CN106967731A - A kind of Chinese tamarisk salt stress response key gene TcARF6 and its application - Google Patents

Abstract

The invention discloses a kind of Chinese tamarisk salt stress response key gene TcARF6 and its application, key gene TcARF6 nucleotide sequence is as shown in SEQ ID NO.1.Chinese tamarisk salt stress response key gene TcARF6 expressing protein, its amino acid sequence is as shown in SEQ ID NO.2.The present invention has cloned TcARF6 genes using the Chinese tamarisk of Chinese tamarisk salt stress processing as material by RACE technologies.By Quantitative Real-Time TaqMan PCR Technique, expression pattern of the detection TcARF6 genes after Chinese tamarisk is forced verifies the key of its response stress.Meanwhile, using its Chinese tamarisk Overexpression vector pH35GS TcARF6 of gateway technique constructions, under promoter P35GS driving, TcARF6 can in transgenosis high efficient expression.TcARF6 relative quantifications in salt stress response process, showing TcARF6, only rapid lower of the specificity in root is expressed, and demonstrates gene key effect in stress response, turning resistance breeding field in forest has significant application value.

Description

A kind of Chinese tamarisk salt stress response key gene TcARF6 and its application

Technical field

The invention belongs to field of plant genetic, and in particular to a kind of Chinese tamarisk salt stress responds key gene TcARF6 and its application.

Background technology

China there are about more than 20 ten thousand square kilometres of salt-soda soils and in continuous increase, and salt-soda soil can not be utilized in agricultural production The contradiction that population increased requirement can not be met by causing China's grain-production is becoming increasingly acute, the plant in active demand High Efficiency Reform salt-soda soil Goods and materials source；Chinese tamarisk is China's indigenous tree, belongs to one of salt tolerance most strong xylophyta, is maintaining the beach such as the Huanghe delta Important function weight has been played in the Ecological Stabilization of wetland Saline Region, has been also the valuable source that China coast shelter-forest builds. Although in willow, paddy rice isotype species, there is the correlative study of many resistant gene of salt, part resistant gene of salt passes through transgenosis skill Art high efficient expression, but the effect that the genetically modified plants salt tolerance of these genes of application is improved at present is unsatisfactory, it is impossible to meet Demand in actual production and be difficult to promote, this may not evolve the special salt tolerant of similar halophytes with glycophyte Mechanism is relevant.There is Chinese tamarisk uniqueness to secrete molecules of salt mechanism and corresponding stress response gene, to plant salt tolerance study mechanism meaning It is great, can be provided fundamental basis and resistant gene resource for salt lick desalination, adversity resistant plant breeding, at present Tamarix other Cloned in plant species and obtained tens important resistant gene of salt and transcription factor, weight is provided for plant salt tolerance breeding Want molecule resource, but not yet have the report of resistant gene of salt in China's distribution area most wide Chinese tamarisk, in the urgent need to exploitation Chinese tamarisk Resistant gene of salt.

ARF transcription factors are the distinctive transcription factor families of plant, and are widely present and have between different plant species Necessarily conservative property, goes in approach in the auxin signal of plant and plays a significant role, and adjusts the table of the lots of genes in downstream Reach.ARF functional studies are mostly derived from growing and morphogenesis research, mesh for the native plant in the fields such as arabidopsis, paddy rice, willow Before, numerous studies show that ARF families are coerced by the post-transcriptional control mechanism regulating such as microRNA and trans-acting siRNA s Expression under compeling, and then induce a large amount of plant salt tolerance genes in downstream so that plant improves salt tolerance.Meanwhile, ARF family Race causes family member's substantial amounts due to gene expansion event, and member has obvious functional redundancy between, increases pair The parsing difficulty of specific ARF gene functions, certain interference is caused to the resistant gene of salt selection in follow-up transgenic research.Enter The research of row Chinese tamarisk ARF families will be helpful to people and deeply understand Auxin Signal Tranducation approach under halophytes environment stress Mechanism of action, it helps heighten people and the not high redundancy gene of salt tolerance is removed in breeding for stress tolerance, important resistance to alkali is screened The identification capability of cause.

