CN107177603A - Tobacco growing element transport protein NtPIN4 and its application - Google Patents

Abstract

The invention belongs to field of plant genetic, and in particular to the patent application of a tobacco growing element transport protein NtPIN4 and its application.The plain transport protein encoding gene of the tobacco growingNtPIN4Gene, including 1953bp, base sequence is as shown in SEQ ID NO.1.The plain transport protein NtPIN4 albumen of the tobacco growing, its amino acid sequence is as shown in SEQ ID NO.2.WillNtPIN4After gene silencing, in gene silencing plant, tobacco side shoot showed increased, the growth population of axillary bud substantially increases, so as to adjust tobacco plant type.In normal tobacco plant,NtPIN4Gene is higher in the middle expression quantity of stem and axillary bud.NtPIN4The mutating strain series of gene knockout, its branch showed increased, axillary bud increases active.By rightNtPIN4The further investigation of gene, has important application value for tobacco growing regulation and control, regulation of plant form.

Description

Tobacco growing element transport protein NtPIN4 and its application

Technical field

The invention belongs to field of plant genetic, and in particular to a tobacco growing element transport protein NtPIN4 and Its patent application applied.

Background technology

Auxin（Auxin, IAA）It is one of most important hormone of plant, is also that earliest discovery can regulate and control plant branching One of hormone, in coordinate plant growth growth course, for example：In cell division, cell elongation, the differentiation of dimension pipe, embryogenesis, root In numerous biological process such as Morphogenesis, play an important role.

In growing process, phenomenon of the branch development with apical dominance, i.e. terminal bud can suppress the generation of lateral bud, go Except terminal bud（Pinch）The generation of lateral bud can then be promoted.All the time, it is believed that it is this suppression lateral bud development the reason for be exactly raw Long element.Its main cause is：Research is found, is removed after terminal bud, the IAA contents in plants stems are decreased obviously, and lateral bud occurs, now Additional IAA can not completely inhibit the generation of lateral bud；In addition, applying to plant in polar auxin transport inhibitor reduction stem Cellulose content is grown, can not also promote the generation of lateral bud.Thus speculate also have other factors to participate in apical dominance in addition to auxin Foundation.

Research thinks that auxin is transported up entering lateral bud to the transport of basal part of stem while have impact on second messenger.Cell Mitogen is considered as the first choice of auxin second messenger.Main cause is：The basic element of cell division has direct rush to axillary bud growth Enter effect, the synthesis and transport that auxin may be by preventing the basic element of cell division in stem and in root suppress to play its Effect.It is another studies have found that, witchweed lactone with auxin in the development of regulation and control branch, build branch developmental regulation in terms of there is weight The interaction wanted, witchweed lactone is newfound can to suppress the hormone of plant branching.

For the synthesis and transhipment of auxin, auxin is mainly in the phyllopodium of aboveground vegetation part, vegetative bud, tender leaf Middle synthesis, then transports specific site of action.The key protein for adjusting Polar Transport of Auxin is a kind of PIN-FORMED （PIN）Albumen, belongs to a class transmembrane protein specific to plant, and the orientation transport of auxin is adjusted on cell membrane.In plant First PIN GFP cloned in arabidopsis, research shows, arabidopsispin-formed1Mutant has different Normal inflorescence, willAtPIN1After gene mutation, the missing of Auxin transport albumen causes the Auxin transport energy from stem into root Power is reduced.There is research to also found that in the phyllopodium newly formed, it is thin that auxin polarity is transported downwards subcuticle by PIN1 Born of the same parents.

It is right in paddy rice, arabidopsis, sorghum, corn and tomato with the completion of each species gene group examining orderPIN Gene has carried out the identification and analysis of full-length genome scope.Tobacco, as a kind of important Model of Scientific Research crop, is also important Industrial crops and crops, branch development and the yield of tobacco and its interior quality of tobacco have very close relationship, still In the prior art, there is not yet on the plain transport protein of tobacco growing and the play-by-play of its branch development.Thus, for The detailed research of Auxin transport albumen in tobacco, with highly important research theory significance and practical value.

The content of the invention

The application purpose is to provide a tobacco growing plain transport protein encoding geneNtPIN4Gene, and its it is coded Tobacco growing element transport protein NtPIN4 albumen, the albumen is related to auxin transhipment in tobacco, in tobacco growing development, branch There is highly important application value in development.

Details are as follows for the technical scheme that the application is taken.

The plain transport protein encoding gene of one tobacco growingNtPIN4Gene, the base sequence such as SEQ ID of the gene Shown in NO.1.

