CN109082429A - A kind of key gene PeRBR and its expression albumen and the application of poplar adjusted and controlled adventitious root and xylem development - Google Patents

Abstract

The key gene PeRBR and its expression albumen that develop the invention discloses a kind of poplar adventitious root and xylem and application, the nucleotide sequence of key gene PeRBR is as shown in SEQ ID NO.1.The present invention is by being transferred to poplar for PeRBR gene, and the transgenic poplar adventitious root length of overexpression PeRBR gene increases, lateral root number increases, and the xylem thickness of adventitious root and stem all obviously increases.This shows that PeRBR gene is the key regulator of poplar adventitious root and xylem development, has important application value in terms of trees clone breeding and raising.

Description

A kind of the key gene PeRBR and its table of poplar adjusted and controlled adventitious root and xylem development Up to albumen and application

Technical field

The invention belongs to field of plant genetic project technology, and in particular to a kind of poplar adjusted and controlled adventitious root and xylem development Key gene PeRBR and its expression albumen and application.

Background technique

Poplar is the important fast-growing industrial cut stock tree species and afforestation tree species in China, although most of poplars all compare appearance Easy cuttage root-taking, however still there are many choiceness cuttage root-taking difficulties.Therefore carry out point of poplar adventitious root genesis and development Sub- mechanism study not only has important theory significance, but also contains potential using value.However, the development tune of plant adventitious root It controls sufficiently complex, can be influenced by multiple endogenous hormones or environmental factor.Therefore, only a small amount of gene is identified at present participates in adjusting Adventitious root is controlled, the molecule mechanism of entire indefinite root development is also very weak.In addition, formation and the direct shadow of development of xylem The lumber quality and yield for arriving forest are rung, therefore related xylem is formed and the research of development is also always the research of forest genetics Emphasis, but due to xylem formed and develop mechanism it is sufficiently complex, up to the present, the correlative study in poplar is also It is very limited.

Retinoblastoma protein (retinoblastoma, RB) is a kind of tumour expressed in kinds of tumors tissue Inhibit albumen, is isolated and identified in human body at first.RB albumen has there are two highly conserved structural domain, respectively A, B frame, It can interact between them, form a kind of structure similar to pocket-like in C-terminal, this pocket region is RB albumen and transcription The bond area of the factor, therefore RB albumen is also referred to as " pocket albumen ".In the different phase of cell development, RB albumen is all risen Highly important effect, be the important attemperator of many cell events such as cell differentiation, gene expression control and Apoptosis.

With going deep into for research, researcher has found that RB albumen is not only present in vertebrate, in a variety of eukaryocytes Their presence is had found in biology.In plant, RBR (RB-related) gene most early in corn discovery and successfully It clones and.Then, RBR gene is also cloned in succession in the plants such as arabidopsis, tobacco, rice.Amino acid sequence the area A with In the area B, the homology of plant RBR gene and animal RB gene is 20-35%, has certain conservative, this illustrates that the two exists There is similitude on biochemical characteristic.In addition, have found RBR gene in each tissue of plant, but expression quantity is respectively not Identical and different development developmental stage expression quantity is also different, this point is identical as RB albumen, and it is higher to illustrate that RB/RBR albumen has Conservative, the research of RBR function can use for reference RB albumen.

The study found that RBR/rbr heterozygosis arabidopsis is only and normal paternal hybrid could generate offspring, but abortion rate compared with Height illustrates that the mutation of RBR gene has great influence to the generation of maternal plant pollen.And by the research to abortion ovule, It was found that the missing of RBR gene function can cause oogamete to develop disorder, this shows before fertilized eggs are formed, and RBR gene has been sent out Wave important function.During Development of Female Gametophyte, mother cell is will form after mitosis containing seven cells and eight The blastular structure of a nucleus, and the nucleus in the blastular that prefecundation, the oogamete of rbr genotype are formed is more than eight, Cell division is incomplete, and the nucleus having more, which is gathered in the hole of bead, hinders pollen tube to enter.In addition, in oogamete development, rbr There is gene unconventionality expression phenomenon in genotype., research shows that female organ is in no prefecundation, RBR gene is able to suppress carefully for these Born of the same parents' mitosis.