At present, not yet there is the ARF families relevant report of Tamarix, clone and develop these genetic resourceses, no Only help to illustrate forest salt tolerant molecular Regulation Mechanism, and forest molecular breeding process can also be promoted, salt-soda soil is utilized and The development of agricultural has immeasurable value.

The content of the invention

Goal of the invention：For the deficiencies in the prior art, it is an object of the invention to provide a kind of Chinese tamarisk salt stress sound Key gene TcARF6 is answered, use demand is met.Closed it is a further object of the present invention to provide a kind of above-mentioned Chinese tamarisk salt stress response Key gene TcARF6 application.

Technical scheme：In order to realize foregoing invention purpose, the technical solution adopted by the present invention is as follows：

A kind of Chinese tamarisk salt stress responds key gene TcARF6, and its nucleotide sequence is as shown in SEQ ID NO.1.

Described Chinese tamarisk salt stress response key gene TcARF6 expressing protein, its amino acid sequence such as SEQ ID Shown in NO.2.

Carrier containing described Chinese tamarisk salt stress response key gene TcARF6.

5 ' end assembling composing type strongly expressed promoter P35Ss of the carrier in TcARF6 genes.

The carrier assembles strong terminator NOS at 3 ' ends of TcARF6 genes.

The carrier assembles HPT expression casettes, and as the selection markers of genetically modified plants, transgenosis is carried out with hygromycin The screening of plant.

The carrier assembles LB and RB sequences, promotes to assemble TcARF6 gene expression constructs and selection markers base therebetween Because HPT is integrated into dicotyledon recipient cell chromosome.

Applications of the described salt stress response key gene TcARF6 in plant resistance to environment stress breeding.

Beneficial effect：Compared with prior art, the present invention passes through RACE skills using the Chinese tamarisk of Chinese tamarisk salt stress processing as material Art has cloned TcARF6 genes.By Quantitative Real-Time TaqMan PCR Technique, detection TcARF6 genes are after Chinese tamarisk is forced Expression pattern, verifies the key of its response stress.Meanwhile, using its Chinese tamarisk Overexpression vector of gateway technique constructions PH35GS-TcARF6, under promoter P35GS driving, TcARF6 can in transgenosis high efficient expression.Salt stress response process In TcARF6 relative quantifications, show TcARF6 only the specificity in root be rapid to lower expression, demonstrate the gene and rung in stress Key effect in answering, turning resistance breeding field in forest has significant application value.

Brief description of the drawings

Fig. 1 is the expression pattern figure under the salt stress of TcARF6 genes；

Fig. 2 is plant expression vector pH35GS structure charts.

Embodiment

With reference to specific embodiment, the present invention is described further.

Embodiment 1

1st, TcARF6 genes are cloned by RACE technologies

Result of study is sequenced based on existing Chinese tamarisk salt stress transcript profile, the RACE primers of 5 ' and 3 ' ends are designed, obtained Responsively PCR primer, is cloned into T- carriers, is sequenced after carrying out positive-selecting to Insert Fragment, sequencing result sequence passes through Overlay region is spliced, and obtains cNDA total lengths.RNA is derived from

TcARF6RACE primers：

RACE Adaptor contain unique design Adaptor Primer sequences (5 '- CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ') it is easy to design primer.

3 ' RACE forward primers：

Outer Primer：5’-TACAGCATCCTCAACAGCAAATGGT-3’；

Inner Primer：5'-TAGACATGGTTGGTACAGACTC-3’；

3 ' RACE reverse primers：

Outer Primer：5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’；

Inner Primer：5-'CTAATACGACTCACTATAGGGC-3’；

5 ' RACE forward primers：

Outer Primer：5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’；

Inner Primer：5'-CTAATACGACTCACTATAGGGC-3’；

5 ' RACE reverse primers：

Outer Primer：5'-ACCATCGGGCTCTTCCAGTGTAGTTA-3’；

Inner Primer：5'-AATCTGTCTTTCGGCTATCTCC-3’.