The plain transport protein encoding gene of the tobacco growingNtPIN4Coded by said gene tobacco growing element transport protein NtPIN4 albumen, its amino acid sequence is as shown in SEQ ID NO.2.

Application of the plain transport protein NtPIN4 albumen of the tobacco growing in tobacco, can transport tobacco growing element, tool Play the role of to suppress side shoot and axillary bud growth；By the encoding gene of NtPIN4 albumenNtPIN4After gene silencing, gene silencing is planted In strain, tobacco side shoot showed increased, the growth population of axillary bud substantially increases；By to the plain transport protein NtPIN4 eggs of tobacco growing White regulation and control, regulation and control and raising yield of tobacco for tobacco plant type have important application value.

For knocking out（Silence）Tobacco growing element transport protein in tobaccoNtPIN4The recombinant C RISPR/Cas9 expression of gene Carrier, builds obtain as follows：

It is as follows that design knocks out primer sequence PIN4-K-F and PIN4-K-R：

PIN4-K-F:5 '-GATTGCATATGGTTCTGTCCGATGG-3 ',

PIN4-K-R: 5’-AAACCCATCGGACAGAACCATATGC-3’；

Using above-mentioned primer sequence, the DNA double chain of target site is obtained（Annealing）；

By the DNA double chain of obtained target site（Annealed product）It is attached with the CRISPR/Cas9 carriers after the digestions of Bsa I, and Conversion, screening, identification, are obtained for knocking out tobacco growing element transport protein in tobaccoNtPIN4The recombinant C RISPR/ of gene Cas9 expression vectors.

It is described to be used to knock out（Silence）Tobacco growing element transport protein in tobaccoNtPIN4The recombinant C RISPR/Cas9 of gene Expression vector is applied in tobacco, after the carrier transformation of tobacco, can knock out tobacco growing element transport protein in tobaccoNtPIN4Gene, makes tobacco growing element transport protein NtPIN4 expressing quantities in transformed plant substantially reduce, does not express even.

Using the method for the CRISPR/Cas9 expression vector establishments transfer-gen plant, agriculture bacillus mediated gene is utilized Method for transformation, converts Agrobacterium, screening obtains conversion engineering bacteria, with reference to tissue culture mode, profit by CRISPR/Cas9 expression vectors Plant tissue culture tissue is contaminated with engineering bacteria, and obtained through tissue culture, identificationNtPIN4The new variety of plant of gene delection.

By designing specific primer, inventor clone obtains the volume to the plain transport protein NtPIN4 albumen of tobacco growing Code geneNtPIN4Gene, Real-time PCR Analysis shows, in normal tobacco plant,NtPIN4Gene is in stem and axillary bud Expression quantity is higher.By carrying out under hormon treatment conditionsNtPIN4Gene expression analysis, inventor turns to tobacco growing element Transporting effect of the albumen NtPIN4 albumen in tobacco growing has preliminary understanding.For further researchNtPIN4Gene（NtPIN4 Albumen）Effect in tobacco growing, inventor constructs CRISPR/Cas9 carriers, after the carrier transformation of tobacco plant, structure Build and obtainNtPIN4The mutating strain series of gene knockout.Phenotypic Observation result shows,NtPIN4The mutating strain series of gene knockout, its Branch showed increased, axillary bud increases active.Therefore, utilizeNtPIN4Gene（NtPIN4 albumen）Effect in tobacco growing, NtPIN4 albumen or overexpression are applied by external sourceNtPIN4Gene, and then suppress the growth of tobacco branch and axillary bud；Also may be used By buildingNtPIN4The transgenosis mutant strain of gene delection, so as to promote tobacco branch and axillary bud to increase.In a word, by rightNtPIN4Gene（NtPIN4 albumen）Further investigation, for tobacco growing regulation and control, regulation of plant form there is important theory significance And actual application value.

Brief description of the drawings

Fig. 1 isNtPIN4Expression characteristic of the gene under the processing of different exogenous plant hormones（10 μm of ol/L auxin IAA, 10 μm of ol/L witchweed lactone analogues GR24,10 μm of ol/L basic element of cell division 6-BA, 10 μm of ol/L abscisic acid ABA）；

Fig. 2 isNtPIN4Expression characteristic of the gene in different tissues；

Fig. 3 isNtPIN4The target position point selection schematic diagram of gene knockout（It is PAM regions behind 20 bp target site ,+display is just Adopted chain, asterisk indicates sgRNA relative position）；

Fig. 4 is that T0, T1 knock out target site sequencing result for transgenosis system；

Fig. 5 is T0, T1 generationpin4The phenotype of mutant, wherein A figures are that T0 observes result for plant phenotype, and B figures are T1 for plant Phenotypic Observation result.