In arabidopsis after RBR gene lacks functionality, it may appear that the disorder of mitosis rule, male and female ligand dysplasia, this A little phenomenons show that RBR gene plays a significant role in cell division, proliferation.The study found that in RBR-RNAi (rRBr) plant After inhibiting RBR to transcribe, root cap portion has had more undifferentiated cell, and due to no amyloplaste, they are protected this part cell There is the characteristics of stem cell.After the tip of a root quiescent center cell for cutting off rRBr plant, undifferentiated cell is broken up, to wild The cell of stem cell surrounding also will do it differentiation after type plant carries out the excision of same area, this mitogenetic group of tip of a root as the result is shown After knitting the function of having lacked RBR gene, the area of tip of a root stem cell can be increased, enhance the signal of quiescent center, keep dry The characteristic of cell.Having studies have shown that part that can allow, cell remain stationary the gene of center situation and RBR gene carries out phase Interaction, to maintain the Stem Cell Niche in Root apical meristem, it is suppressed that stem cell is broken up ahead of time.By studying above It can be concluded that RBR gene is for maintaining stem cell division differentiation, proliferation etc. to play a key role.

Leaf development is an important link in plant growth and development.It is leaf at early stage, phyllopodium is by first Raw meristematic cell regulates and controls to generate through the transcription factors networks of a series of complex, then under the regulation of multiple transcription factors, Phyllopodium is grown along rachis, is proliferated, is divided etc. after processes, blade ultimately forms the leaf of stretching, extension.The study found that in tobacco After the expression for inhibiting RBR1 gene, tobacco can generate phenomena such as development slows down, plant is short and small, leaf curl is obvious, deeper into Result of study shows that cell number increases in blade, thus it is speculated why blade is abnormal development, the reason is that cell Proliferation activity In vigorous.In addition, also affecting the arrangement of stomata on epidermis.Studies have shown that RBR gene in arabidopsis rises in leaf development Inhibit the effect of cell Proliferation.

Although peering RBR gene has carried out more in-depth study in the draft model plant such as arabidopsis, Few related report with specific aim, systematic further investigation illustrate its mechanism of action in xylophyta.

Summary of the invention

Goal of the invention: the deficiencies in the prior art are directed to, the object of the present invention is to provide a kind of poplar adventitious root hairs Key gene PeRBR is educated, the use demand of poplar adventitious root and xylem development is met.It is a further object of the present invention to provide one Kind is the expression albumen of the indefinite root development key gene PeRBR of above-mentioned poplar.Further object of the present invention is to provide a kind of above-mentioned The application of the indefinite root development key gene PeRBR of poplar.

Technical solution: in order to achieve the above-mentioned object of the invention, The technical solution adopted by the invention is as follows:

A kind of poplar adjusted and controlled adventitious root and xylem develop key gene PeRBR, nucleotide sequence such as SEQID NO.1 It is shown.

The expression albumen of the poplar adventitious root and xylem development key gene PeRBR, amino acid sequence such as SEQ Shown in NO.2.

Carrier containing the poplar adventitious root and xylem development key gene PeRBR.

The carrier, in 5 ' end assembling composing type strongly expressed promoter P35S of PeRBR gene.

The carrier assembles strong terminator NOS at 3 ' ends of PeRBR gene.

The carrier assembles HPT expression casette, as the selection markers of transgenic poplar, can with hygromycin into The screening of row transgenic poplar.

The carrier assembles LB and RB sequence, promotes to assemble PeRBR gene expression construct therebetween and selection markers Gene HP T is integrated into poplar recipient cell chromosome.

Host cell containing the poplar adventitious root and xylem development key gene PeRBR.

The poplar key gene PeRBR is in poplar adjusted and controlled adventitious root and the developmental application of xylem.