1) 3'RACE courses of reaction：

(1) in reverse transcription, the small centrifuge tube that following component is added to a RNase-free being placed on ice：1μg Total RNA (are extracted from Chinese tamarisk blade and obtained), 4 μ L dNTP Mix, 2 μ L 3'RACEAdapter, 2 μ L 10X RT Buffer, 1 μ L RNase Inhibitor, 1 μ L M-MLV Reverse Transc riptase, Nuclease-free Water is mended to 20 μ L.

(2) gently mix, of short duration centrifugation, 42 DEG C of incubation 1h；Into PCR steps, or -20 DEG C of preservation reactants；

(3) 3'RACE nest-type PRCs；

Reaction system is：50 μ L Outer 3'RACE compositions：5.0μL 10×LA PCR Buffer(Mg²⁺Free), 5.0μL MgCl₂(25mM), 8.0 μ L dNTP Mixture (each 2.5mM), 2.0 μ L 3'RACE gene-specific Outer primer, 2.0 μ L 3'RACE Outer Primer (10 μM), 1 μ L RT reaction product, 0.5 μ L TakaRa LA Taq (5U/ μ L), 26.5 μ L Nuclease-free Water.

50 μ L Inner 3'RACE compositions：5.0μL 10×LA PCR Buffer(Mg²⁺Free), 5.0 μ L M gCl₂ (25mM), 8.0 μ L dNTP Mixture (each 2.5mM), 2.0 μ L 3'RACE gene specific in ner The μ L Outer 3'RACE PCR of primer, 2.0 μ L 3'RACE Inner Primer (10 μM), 1 product, 0.5 μ L TakaRa LA Taq (5U/ μ L), 26.5 μ L Nuclease-free Water.

Response procedures：94℃3min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1min, 35cycles；72℃7min.

(4) coupled reaction of purified fragments and cloning vector

Target DNA molecule is cloned using the pMD19-T simple Vetor of TaKaRa companies, reference explanation book, connection is anti- System is answered slightly to be improved with program.

Reaction system (5 μ L)：The PCR primer that 2.2 μ L purifying is reclaimed, 0.3 μ L pMD-19Simple Vector, 2.5 μ L Solution I。

Reaction condition：16℃30min；4 DEG C overnight.

(5) Escherichia coli convert：By fresh preparation or -70 DEG C of Escherichia coli TOP10 competent cells frozen on ice Melt；The connection product of 5 μ L purified fragments and cloning vector is taken, is added in 100 μ L competent cells, and gently mix, ice Bathe 30min or so；Thermal shock 90sec in 42 DEG C of water-baths, is immediately placed in 3-5min on ice；Add 800 μ L LB fluid nutrient mediums, 37 DEG C ＆100rmp shakes bacterium 1h；4000rmp centrifuges 3min, sops up the μ L culture mediums of upper strata 800, mixes remaining bacterium solution；Bacterium solution is applied to On LB sifting motion cultivation plates containing Amp, 37 DEG C of inversion overnight incubations.

(6) positive colony screening and sequencing analysis

From picking individual colonies on sifting motion cultivation plate are inoculated in LB fluid nutrient mediums, 37 DEG C ＆250rmp shake bacterium and stayed overnight；Directly The PCR detections of recombinant conversion are carried out by template of the bacterium solution of overnight incubation.

Reaction system (20.0 μ L)：2.0μL 10×PCR Buffer(Mg²⁺Free), 1.5 μ L MgCl₂(25mM), 1.3 μ L dNTP Mixture (each 2.5mM), 1.0 μ L 3'RACE gene specific inner primer (10 μM), 1.0 μ L 3'RACE Inner Primer (10 μM), 0.1 μ L bacterium solutions, 1.0 μ L rTaq, 12.1 μ L Milli-Q Water,

Response procedures：94℃3min；94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1min, 28cycles；72℃7min.

Clone Song Ying fine horses biotech company (Shanghai) sequencing identification of bacterium solution PCR test positive.