Embodiment

Explanation is further explained to the application with reference to embodiment, before specific embodiment is introduced, with regard to following realities Applying situations such as being related to part biological material, experiment reagent, experimental facilities in example is briefly discussed below.

Biomaterial：

Tobacco bred：Seed is in countries tobacco gene studies employed in the big gold dollar of safflower, a kind of commercialization tobacco, embodiment The heart, which is preserved, to be provided；

Carrier：PEASY-T1 Simple carriers, purchased from Beijing Quanshijin Biotechnology Co., Ltd；

CRISPR/Cas9 carriers are provided by domestic silkworm gene group biology National Key Laboratory of Southwestern University friendship；

Bacterial strain：

Trans1-T1 Competent cells, purchased from Beijing Quanshijin Biotechnology Co., Ltd；

LBA4404 agrobacterium strains, commonly use bacterial strain in Bioexperiment, can disclose and obtain；

The synthesis of primer and DNA sequencing provide completion by Beijing Liuhe Huada Genomics Technology Co., Ltd；

Experiment reagent：

RNA extracts kits, SuperPure Plant polyRNA Kit；

Quantitative fluorescent PCR enzyme（SYBR qPCR kit）, purchased from Zhengzhou An Sai bio tech ltd；

Reverse transcription reagent box, T4 ligases, purchased from precious bioengineering（Dalian）Co., Ltd；

Restriction enzyme BsaI, purchased from NEB companies；

DNA cloning enzyme, purchased from Beijing Quanshijin Biotechnology Co., Ltd；

Plant Genome extracts kit, DNA purification kits are purchased from QIAGEN companies；

Experimental facilities：

PCR synthesizer TProfessional Thermocycler, Biometra companies；

Quantitative PCR apparatus CFX96, Bio-Rad companies；

Ultraviolet gel imaging system BioSpectrum, UVP companies.

Embodiment 1

The present embodiment is main justNtPIN4The clone of gene obtains process and is briefly discussed below.

（1）Prepare cDNA and be used as cloned template

Take prosperous long-term tobacco（The big gold dollar of safflower）Stem 100mg as sample, be fully ground in liquid nitrogen, with reference to RNA extract examination Agent box specification extracts total serum IgE, and then reverse transcription is that cDNA is standby；

（2）Primer is designed, enters performing PCR amplification

Designed for the plain transporter gene of amplification tobacco growingNtPIN4Primer sequence it is as follows：

NtPIN4-F:5 '-ATGATCACTTGGCACGATCTA-3 ',

NtPIN4-R: 5’-TTATAATCCAAGAATGATGTAGTACAC-3’；

With step（1）In prepared cDNA be template, enter performing PCR amplification using above-mentioned primer, PCR amplification conditions are：94 DEG C pre- It is denatured 4min；94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 40s, totally 25 circulations；72 DEG C extend 10min eventually again；PCR 4 DEG C of amplification is saved backup, or directly carries out electrophoresis detection analysis.

With reference to product after the purifying PCR amplifications of glue reclaim kit specification, purified product is then connected to pEASY-T1 carriers On, linked system is as follows：

DNA cloning product, 6 μ L；

PEASY-T1 carriers, 1 μ L；

After mixing, 25 DEG C of 25 min of connection.

Connection product is converted into competent escherichia coli cell, specific conversion process is briefly discussed below：

Competent cell is taken out from -80 DEG C of refrigerators, be placed in dissolves it on ice, plus connection product is in 50 μ L Trans1-T1 In competent cell, mixing, the min of ice bath 30 are flicked；

Heat shock 30s in 42 DEG C of water-baths, is immediately placed on 2min on ice；250 μ L are added to balance to the LB of room temperature（Without antibiotic）, 37 DEG C sway culture 1h；

8 μ L 500mM IPTG, 40 μ L 20 mg/mL X-gal is taken, LB solid plates are uniformly applied to after mixing（Contain 60 μ g/ μ L ampicillins）On flat board, culture dish, 37 DEG C of overnight incubations are inverted.

After picking hickie amplification cultivation, each DNA is extracted, recombinant plasmid is identified by plasmid PCR amplification, By the sequencing of corresponding positive colony sample presentation, obtainNtPIN4Gene order.

Sequencing analysis result shows,NtPIN4Gene code section length is 1953bp nucleotides, specific such as SEQ ID Shown in NO.1；To being understood after the genetic analysis, the amino acid sequence such as SEQ ID NO.2 institutes of the NtPIN4 albumen coded by it Show.