895 poplar of woods comes into being adventitious root as material on the south the present invention, has cloned PeRBR gene by RACE technology.Meanwhile it adopting Its poplar Overexpression vector pH35GS-PeRBR is constructed with Gateway clone technology, which is located at after promoter P35S, Under the driving of promoter P35S, PeRBR can in poplar body high efficient expression, thus regulate and control adventitious root and xylem formation and Development.Wherein, institute's PeRBR gene is poplar adventitious root genesis and development key gene.

The utility model has the advantages that compared with prior art, the present invention is by being transferred to poplar, overexpression PeRBR base for PeRBR gene The transgenic poplar adventitious root length of cause increases, lateral root number increases, and the xylem thickness of adventitious root and stem all obviously increases Add.This shows that PeRBR gene is the key regulator of poplar adventitious root and xylem development, in trees clone breeding and mentions High timber yield etc. has important application value.It is not only that poplar molecular breeding provides theoretical basis, and can also promote Into the breeding of the excellent strain of poplar, there is huge economic value to forest development.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of plant expression vector pH5GS；

Fig. 2 is the real-time quantitative Molecular Detection result figure of overexpression PeRBR gene；

Fig. 3 is the transgenosis poplar of overexpression PeRBR gene and the configuration comparison result figure of non-transgenosis poplar (CK)；

Fig. 4 is the transgenosis poplar of overexpression PeRBR gene and the root morphology comparison result figure of non-transgenosis poplar (CK)；

Fig. 5 is the transgenosis poplar of overexpression PeRBR gene and the adventitious root length comparison result of non-transgenosis poplar (CK) Figure；

Fig. 6 is root, the stem structure comparison result figure for being overexpressed PeRBR transgenosis poplar and non-transgenosis poplar (CK)；4WR in figure Refer to that the root of 4 weeks and 5 weeks, 5WS refer to 5 weeks stems with 5WR.

Specific embodiment

The present invention is described further combined with specific embodiments below.

Embodiment 1 clones PeRBR gene by RACE technology

Based on applicant's poplar early period adventitious root chip of expression spectrum result of study, 3 ' ends are designed using Oligo 6 RACE primer carries out 3 ' RACE, obtains 3 ' cDNA terminal fragments, is cloned into T- carrier, and it is laggard to carry out PCR screening to Insert Fragment Row sequencing, Blast confirm above-mentioned segment DNA homolog relevant to other plants.In the same way, according to the correct of acquisition 3 ' cDNA terminal fragment sequence design, 5 ' end RACE primer, carry out 5 ' RACE, obtain 5 ' cDNA terminal fragments, be cloned into T- carrier is sequenced after carrying out PCR screening to Insert Fragment.Specific primer and main process are as follows:

PeRBR-3 ' outer-F:5 '-CCGCAGTGTGTTTGTGGATTGGTCATC-3 '；

PeRBR-3 ' outer-R:5 '-ACTCTGCGTTGATACCACTGCTTGCCCTATAGTGAGTCGTATTAG-3 '；

PeRBR-3 ' inner-F:5 '-CCAGTAATGTTCCTGAAGTTGGT-3 '；

PeRBR-3 ' inner-R:5 '-GCCCTATAGTGAGTCGTATTAG-3 '；

PeRBR-5 ' outer-F:5 '-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 '；

PeRBR-5 ' outer-R:5 '-CAGCTCCTTTGCCTCAAGTCTGTTCTC-3 '；

PeRBR-5 ' inner-F:5 '-CTAATACGACTCACTATAGGGC-3 '；

PeRBR-5 ' inner-R:5 '-CATCTCTATTCTTCTCGCTCAAC-3 '.

1, prepare RACE-Ready cDNA

1) prepared by Buffer Mix: 5 × First-Strand Buffer 4.0 μ L, DTT (100mM) 0.5 μ L, dNTP (20mM)1.0μL。

2) prepared by Master Mix: 5.5 μ L, RNase Inhibitor of Buffer Mix from step1,0.5 μ L, SMART ScribeTM Reverse Transcriptase 2.0μL。

3) prepared by 5 '-RACE-Ready cDNA: 1.0 1.0 μ L, Sterile H of μ L, 5 '-CDS PrimerA of RNA₂O 9.0μL.PCR reaction: 72 DEG C of 3min, 42 DEG C of 2min.After reaction, 1200rpm is centrifuged 10S, and 8 μ L Master are added Mix.PCR reaction: 42 DEG C of 90min, 70 DEG C of 10min.