2) 5'RACE courses of reaction

(1) RNA processing：CIP is reacted, and following composition is added in RNase-free small centrifuge tube：10μg Total RNA, 2 μ L 10X CIP buffer, 2 μ L Calf Intestine Alkaline Phosphatase (CIP), Nuclease- Free Water to 20 μ L.

(2) gently mix, of short duration centrifugation；37 DEG C of incubation 1h；

(3) add following reagent and react centrifuge tube to CIP：15 μ L Ammonium Acetate Solution, 115 μ L Nuclease-free Water, 150 μ L acid phenol:chloroform.

(4) fully it is vortexed, room temperature high speed centrifugation (≤10000g) 5min；

(5) transfer upper strata aqueous phase adds 150 μ l chloroforms, is fully vortexed into a new centrifuge tube, room temperature high speed centrifugation (≧10000g)5min；

(6) transfer upper strata aqueous phase adds 150 μ l isopropanols, is fully vortexed into a new centrifuge tube, ice bath 10min；

(7) maximum (top) speed centrifugation 20min, is precipitated with 70% alcohol flushing of 0.5ml precoolings, maximum (top) speed centrifugation 5min, It is careful to abandon ethanol, the dry precipitation of gas；

(8) it is resuspended and is precipitated with 11 μ L Nuclease-free Water, produce CIP ' RNA, is placed be further used on ice TAP reacts, or -20 DEG C of preservations；

(9) TAP reacts, in the small centrifuge tube for following component being added to a RNase-free：5μL CIP’d RNA (from f above), 1 μ L 10X TAP buffer, 2 μ L Tobacco Acid Pyrophosphatase (TAP), 2 μ L Nuclease-free Water；

(10) gently mix, of short duration centrifugation, 37 DEG C of incubation 1h produce CIP/TAP-treated RNA；Into joint connection Step, or -20 DEG C of preservation reactants；

(11) 5'RACE joints are connected, in the small centrifuge tube for following component being added to a RNase-free：2μL CIP/TAP-treated RNA, 1 μ L 5'RACE Adapter, 1 μ L 10 × RNA Ligase Buffer, 2 μ L T4RNA Ligase (2.5U/ μ l), 4 μ L Nuclease-free Water.

(12) gently mix, of short duration centrifugation, 37 DEG C of incubation 1h produce Ligated RNA；Into reverse transcription step, or -20 DEG C preserve reactant.

3) reverse transcription

(1) following component is added in a RNase-free being placed on ice small centrifuge tube：2μL Ligated RNA, 4 μ L dNTP Mix, 2 μ L Random Decamers, 2 μ L 10X RT Buffer, 1 μ L RNase Inhibitor, 1 μ L M-MLV Reverse Transcriptase, Nuclease-free Water are mended to 20 μ L.

(2) gently mix, of short duration centrifugation；42 DEG C of incubation 1h, produce RT reaction；Into PCR steps, or -20 DEG C of guarantors Deposit reactant.

(3) 5'RACE nest-type PRCs：Reaction system, reaction condition are consistent with 3'RACE nest-type PRC.

(4) PCR primer cloning and sequencing, operation is consistent with 3'RACE clones.

Splicing is compared to 3'RACE and 5'RACE sequences, and predicts that it is read using NCBI-ORFfinder instruments Frame.According to full length gene primers (amplicon includes initiation codon and terminator codon), TcARF6 genes are carried out Full-length clone.

TcARF6ORF forward primers：5'-ATGAAGCTTTCCTCAGCAGGT-3’；

TcARF6ORF reverse primers：5'-CTAAAACTCAAGTGAATCCAG-3'.

TcARF6 full length cDNA sequences are 3704bp, and its sequence is complete comprising 2631bp as shown in SEQ ID NO.1 Whole reading frame (such as Fig. 1), the amino acid sequence of corresponding TcARF6 albumen is 877AA, and its sequence is as shown in SEQ ID NO.2.