Embodiment 2

In the present embodiment, inventor is handled and gathered different tissues, organ by different exogenous plant hormones, utilizes fluorescent quantitation PCR pairsNtPIN4The expression pattern of gene is analyzed, and related experiment is briefly discussed below.

Tobacco seed is placed in culture dish, Hoagland solution is added and is cultivated（Illumination/dark=18/6 h, 23 DEG C ~28℃）, after culture to germination 2 weeks, in the solution that seedling is moved to hormon, and in blade spraying respective concentration hormone, place The reason time is 5h, and collection sample is placed in -80 DEG C of refrigerators after liquid nitrogen flash freezer, saved backup；In addition after normal cultivation, collection Root, stem, leaf, axillary bud and the flower of squaring period tobacco are placed in -80 DEG C of refrigerators after liquid nitrogen flash freezer, saved backup as sample.

The specific processing mode of experimental group is as follows：

10 μm of ol/L auxin IAA,

10 μm of ol/L witchweed lactone analogue GR24,

10 μm of ol/L basic element of cell division 6-BA,

10 μm of ol/L abscisic acid ABA；

Control group is set simultaneously, and control group tobacco seedling uses 1%（v/v）DMSO solution processing.

Tobacco seedling material after processing is respectively placed in 1.5mL centrifuge tubes, liquid nitrogen flash freezer, -80 DEG C save backup.

By the material extraction RNA preserved, synthesized using reverse transcription reagent box after cDNA（Reference reagent box specification is operated ）, with tobaccoNtL25Gene is internal reference, carries out fluorescence quantitative PCR detection, during detection, and primer sequence design is as follows：

The fluorescent quantitation primer of NtPIN4 genes is detected, primer sequence is：

NtPIN4-q-F:5 '-GCAGTCCCTTTACTTTCCTTTCA-3 ',

NtPIN4-q-R: 5’-AAGTGTATTAGGAAGTGTTGAAAGTGAG-3’；

When detecting tobacco NtL25 genes, specific primer is：

NtL25-F:5 '-CAAAAGTTACATTCCACCG-3 ',

NtL25-F: 5’-TTTCTTCGTCCCATCAGGC-3’ ；

The condition of quantitative fluorescent PCR is：First step pre-degeneration, 95 DEG C of 10 s；Second step PCR reacts, 95 DEG C of 5 s, 57-60 DEG C 30 s, 39 circulations；3rd step solubility curve.

Each sample carries out 3 secondary pollutants and repeated, and passes through 2^-△△CTMethod analyzes Relative gene differential expression.Analysis result As shown in figure 1, compared with control group,NtPIN4The relative expression quantity of gene is significantly raised by the induction of auxin, but by de- The induction for falling acid, the basic element of cell division and witchweed lactone is significantly lowered.

Different tissues expression analysis result as shown in Fig. 2NtPIN4It is secondary in expression quantity highest of the gene in stem, axillary bud It, it is relatively low in root and blade.

Embodiment 3

To further appreciate thatNtPIN4Adjustment effect of the gene in plant strain growth, inventor is constructed for knocking outNtPIN4Base The CRISPR/Cas9 expression vectors of cause, are briefly discussed below with regard to building process below.

First according to known to embodiment 1NtPIN4Genome and coding region sequence, the standard designed according to target site,NtPIN4The target site of 20bp length is designed in the First Exon sequence of gene（Schematic diagram is as shown in Figure 3）, design knockout and draw Thing sequence PIN4-K-F and PIN4-K-R is as follows：

PIN4-K-F:5 '-GATTGCATATGGTTCTGTCCGATGG-3 ',

PIN4-K-R: 5’-AAACCCATCGGACAGAACCATATGC-3’；

Reaction system is designed, to obtain the DNA double chain of target site（Annealing）, 20 μ L reaction systems design as follows：

Annealing Buffer for DNA OLigos (5 ×), 4 μ L；

Upstream and downstream primer（PIN4-K-F、PIN4-K-R）, each 4 μ L（50 μmoL/μL）；

Nuclease-free water are supplemented to 20 μ L；

Response procedures are：95 DEG C of 5 min, every 8 s declines 0.1 DEG C, is down to 25 DEG C；4 DEG C of reaction product is saved backup, Huo Zhezhi Tap into row subsequent reactions.

Annealed product is attached with the CRISPR/Cas9 carriers after the digestions of Bsa I by above-mentioned, and screens and is used for Knock outNtPIN4The CRISPR/Cas9 expression vectors of gene, 20 μ L linked systems design is as follows：

Annealed product, 6 μ L；

Digestion products（CRISPR/Cas9 carriers after the digestions of Bsa I）, 3 μ L；

10 × T4 DNA Ligase Buffer, 2 μ L；

T4 DNA Ligase, 1 μ L；

Aqua sterilisa is supplemented to 20 μ L, 37 DEG C of connection 3h.