4) prepared by 3 '-RACE-Ready cDNA: 1.0 1.0 μ L, Sterile H of μ L, 3 '-CDS PrimerA of RNA₂O 10.0μL.5 '-RACE-Ready cDNA of trim: 1 μ L SMARTer of addition | | A oligonuclevtide to 5 '-RACE- In Ready cDNA.PCR reaction: 72 DEG C of 3min；42℃ 2min.After reaction, 1200rpm is centrifuged 10S, and 8 μ L are added Master Mix.PCR reaction: 42 DEG C of 90min, 70 DEG C of 10min.

5) be added 90 μ l Tricne-EDTABuffer dilution steps 3), 4) final product.

2, RACE-PCR reacts

1) first round reacts: reaction system: PCR-Grade H₂15.5 μ L, 2 × SeqAmpTM Buffer of O, 25.0 μ L, 1.0 μ L, 5 '-or3 '-RACE-Ready cDNA of SeqAmp DNA Polymerase 2.5 μ L, 10 × UPM 5.0 μ L, 5 ' or3’GSP(10μm)1.0μL。

Response procedures: 94 DEG C of 3min, 94 DEG C of 30S, 5Cycles；72 DEG C of 3min, 94 DEG C of 30S, 72 DEG C of 30S, 5Cycles；72 DEG C 3min, 94 DEG C of 30S, 68 DEG C of 30S, 25Cycles；72 DEG C of 3min, 72 DEG C of 10min.

2) the second wheel reaction: reaction system: PCR-Grade H₂15.5 μ L, 2 × SeqAmpTM Buffer of O, 25.0 μ L, SeqAmp DNA Polymerase 1.0 μ L, wheel 5.0 μ L, a Universal Primer short of PCR product 1.0 μ L, 5 ' or3’Gsp(10μM)1.0μL。

Response procedures: 94 DEG C of 3min, 94 DEG C of 30S, 60 DEG C of 30S, 72 DEG C of 3min, 30Cycles.

The final PeRBR full length cDNA sequence that obtains is 3638bp, and sequence includes one as shown in SEQ ID NO.1 The entire reading frame of 3108bp, expressed by albumen amino acid sequence as shown in SEQ ID NO.2.

2 PeRBR gene plant expression vector establishment of embodiment

Utilize the Overexpression vector of gateway cloning technology building PeRBR gene.Using specific PCR primers (embodiment 1 PeRBR ORF primer), using cDNA as template, PCR amplification is carried out, PeRBR gene ORF is building up to entry vector.Entry vector For pCR^TM8/GW/TOPO^TMvector(Invitrogen).Reaction system are as follows: Fresh PCR product (purified) 10- 20ng；Salt solution 1μL；pCR^TM8/GW/TOPO^TMvector 1μL；Add sterile ddH₂O supplies 6 μ L.Response procedures Are as follows: it is stored at room temperature 30min.

Picking positive colony carries out PCR detection and sequence verification, the entry vector with PeRBR gene from sifting motion cultivation plate LR is carried out with plant expression vector pH35GS to react.Vector plasmid is as shown in Figure 1.Reaction system are as follows: linearized entry clone 100ng；purified destination vector(100ng/μL)1.5μL；LR Clonase II enzyme mix 2μL；TE (pH 8.0) is added to supply 10 μ L.Reaction condition: 25 DEG C of 1h.PeRBR gene transfered plant is expressed after LR reacts In carrier pH35GS, in 5 ' end assembling composing type strongly expressed promoter P35S of PeRBR gene, it can make PeRBR gene in poplar High efficient expression in tree body；Strong terminator NOS is assembled at 3 ' ends of PeRBR gene, can effectively terminate the transcription of PeRBR gene； In vector plasmid over-assemble HPT expression casette transgenosis can be carried out with hygromycin as the selection markers of transgenic poplar The screening of poplar；LB and RB sequence is assembled in vector plasmid, promotes to assemble PeRBR gene expression construct and screening mark therebetween Note gene HP T is integrated into poplar recipient cell chromosome.By PCR detection and sequence verification, Overexpression vector structure is confirmed Function is built up, pH35GS-PeRBR is named as, which is located at after promoter P35S, under the driving of promoter P35S, PeRBR Can in poplar body high efficient expression.