Embodiment 2

Mutually being responded in salt stress for TcARF6 genes, the ORF based on TcARF6 genes are verified by fluorescent quantitative pcr technology Fluorescence quantification PCR primer is designed in region, and the TIFY genes design internal control primer based on Chinese tamarisk, sequence is as follows：

TcARF6 forward primers：5’-TCTGGGCATTCGGCGAGCTA-3’；

TcARF6 reverse primers：5'-GCGGCTATTTGTCGCTGCTG-3’；

TIFY forward primers：5’-TGGAGTAACTGAACCAGGGAGGAG-3’；

TIFY forward primers：5’-GGCTGTAGGTGCCTGAACTGG-3’.

Using saturable dye evagreen (Biotium companies) and quantitative real time PCR Instrument Viia7 (ABI companies), inspection in real time Survey the fluorescence intensity of PCR processes, specific PCR system is with reference to evagreen specifications, by comparing TcARF6 genes and internal reference The period of fluorescence threshold is reached to calculate the relative expression quantity of determination TcARF6 genes.Wherein, pcr template is different tissues The cDNA that mRNA reverse transcriptions are obtained, sample of tissue is from Chinese tamarisk cuttage seeding without processing, clear water processing, salt treatment 0.5 hour, salt treatment 1 hour with the salt treatment root of 4 hours, stem, leaf, obtained quantitative result as shown in figure 1, TcARF6 genes are special in Chinese tamarisk root Property expression (salt treatment 0.5 hour and 1 hour expression quantity are adjusted downward to 0.5 or so, salt treatment 4 hours is notable be lowered to 0.04), Constitutive expression (expression quantity faint change between 0.81-1.11) in leaf and stem, illustrates that the gene is specific in Chinese tamarisk root Ground responds rapidly to the salt stress in environment.

Embodiment 3TcARF6 gene plant expression vector establishments

Utilize the Overexpression vector of gateway technique construction TcARF6 genes.Use specific PCR primers (TcARF6ORF Primer), using cDNA as template, enter performing PCR amplification, TcARF6 genes ORF is building up to entry vector.Entry vector is pCRTM8/GW/TOPOTM vector(Invitrogen).Reaction system is：Fresh PCR product(purified)10- 20ng；Salt solution 1μL；pCRTM8/GW/TOPOTM vector 1μL；Plus sterile ddH₂O supplies 6 μ L.Reaction interval Sequence is：It is stored at room temperature 30min.

Picking positive colony enters performing PCR detection and sequence verification from sifting motion cultivation plate, and the introduction with TcARF6 genes is carried Body carries out LR reactions with plant expression vector pH35GS.Vector plasmid is as shown in Figure 2.Reaction system is：linearized entry clone 100ng；purified destination vector(100ng/μL)1.5μL；LR Clonase II enzyme mix 2μL；Plus TE (pH 8.0) supplies 10 μ L.Reaction condition：25℃1h.TcARF6 channel genes after being reacted through LR In plant expression vector pH35GS, in 5 ' end assembling composing type strongly expressed promoter P35S of TcARF6 genes, it can make TcARF6 genes high efficient expression in plant；Strong terminator NOS is assembled at 3 ' ends of TcARF6 genes, can effectively be terminated The transcription of TcARF6 genes；, can be with as the selection markers of genetically modified plants in vector plasmid over-assemble HPT expression casettes The screening of transfer-gen plant is carried out with hygromycin；LB and RB sequences are assembled in vector plasmid, promote to assemble TcARF6 therebetween Gene expression construct and riddled basins HPT are integrated into recipient plant cell chromosome.Detected and be sequenced by PCR and tested Card, confirms the success of overexpression vector construction, is named as pH35GS-TcARF6, the gene is located at after promoter P35S, is opening Under mover P35S driving, TcARF6 can in plant high efficient expression.