Then connection product is converted into competent escherichia coli cell, by picking positive colony, expands and cultivate, carry Take after plasmid, after confirming vector construction success through PCR, Cord blood, for Agrobacterium-mediated Transformation.

Embodiment 4

Constructed CRISPR/Cas9 expression vectors in embodiment 3 are converted into Agrobacterium, and and then transformation of tobacco plant, structureNtPIN4The transfer-gen plant of gene knockout, specific experiment process is briefly discussed below.

（1）The conversion of Agrobacterium

Agrobacterium competent cell is taken out from -80 DEG C of refrigerators, in freeze thawing on ice, 6 μ L embodiments 3 are added when will thaw Prepared CRISPR/Cas9 expression vectors, flick mixing；Mixture is placed in the electric revolving cup of precooling, 5 min are placed on ice；

Electroporation parameter is adjusted to：The kV of voltage 2.5, electric capacity 25 μ F, the Ω of resistance 200；Then with blotting paper by electric revolving cup outer wall On water droplet absorb it is clean after, electric revolving cup is put into electric shock tank, 5 ms of electric shock；

It is rapidly added the 800 μ L YEB fluid nutrient mediums for being preheated to 28 DEG C, 220 rpm, 28 DEG C of 3 h of vibration recovery；

Then the rpm of bacterium solution 4500 is centrifuged into 1 min, abandons the supernatant of half volume, be uniformly applied to after suspending again containing Rif （100 μg/mL）、Str（50 μg/mL）And Kan（50 μg/mL）YEB solid mediums on, 28 DEG C be inverted culture about 2 ~ 3 D, until single bacterium colony is formed；

Picking single bacterium colony, performing PCR identification is entered to bacterium solution, correct positive colony bacterial strain is identified after spreading cultivation, as converted correct Engineering bacteria.

（2）Transformation of tobacco plant

The tobacco tests for sterility of growth one month or so is taken, blade is processed into the leaf of the cm sizes of diameter 0.5 with card punch Disk, by leaf dish after processing on MS solid mediums the d of preculture 3；

By Agrobacterium engineering bacteria, culture to OD after above-mentioned prepared conversion₆₀₀=0.6 or so, 4000 rpm centrifuge 5 min and collect bacterium Body, then with 20 mL MS fluid nutrient medium suspension thallines；

Then leaf dish after above-mentioned preculture is placed in bacterium solution, infects 10 min；

Bacterium solution unnecessary around leaf dish after contaminating is blotted with sterile filter paper, in MS+6-BA（2 mg/L）+NAA（0.5 mg/L） Solid medium on the d of light culture 3；

With containing Cef（400 mg/L）Sterile water wash leaf dish, and suck unnecessary liquid with aseptic filter paper, leaf dish gone to Contain 6-BA（2 mg/L）、NAA（0.5 mg/L）、Cef（200 mg/L）And Kan（50 mg/L）MS solid screening and culturing mediums On, 28 DEG C of illumination cultivations；

When adventitious bud grows to 0.5cm, it is transferred to containing Cef（200 mg/L）And Kan（50 mg/L）MS solid mediums on it is raw Root.

One month to be grown or so, a small amount of blade is taken, DNA is extracted with reference to Plant Genome extracts kit specification, is led to Cross PCR amplifications, clone, the method for sequencing, detection positive transgenic strain and mutant form.Specifically authentication method is：

On NtPIN4 genomes, a pair of detection primers are designed, the primer is located at the both sides for knocking out target site, is specially：

PIN4-J-F:5 '-TCAGAGATAAGTCAGCACCACAC-3 ',

PIN4-J-R: 5’-TAAAGAAACAACATCAGATTCCACT-3’；

With T0 for transgenic line DNA profiling, enter performing PCR amplification；PCR conditions are：94 DEG C of min of pre-degeneration 4；94 DEG C of denaturation 30 S, 56 DEG C of annealing 30 s, 72 DEG C of 40 s of extension, totally 25 circulations；72 DEG C extend 10 min eventually again；

After pcr amplification product is purified, it is connected on pEASY-T1 carriers, by converting competent escherichia coli cell, chooses Take single bacterium colony to enter performing PCR amplification checking, and send sequencing.