The genetic transformation of 3 PeRBR gene of embodiment

Constructed pH35GS-PeRBR Overexpression vector is transferred to agrobacterium strains EHA105 by frozen-thawed method (Invitrogen), PeRBR gene is transferred to by poplar by mediated by agriculture bacillus.As a result as figures 2-6, wherein Fig. 2 was The real-time quantitative Molecular Detection of amount expression PeRBR gene；Transgenosis poplar and non-transgenosis of the Fig. 3 for overexpression PeRBR gene The configuration of poplar (CK) compares；Fig. 4 is the transgenosis poplar of overexpression PeRBR gene and the root morphology of non-transgenosis poplar (CK) Compare；Fig. 5 is the transgenosis poplar of overexpression PeRBR gene compared with the adventitious root length of non-transgenosis poplar (CK)；Fig. 6 was PeRBR transgenosis poplar is expressed compared with the root of non-transgenosis poplar (CK), stem structure；4WR and 5WR refers to the root of 4 weeks and 5 weeks in figure, 5WS refers to 5 weeks stems.It can be apparent from from result, the transgenic poplar adventitious root length increase of overexpression PeRBR gene, Lateral root number increases, and the xylem thickness of adventitious root and stem all obviously increases.Show PeRBR gene be poplar adventitious root and The key regulator of xylem development has important application valence in terms of trees clone breeding and raising Value.

Sequence table

<110>Nanjing Forestry University

<120>the key gene PeRBR and its expression albumen of a kind of poplar adjusted and controlled adventitious root and xylem development and application

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gaaaagtgat agaaagagaa agggagatag agagagagcc tgactgataa gggtttcccc 60