SEQUENCE LISTING

<110>Nanjing Forestry University

<120>A kind of Chinese tamarisk salt stress response key gene TcARF6 and its application

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<170> PatentIn version 3.3

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Val Ser Leu Pro Ala Val Gly Ser His Val Val Tyr Phe Pro Gln Gly

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His Ser Glu Gln Val Ala Ala Ser Thr Asn Arg Glu Val Asp Ala Gln

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Thr Pro Asn Tyr Pro Thr Leu Leu Pro Gln Leu Ile Cys Gln Leu His

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Asp Val Thr Met His Ala Asp Ile Glu Thr Asp Glu Val Tyr Ala Gln

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Pro Ala Glu Met Gly Met His Asn Lys Gln Pro Thr Asn Tyr Phe Cys

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Pro Arg Arg Ala Ala Glu Lys Val Phe Pro Pro Leu Asp Phe Ser Leu

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Gln Pro Pro Ala Gln Glu Leu Ile Ala Arg Asp Leu His Asp Asn Glu

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Trp Lys Phe Arg His Ile Phe Arg Gly Gln Pro Lys Arg His Leu Leu

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Thr Thr Gly Trp Ser Val Phe Val Ser Ala Lys Arg Leu Val Ala Gly

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Asp Ala Val Leu Phe Ile Trp Asn Glu Lys Asn Gln Leu Leu Leu Gly

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Ile Arg Arg Ala Asn Arg Pro Gln Ala Val Met Pro Ser Ser Val Ile

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Ser Ser Asp Ser Met His Leu Gly Leu Leu Ala Ala Ala Ala His Ala

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Ala Ala Thr Asn Ser Arg Phe Thr Ile Phe Tyr Asn Pro Arg Ala Cys

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Pro Ser Glu Phe Val Ile Pro Leu Ala Lys Tyr Ile Lys Ala Val His

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His Thr Arg Ile Ser Val Gly Met Arg Phe Arg Met Leu Phe Glu Thr

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Glu Glu Ser Ser Val Arg Arg Tyr Met Gly Thr Ile Thr Gly Ile Ser

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Asp Tyr Asp Pro Val Lys Trp Pro Asn Ser His Trp Arg Ser Val Lys

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Val Gly Trp Asp Glu Ser Thr Ala Gly Glu Arg Gln Pro Arg Val Ser

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

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Phe Thr Ile Gly Leu Lys Arg Pro Trp Pro Ser Gly Ile Arg Asp Asp

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Asp Leu Thr Met Asn Arg His Pro Trp Leu His Gly Asp Gly Ala Asp

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Arg Met Met Gln Ser Leu Asn Phe Gln Gly Met Gly Val Ser Pro Trp

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Met Gln Pro Arg Phe Asp Leu Ser Val Leu Gly Gln Arg Thr Asp Met