Sequencing result is as shown in figure 4, in 9 plants of T0 in plant, detectNtPIN4Gene has the mutation of form in 6, point Be not 1, the deletion of 2,5,17,20 bases（delete）With the insertion of 1 base（insert）, these mutation occur striking On the target site removed, and WT linesNtPIN4Any mutation that gene is not detected.This illustrates in T0 is for plant It is successfully realized pairNtPIN4Gene is mutated（In other words, deleted or base infix form pair with baseNtPIN4Gene Silence is carried out）.

Transgenic line character mutation situation：

Respectively by the T0 of wild type and transgenic line for plantlet of transplant into basin, hot-house culture 7 weeks or so, Phenotypic Observation result It has been shown that, compared with wild-type tobacco, the axillary bud growth of positive plant is accelerated.When growing to 12 weeks（Fig. 5 A）, positive strain Branch phenotype is more notable, and substantially, blade increases axillary bud growth.

In order to verifypin4Can mutant mutational site and branch phenotype stablize heredity to the next generation, and research have collected T0 For the seed of positive strain, continue to obtain T1 after planting for plant, carry out PCR amplifications, monoclonal sequencing, mutational site and phenotype point Analysis.Analysis shows T0 can stablize heredity to T1 generations for mutational site, and obtain and comprise only the homozygosis that 5 bases delete and dash forward Mutant.Wild type and T1 are cultivated 4 weeks or so for plant in greenhouse simultaneously, Phenotypic Observation result is as shown in Figure 5 B.From figure As can be seen that compared with wild-type tobacco, axillary bud suppression is eliminated in mutant, axillary bud increase, branch phenotype significantly increases Plus.

To sum up, regulation and control are passed throughNtPIN4The expression of gene can control the axillary bud of tobacco and the proliferative conditions of branch, So as to be that tobacco regulation of plant form and final tobacco production establish application foundation.