aagaagaggt ctctcagaga gagagagaga gaaacagaga gatatagaga gagaaagaag 120

gagggaaatt tttaattttt aatttttagg gtttcacgag ttaaacaaag tcttgtggtt 180

ttttgaattc gatctggtaa aaacggtggc tttttttcct tgtggtattg aaaggtagaa 240

ggttttgttg ttggatctgc agatcagtga gatttgaaag atctggagat aagtattggt 300

tttgaatgag tccagctgct ctgaagaata tggaagaaaa caaaactaca gttatgacaa 360

ccagtcattc gagtaatgat ggaggggaaa ctgtgaaagg gtatagtgat gcggttgaag 420

ttcgattttc tgacttttgc aagagtggat tagcattgga tgagaacact tgtacacagg 480

ctattaagct tttcaaagac acgaaacatc ttttgatgac aaatgtttcg tctattggga 540

atggcacgtc ggaagaagca gagaggtttt ggtttgcatt tgtttcgtac tctgttaaga 600

ggttgagcga gaagaataga gatgatgcac agcagaagtc tgatgatcct gggcttactt 660

tatgccaaat attgagatta gcaaagctga acatcatgga cttctttaaa gagctacccc 720

attttattgt caaggctggt ccaatcttaa gcaatatata tggtgcagac tgggagaaca 780

gacttgaggc aaaggagctg caagccaatt ttgtgcactt gagcatttta agcaggcact 840

acaagcgtgc atgcagggaa cttttcttga caagtgatgc aagttctgat aaacagccag 900

caatatccaa tgaagcaaca catgtgtcag accaccatcg ttttgggtgg ttgctgtttc 960

tggctctccg ggtacatgca ttcagccgtt ttaaagatct ggtcacttgc acaaatggtc 1020

tggtttctgt actggctgtt ctgattatac atgttccagt tcgcttcaga aattttagtt 1080

tcaatgactc tcaatggttt gttaggaaag gagacaaagg tgctgatttg cttgcttcac 1140

tctgcaataa atatgacacc tcagaagaag tgttgaggaa atcgatggaa actaccaatg 1200

atttaatagc caatatcttg aagaagaagc cacattcggc ctctgagtat aaaaatgaaa 1260

acctagtgaa tatcaaccca gaaggtttga tctattatga agatttgatg gaggaatcat 1320

ccctgcaatc cagtttgaat attctagaga aggattatga tgatgcaatt cgtaacaagg 1380

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caacaaagac aatcacaagt ccactatctc ctcatcgttc tcctgcatct catgcaaatg 1560

gaattcctgg tagtgcaaac tcgaagatgg cagccacacc tgtaagcact gcaatgacaa 1620

ctgcaaagtg gcttcggacc atcatttccc cacttccatc aaaaccctca gcacagttgg 1680

agcgcttcct ggtgtcatgt gataaggatg taactaatga tgtcattcgt agagcacaga 1740

taatattgga ggctatattt ccaagtagtt ctcttggaga acgctgtgtg aatggaagtc 1800

tgcaaagtac aaacctaatg gacaacatat gggcagaaca aagaagattg gaggcactca 1860

agttatatta cagggttttg gaatcaatgt gcacagcaga ggcccaaata ctgcatgcaa 1920

ctaacttgac ctctttatta actaatgaga ggttccatag atgcatgcta gcgtgttctg 1980

ctgagctagt tgtggcaact tataagacag tgacaatgtt gtttcctgca gttttggaga 2040

gaacaggcat tacagctttt gatcttagca aggtgataga gagtttcatt aggcatgagg 2100

agtccctccc acgggagttg agacggcatt tgaattcctt ggaagagcga cttttggata 2160

gcatggtgtg ggaaaaagga tcctcactct acaattcttt gacagttgca agaccggctc 2220

tttctgcaga gatcaatcga cttggattat tagcagaacc aatgccatca ttggatgcaa 2280

ttgccatgca tattaatttt tcatctggat gcttgcctcc tgtgccttct ttgcaaaagc 2340

acgagacttc tccaggatca ggtcagaatg gagatctcag gtctccaaag agaccttgca 2400

cagacttccg aagtgtgttg gtagagcgaa attccttcac atcaccagtg aaagaccgcc 2460

tattgggtaa tcttaaatca aagctaccac ctcctccttt gcagtctgcc tttgccagtc 2520

caacacgtcc aaaccctgga ggaggagggg aaacatgtgc agaaactggg atcaatgtat 2580

tttttacgaa gattaataag ttggctgctg tcagaattaa tggtatgatt gaaaagctac 2640

aaccgtctca gcagcatatt agagaaaatg tctatcgtct ttttcaactt atactaagcc 2700

accagacatc tctcttcttc aaccgtcata ttgaccagat cattctttgc tgtttctatg 2760

gagttgcaaa gatttctaaa ttgaatctga ccttcaggga aattatatac aattatagga 2820

ggcagccaca ttgtaaaaca ctagttttcc gcagtgtgtt tgtggattgg tcatctgcac 2880

gccataatgg gagaacgggg caggatcatg tggatattat tacgttttac aatgaaattt 2940

ttatccctgc tgcaaagcct ttgctggtgg atgttggttc tgctggaaca acagtgaaag 3000

ccagtaatgt tcctgaagtt ggtaataata aagatggtca atgtcctgca tcacctaaag 3060

tatcaccttt tccaagtctc cctgatatgt ctcctaagaa agtatcgtca gcacataatg 3120

tgtatgtctc tccattgcgg tcatccaaga tggatgcttt aatctcaaat agctcaaaaa 3180

gctattatgc ttgtgttgga gagagcaccc atgcttacca gagcccttca aaagacctaa 3240

atgctatcaa taaccgcctg aacggtaacc ggaaggccag aggaaccctc aacttggata 3300

atgatgttgg gttggttagt gattctatgg tggccaacag cctcggcctt caaaatggga 3360

attgtgcatc tacatcgggt gcagctttga aatctgagca gtctgactcc taattcaaac 3420

cgtggcatac tttgtacatc ctagttctcc atccatttct tcgtatgctt atttatactt 3480

agaagtttgt gactcggggc aatgtacgag cagcgagttg tataatgcat gccatcaccc 3540

ccatgtcttt tgtatgtttg ttagcgcacc cagttattga atagaatagg ttccaactaa 3600

tctaataaaa tttactattt ttgctgctaa aaaaaaaa 3638

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<213>895 poplar of Nan Lin (P. deltoides × P. euramericana ' Nanlin895 ')

