggttaatgct 2160

tgctctagca aagattacac aaacaacatt gttggtgttt gttttgttgg ccaggatgtt 2220

actgggcaaa aagttgtaat ggacaagttt attcacatcc aaggtgatta caaggccatt 2280

gtgcacagcc ccaatcctct gatcccaccc atatttgcgt cagatgagaa cacttgttgc 2340

tctgagtgga acactgccat ggaaaagctc actggttggt ccagagggga gatcattgga 2400

aaaatgttag ttggtgagat ttttggaagt tgctgtcggc tcaagggtcc agacgccatg 2460

acaaagttca tgatcgtgtt gcataatgcg attggagtcc aggatacgga caagtttcca 2520

ttttcctttt ttgaccgaaa tgggaaatat gtgcaagctc ttttgactgc gaacaagaga 2580

gtcaatatgg agggccagat tatcggggct ttctgtttca tacagatagc cagtcctgaa 2640

ttgcagcaag ctctaagagt tcaaaggcaa caggaaaaga agtgttattc tcagatgaaa 2700

gagttggcat acctttgtca ggaaataaag agtcctttga atggtatacg ctttacaaat 2760

tcattgttgg aagcgacaga tttgacagaa aaccagaagc agtatctgga gacaagtgct 2820

gcttgtgaga ggcagatgtc taagatcata agggatgttg acctggaaaa cattgaggat 2880

ggctcactga cccttgagaa agaagaattt ttccttggga gtgcaataga tgctgttgtt 2940

agccaagtga tgttattgct gagggaaaga agtgtgcaat taatcaggga tattccagag 3000

gaaattaaga ccttaacagt acatggtgat caagtgagaa ttcaacaggt cttggcagat 3060

ttcttgctta acatggtacg gtatgcacca tcacctgatg gttgggtagg gatccaactt 3120

cagccaaata tgaagcaaat atctgatgaa gtaactgttg tgcatattga attcaggatt 3180

gtatgccctg gtgaagggct tcctcctgaa ttggttcaag acatgttcca cagcagtcgg 3240

tgggtaacca aggaaggcct aggactgagc atgtgcagaa aaatcttaaa gcttatgaat 3300

ggagatatcc agtatatcag agaatcagaa agatgttatt tcctgatcat ccttgaccta 3360

ccaatgaccc gcagaggttc aaagagtctt ggctag 3396

<210> 3

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223> NtPHYB1Clone forward primer

<400> 3

atggcttctggaagtagaacaaag 24

<210> 4

<211> 25

<212> DNA

<213>Artificial sequence

<220>

<223> NtPHYB1Clone reverse primer

<400> 4

ctagccaagactctttgaacctctg 25

<210> 5

<211> 22

<212> DNA

<213>Artificial sequence

<220>

<223> NtPAL4 Real-time PCR sense primers

<400> 5

ttatttcccctcaacctaccca 22

<210> 6

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223> NtPAL4 Real-time PCR downstream primers

<400> 6

catcatctcccatcattttcaagt 24

<210> 7

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223> NtCHS Real-time PCR sense primers

<400> 7

ctggcggcactgttctcc 18

<210> 8

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223> NtCHS Real-time PCR downstream primers

<400> 8

agggcttgtccgaccatacta 21

Claims

1. grow tobacco NtPHYB1 albumen, it is：The protein that amino acid sequence forms shown in SEQ ID No.1.

2. a kind of tobacco NtPHYB1 genes of coding albumen described in claim 1.

3. tobacco NtPHYB1 genes according to claim 2, it is characterised in that：With nucleosides shown in SEQ ID No.2 Acid sequence.

4. the method for obtaining conversion plant cell or genetically modified plants using gene described in Claims 2 or 33.

5. application of the tobacco NtPHYB1 genes as claimed in claim 2 or claim 3 in adjusting the metabolism of tobacco leaf polyphenols.

6. application according to claim 5, it is characterised in that：The overexpression NtPHYB1 genes in tobacco body, or reduce The expression of NtPHYB1 genes, to change the polyphenol content in tobacco leaf.