SEQUENCE LISTING

<110>Zhengzhou Tobacco Research Institute of CNTC

<120>Tobacco growing element transport protein NtPIN4 and its application

<130> none

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1953

<212> DNA

<213> Nicotiana tabacum L.cv.Honghua Dajinyuan

<400> 1

atgatcactt ggcacgatct atatgttgtg ttaacagcag ttgttccttt gtatgttgct 60

atgatcttgg catatggttc tgtccgatgg tggaaaatct tttcccctga tcaatgctct 120

ggtataaaca gatttgtggc tatttttgca gtccctttac tttcctttca ctttatagcc 180

atgaacaacc cgtatgaaat gaacttccgt ttcattgctg ctgattcttt acaaaaggtt 240

attatgttag tggtgctttc attatgggct aatttgacaa aaaatggtag cctagaatgg 300

agcattacaa ttttctcact ttcaacactt cctaatacac ttgttatggg aattcctttg 360

ttaattgcta tgtatggtga gtactctggt agtttaatgg tacaagttgt agtgttacag 420

tgtattattt ggtacaccct tttgcttttc ttgttcgaat atcgtggggc taagatgctt 480

attatggaac aatttcctga aactgctgct tccattgttt catttaaagt ggaatctgat 540

gttgtttctt tagatggaca tgattttctt gaaactgacg ctgaaattgg ccaagatggt 600

aaacttcatg ttactgtaag aaaatcaaat gcgtctagga gatcatttgc tatggaccat 660

agaccatcaa atctaactgg agctgaaatt tatagcttaa gttcttcaag aaatccaact 720

cctagaggat ctaattttaa tcataatgat ttttactcta tgatgggttt tcctggagga 780

agattatcca attttggtcc tgcggataat atgtattctg ttcaatcgtc tcggggtcca 840

accccgagac cgtctaattt tgaagaaaat tgtgctccgg gaggtctgat tcagaactct 900

ccgaagtttg gatttttccc ggcgcagact gctaccccag gttattatcc tgcaccgaat 960

cccgaaattg cttcgacagt acctaagaat acaaaacctc agcaacaaca aaatgttcaa 1020

gtacaaaagc aagatgtaca acaacagctg aatgctaaag gtaataatca tgatgctaag 1080

gaacttcaca tgtttgtttg gagctcgagt aattcgccgg tgtcggaagc cggcggcctc 1140

catgtgttcg gcggcaatga tttcagtgct aacgaacaat ctggtcggtc cgatggtgct 1200

aaagaaatta ggatgttggt ttctgatcat cctcaaaatg gggataatac taaagccatt 1260

ccgcaaagtg gggactttgt tagggaagat tttagctttg gaggtgccaa tggtggtgga 1320

aaagatggag atgaagaaaa aggagaaaaa gagggaccca ctggactgac taaactgggg 1380

tccagttcca catcggagct acacccgaaa accgccggag ttcaagattc cggtaccgga 1440

aaacagatgc caccggcgag tgttatgact cgtttaatct taatcatggt ttggcgtaag 1500

cttatccgta acccgaacac gtattcgagc ctcattggtc tgatttggtc actaatctca 1560

tttaggtggc atgtgcatat gcccaaaatc atagagaaat caatctctat tctctcagac 1620

gctggtcttg gaatggctat gtttagctta ggtctgttta tggctttgca accaaagatc 1680

attgcatgtg ggaacacagt ggctacattt gcaatggctg tgaggttttt gactggccca 1740

gcagttatgg ctgctgcttc cattgccgtt ggtcttcgtg gtaccctcct ccatgtagcc 1800

attgtacagg ctgcattgcc acaagggatt gtcccatttg tgtttgctaa ggagtacaat 1860

gttcatccag ctattcttag cactgcggtt attttcggga tgttgatagc attgccaatc 1920

acattagtgt actacatcat tcttggatta taa 1953

<210> 2

<211> 650

<212> PRT

<213> Nicotiana tabacum L.cv.Honghua Dajinyuan

<400> 2

Met Ile Thr Trp His Asp Leu Tyr Val Val Leu Thr Ala Val Val Pro

1 5 10 15

Leu Tyr Val Ala Met Ile Leu Ala Tyr Gly Ser Val Arg Trp Trp Lys

20 25 30

Ile Phe Ser Pro Asp Gln Cys Ser Gly Ile Asn Arg Phe Val Ala Ile

35 40 45

Phe Ala Val Pro Leu Leu Ser Phe His Phe Ile Ala Met Asn Asn Pro

50 55 60

Tyr Glu Met Asn Phe Arg Phe Ile Ala Ala Asp Ser Leu Gln Lys Val

65 70 75 80

Ile Met Leu Val Val Leu Ser Leu Trp Ala Asn Leu Thr Lys Asn Gly

85 90 95

Ser Leu Glu Trp Ser Ile Thr Ile Phe Ser Leu Ser Thr Leu Pro Asn

100 105 110

Thr Leu Val Met Gly Ile Pro Leu Leu Ile Ala Met Tyr Gly Glu Tyr

115 120 125

Ser Gly Ser Leu Met Val Gln Val Val Val Leu Gln Cys Ile Ile Trp

130 135 140

Tyr Thr Leu Leu Leu Phe Leu Phe Glu Tyr Arg Gly Ala Lys Met Leu

145 150 155 160

Ile Met Glu Gln Phe Pro Glu Thr Ala Ala Ser Ile Val Ser Phe Lys

165 170 175

Val Glu Ser Asp Val Val Ser Leu Asp Gly His Asp Phe Leu Glu Thr

180 185 190

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

195 200 205

Ser Asn Ala Ser Arg Arg Ser Phe Ala Met Asp His Arg Pro Ser Asn

210 215 220

Leu Thr Gly Ala Glu Ile Tyr Ser Leu Ser Ser Ser Arg Asn Pro Thr

225 230 235 240

Pro Arg Gly Ser Asn Phe Asn His Asn Asp Phe Tyr Ser Met Met Gly

245 250 255

Phe Pro Gly Gly Arg Leu Ser Asn Phe Gly Pro Ala Asp Asn Met Tyr

260 265 270

Ser Val Gln Ser Ser Arg Gly Pro Thr Pro Arg Pro Ser Asn Phe Glu

275 280 285

Glu Asn Cys Ala Pro Gly Gly Leu Ile Gln Asn Ser Pro Lys Phe Gly

290 295 300

Phe Phe Pro Ala Gln Thr Ala Thr Pro Gly Tyr Tyr Pro Ala Pro Asn

305 310 315 320

Pro Glu Ile Ala Ser Thr Val Pro Lys Asn Thr Lys Pro Gln Gln Gln

325 330 335

Gln Asn Val Gln Val Gln Lys Gln Asp Val Gln Gln Gln Leu Asn Ala

340 345 350