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Phe Ser Phe Asn Asp Ser Gln Trp Phe Val Arg Lys Gly Asp Lys Gly

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Val Leu Arg Lys Ser Met Glu Thr Thr Asn Asp Leu Ile Ala Asn Ile

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Leu Lys Lys Lys Pro His Ser Ala Ser Glu Tyr Lys Asn Glu Asn Leu

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Val Asn Ile Asn Pro Glu Gly Leu Ile Tyr Tyr Glu Asp Leu Met Glu

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Glu Ser Ser Leu Gln Ser Ser Leu Asn Ile Leu Glu Lys Asp Tyr Asp

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Asp Ala Ile Arg Asn Lys Ala Glu Leu Asp Glu Arg Val Phe Ile Asn

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Leu Glu Ala Ile Phe Pro Ser Ser Ser Leu Gly Glu Arg Cys Val Asn

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Gly Ser Leu Gln Ser Thr Asn Leu Met Asp Asn Ile Trp Ala Glu Gln

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Arg Arg Leu Glu Ala Leu Lys Leu Tyr Tyr Arg Val Leu Glu Ser Met

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Cys Thr Ala Glu Ala Gln Ile Leu His Ala Thr Asn Leu Thr Ser Leu

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Leu Thr Asn Glu Arg Phe His Arg Cys Met Leu Ala Cys Ser Ala Glu

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Leu Val Val Ala Thr Tyr Lys Thr Val Thr Met Leu Phe Pro Ala Val

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Leu Glu Arg Thr Gly Ile Thr Ala Phe Asp Leu Ser Lys Val Ile Glu

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Ser Phe Ile Arg His Glu Glu Ser Leu Pro Arg Glu Leu Arg Arg His

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Leu Asn Ser Leu Glu Glu Arg Leu Leu Asp Ser Met Val Trp Glu Lys

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Gly Ser Ser Leu Tyr Asn Ser Leu Thr Val Ala Arg Pro Ala Leu Ser

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Ala Glu Ile Asn Arg Leu Gly Leu Leu Ala Glu Pro Met Pro Ser Leu

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Asp Ala Ile Ala Met His Ile Asn Phe Ser Ser Gly Cys Leu Pro Pro

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Val Pro Ser Leu Gln Lys His Glu Thr Ser Pro Gly Ser Gly Gln Asn