Lys Gly Asn Asn His Asp Ala Lys Glu Leu His Met Phe Val Trp Ser

355 360 365

Ser Ser Asn Ser Pro Val Ser Glu Ala Gly Gly Leu His Val Phe Gly

370 375 380

Gly Asn Asp Phe Ser Ala Asn Glu Gln Ser Gly Arg Ser Asp Gly Ala

385 390 395 400

Lys Glu Ile Arg Met Leu Val Ser Asp His Pro Gln Asn Gly Asp Asn

405 410 415

Thr Lys Ala Ile Pro Gln Ser Gly Asp Phe Val Arg Glu Asp Phe Ser

420 425 430

Phe Gly Gly Ala Asn Gly Gly Gly Lys Asp Gly Asp Glu Glu Lys Gly

435 440 445

Glu Lys Glu Gly Pro Thr Gly Leu Thr Lys Leu Gly Ser Ser Ser Thr

450 455 460

Ser Glu Leu His Pro Lys Thr Ala Gly Val Gln Asp Ser Gly Thr Gly

465 470 475 480

Lys Gln Met Pro Pro Ala Ser Val Met Thr Arg Leu Ile Leu Ile Met

485 490 495

Val Trp Arg Lys Leu Ile Arg Asn Pro Asn Thr Tyr Ser Ser Leu Ile

500 505 510

Gly Leu Ile Trp Ser Leu Ile Ser Phe Arg Trp His Val His Met Pro

515 520 525

Lys Ile Ile Glu Lys Ser Ile Ser Ile Leu Ser Asp Ala Gly Leu Gly

530 535 540

Met Ala Met Phe Ser Leu Gly Leu Phe Met Ala Leu Gln Pro Lys Ile

545 550 555 560

Ile Ala Cys Gly Asn Thr Val Ala Thr Phe Ala Met Ala Val Arg Phe

565 570 575

Leu Thr Gly Pro Ala Val Met Ala Ala Ala Ser Ile Ala Val Gly Leu

580 585 590

Arg Gly Thr Leu Leu His Val Ala Ile Val Gln Ala Ala Leu Pro Gln

595 600 605

Gly Ile Val Pro Phe Val Phe Ala Lys Glu Tyr Asn Val His Pro Ala

610 615 620

Ile Leu Ser Thr Ala Val Ile Phe Gly Met Leu Ile Ala Leu Pro Ile

625 630 635 640

Thr Leu Val Tyr Tyr Ile Ile Leu Gly Leu

645 650

Claims (7)

1. the plain transport protein encoding gene of a tobacco growingNtPIN4Gene, it is characterised in that the gene includes 1953bp, alkali Basic sequence is as shown in SEQ ID NO.1.

2. tobacco growing element transport protein encoding gene described in claim 1NtPIN4Coded by said gene tobacco growing element transhipment egg White NtPIN4 albumen, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.2.

3. application of the tobacco growing element transport protein NtPIN4 albumen in tobacco described in claim 2, it is characterised in that the egg It is white to be used to transport tobacco growing element, with the effect for suppressing side shoot and axillary bud growth.

4. tobacco growing element transport protein encoding gene described in claim 1NtPIN4Application of the gene in tobacco, its feature It is, willNtPIN4After gene silencing, in gene silencing plant, tobacco side shoot showed increased, the growth population of axillary bud substantially increases Plus, so as to adjust tobacco plant type.

5. for knocking out tobacco growing element transport protein encoding gene described in claim 1NtPIN4The recombinant C RISPR/ of gene Cas9 expression vectors, it is characterised in that build obtain as follows：

It is as follows that design knocks out primer sequence PIN4-K-F and PIN4-K-R：

PIN4-K-F:5 '-GATTGCATATGGTTCTGTCCGATGG-3 ',

PIN4-K-R: 5’-AAACCCATCGGACAGAACCATATGC-3’；

Using above-mentioned primer sequence, the DNA double chain of target site is obtained；

The DNA double chain of obtained target site is attached with the CRISPR/Cas9 carriers after the digestions of Bsa I, and convert, screen, Identification, is obtained for knocking out tobacco growing element transport protein in tobaccoNtPIN4The recombinant C RISPR/Cas9 expression vectors of gene.

6. it is used to knock out tobacco growing element transport protein encoding gene described in claim 1 described in claim 5NtPIN4Gene Recombinant C RISPR/Cas9 expression vectors applied in tobacco, it is characterised in that for knocking out in tobacco tobacco growing element transhipment AlbumenNtPIN4Gene, makes tobacco growing element transport protein NtPIN4 expressing quantities in transformed plant substantially reduce, not even Expression.

7. using being used to knock out described in claim 1 tobacco growing element transport protein encoding gene described in claim 5NtPIN4 Transfer-gen plant breeding method constructed by the recombinant C RISPR/Cas9 expression vectors of gene, it is characterised in that first with agriculture The gene transformation method of bacillus mediation, Agrobacterium is converted by CRISPR/Cas9 expression vectors, and screening obtains conversion engineering bacteria； Secondly plant tissue culture tissue is contaminated with reference to tissue culture mode, utilizing works bacterium；Most obtained afterwards through tissue culture, identificationNtPIN4Base Because of the new variety of plant of missing.