675 680 685

Gly Asp Leu Arg Ser Pro Lys Arg Pro Cys Thr Asp Phe Arg Ser Val

690 695 700

Leu Val Glu Arg Asn Ser Phe Thr Ser Pro Val Lys Asp Arg Leu Leu

705 710 715 720

Gly Asn Leu Lys Ser Lys Leu Pro Pro Pro Pro Leu Gln Ser Ala Phe

725 730 735

Ala Ser Pro Thr Arg Pro Asn Pro Gly Gly Gly Gly Glu Thr Cys Ala

740 745 750

Glu Thr Gly Ile Asn Val Phe Phe Thr Lys Ile Asn Lys Leu Ala Ala

755 760 765

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

770 775 780

Ile Arg Glu Asn Val Tyr Arg Leu Phe Gln Leu Ile Leu Ser His Gln

785 790 795 800

Thr Ser Leu Phe Phe Asn Arg His Ile Asp Gln Ile Ile Leu Cys Cys

805 810 815

Phe Tyr Gly Val Ala Lys Ile Ser Lys Leu Asn Leu Thr Phe Arg Glu

820 825 830

Ile Ile Tyr Asn Tyr Arg Arg Gln Pro His Cys Lys Thr Leu Val Phe

835 840 845

Arg Ser Val Phe Val Asp Trp Ser Ser Ala Arg His Asn Gly Arg Thr

850 855 860

Gly Gln Asp His Val Asp Ile Ile Thr Phe Tyr Asn Glu Ile Phe Ile

865 870 875 880

Pro Ala Ala Lys Pro Leu Leu Val Asp Val Gly Ser Ala Gly Thr Thr

885 890 895

Val Lys Ala Ser Asn Val Pro Glu Val Gly Asn Asn Lys Asp Gly Gln

900 905 910

Cys Pro Ala Ser Pro Lys Val Ser Pro Phe Pro Ser Leu Pro Asp Met

915 920 925

Ser Pro Lys Lys Val Ser Ser Ala His Asn Val Tyr Val Ser Pro Leu

930 935 940

Arg Ser Ser Lys Met Asp Ala Leu Ile Ser Asn Ser Ser Lys Ser Tyr

945 950 955 960

Tyr Ala Cys Val Gly Glu Ser Thr His Ala Tyr Gln Ser Pro Ser Lys

965 970 975

Asp Leu Asn Ala Ile Asn Asn Arg Leu Asn Gly Asn Arg Lys Ala Arg

980 985 990

Gly Thr Leu Asn Leu Asp Asn Asp Val Gly Leu Val Ser Asp Ser Met

995 1000 1005

Val Ala Asn Ser Leu Gly Leu Gln Asn Gly Asn Cys Ala Ser Thr Ser

1010 1015 1020

Gly Ala Ala Leu Lys Ser Glu Gln Ser Asp Ser

1025 1030 1035

<210> 3

<211> 21

<212> DNA

<213> 3'RACE Outer primer(Artificial)

<400> 3

atatctccca aggctgatta c 21

<210> 4

<211> 24

<212> DNA

<213> 3'RACE Inner primer(Artificial)

<400> 4

caatggaaat gatccataca atga 24

<210> 5

<211> 22

<212> DNA

<213> 5'RACE Outer primer(Artificial)

<400> 5

tttatcccta ccagatgagg aa 22

<210> 6

<211> 31

<212> DNA

<213> 5'RACE Inner primer(Artificial)

<400> 6

gcagttttgt gaagatgaat atattccaga g 31

<210> 7

<211> 20

<212> DNA

<213>PeRBR ORF forward primer (Artificial)

<400> 7

atggggaaga atgtgttggt 20

<210> 8

<211> 20

<212> DNA

<213>PeRBR ORF reverse primer (Artificial)

<400> 8

atgcagagaa ttggttcgag 20

Claims (9)

1. a kind of poplar adjusted and controlled adventitious root and xylem develop key gene PeRBR, nucleotide sequence such as SEQ ID NO.1 institute Show.

2. the expression albumen of poplar adventitious root described in claim 1 and xylem development key gene PeRBR, amino acid sequence Column are as shown in SEQ NO.2.

3. the carrier containing poplar adventitious root described in claim 1 and xylem development key gene PeRBR.

4. carrier according to claim 3, it is characterised in that: 5 ' end assembling composing types of the carrier in PeRBR gene Strongly expressed promoter P35S.

5. carrier according to claim 3, it is characterised in that: the carrier assembles strong end at 3 ' ends of PeRBR gene Only sub- NOS.

6. carrier according to claim 3, it is characterised in that: the carrier assembles HPT expression casette, as transgenosis The selection markers of poplar can carry out the screening of transgenic poplar with hygromycin.

7. carrier according to claim 3, it is characterised in that: the carrier assembles LB and RB sequence, promotes to be assembled in it Between PeRBR gene expression construct and riddled basins HPT be integrated into poplar recipient cell chromosome.

8. the host cell containing poplar adventitious root described in claim 1 and xylem development key gene PeRBR.

9. poplar key gene PeRBR described in claim 1 is in poplar adjusted and controlled adventitious root and the developmental application of xylem.