420 425 430

Tyr Gln Ala Ile Ala Ser Ala Ala Leu Gln Ser Met Arg Val Met Asp

435 440 445

Pro Ser Met Pro Thr Arg Ser Pro Leu Leu Gln Leu Pro Gln Ser Gln

450 455 460

Ile Thr Pro Gly Pro Thr Phe Met Gln Pro Gln Ser Leu Gln Gln Ser

465 470 475 480

Asp Ala His Gln Ala Phe Phe Gln Gly Ala Gln Gly Ser Met Pro Gln

485 490 495

Leu Gln Ser Gln Ala Leu Ser Gln Pro His Val Asp Leu Leu Gln Gln

500 505 510

Gln Leu Gln His Ser Gln Met Ser Leu Ser Ser Lys Leu Gln Gln Pro

515 520 525

Gln Leu Gln His Pro Gln Gln Gln Met Val Asn Ser Gln Arg Leu Ala

530 535 540

Glu Leu Val Ser Thr Val Pro Gln Phe Thr Thr Ala Ala Gln Ser Pro

545 550 555 560

Ala Pro Ser Leu Gln Thr Val Pro Ser Leu Pro His Gln Gln Ser Phe

565 570 575

Leu Asp Tyr Ser Thr Asn Ala Ala Ser Ser Thr Val Phe Ser Ser Gly

580 585 590

His Ser Val Ile Ala Ser Leu Pro Gln Ala Glu Thr Ser Asn Met Leu

595 600 605

Asp Met Val Gly Thr Asp Ser Phe Val Ser Ser Ala Ala Cys Gln Gln

610 615 620

Lys Gln Ala Asp Thr Leu Pro Leu Ile Glu Gln Ser Ser Glu Thr His

625 630 635 640

Tyr Gln Asn Ile Val Ser Leu Pro Pro Phe Pro Gly Lys Glu Cys Ser

645 650 655

Val Gly Glu Asp Gly Ser Thr Val Pro Gln Ser His Val Leu Phe Arg

660 665 670

Gly Ser Val Asp Ser Ser Ser Ile Met Val His Asn Gly Leu Arg Val

675 680 685

Val Gly Thr His Gly Glu Ser Ser Asp Ile Pro Phe Ser Ile Ser Asn

690 695 700

Phe Pro Gly Pro Gly Ala Asp Tyr Gly Pro Asn Pro Ala Thr Thr Ser

705 710 715 720

Ser Ser Phe Val Asp Glu Ala Pro Val Ile His Thr Ser Asp Asn Leu

725 730 735

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

740 745 750

Lys Leu Gly Ser Cys Gly Arg Leu Leu Asp Ile Thr Lys Phe Ser Ser

755 760 765

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

770 775 780

Gln Leu Glu Ser Arg Arg Ser Gly Trp Gln Leu Val Tyr Val Asp Lys

785 790 795 800

Glu Asn Asp Val Leu Leu Leu Gly Asp Asp Pro Trp Thr Gly Phe Val

805 810 815

Asn Ser Val Ser Cys Ile Lys Ile Leu Ser Pro Leu Glu Val Gln Gln

820 825 830

Met Asp Arg Leu Gly Leu Glu Leu Leu Asn Ser Ala Pro Ser Ser Thr

835 840 845

Leu Ser Asn Gly Thr Tyr Glu Asp Tyr Ser Ser Arg Gln Asp Pro Pro

850 855 860

Ser Ala Arg Ile Ala Cys Leu Asp Ser Leu Glu Phe

865 870 875

<210> 3

<211> 45

<212> DNA

<213> Artificial

<220>

<223>Adaptor Primer sequences

<400> 3

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 4

<211> 25

<212> DNA

<213> Artificial

<220>

<223>3'RACE forward primer Outer Primer

<400> 4

tacagcatcc tcaacagcaa atggt 25

<210> 5

<211> 22

<212> DNA

<213> Artificial

<220>

<223>3'RACE forward primer Inner Primer

<400> 5

tagacatggt tggtacagac tc 22

<210> 6

<211> 45

<212> DNA

<213> Artificial

<220>

<223>3'RACE reverse primer Outer Primer

<400> 6

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 7

<211> 22

<212> DNA

<213> Artificial

<220>

<223>3'RACE reverse primer Inner Primer

<400> 7

ctaatacgac tcactatagg gc 22

<210> 8

<211> 45

<212> DNA

<213> Artificial

<220>

<223>5'RACE forward primer Outer Primer

<400> 8

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 9

<211> 22

<212> DNA

<213> Artificial

<220>

<223>5'RACE forward primer Inner Primer

<400> 9

ctaatacgac tcactatagg gc 22

<210> 10

<211> 26

<212> DNA

<213> Artificial

<220>

<223>5'RACE reverse primer Outer Primer

<400> 10

accatcgggc tcttccagtg tagtta 26

<210> 11

<211> 22

<212> DNA

<213> Artificial

<220>

<223>5'RACE reverse primer Inner Primer

<400> 11

aatctgtctt tcggctatct cc 22

<210> 12

<211> 21

<212> DNA

<213> Artificial

<220>

<223>TcARF6 ORF forward primers

<400> 12

atgaagcttt cctcagcagg t 21

<210> 13

<211> 21

<212> DNA

<213> Artificial

<220>

<223>TcARF6 ORF reverse primers

<400> 13

ctaaaactca agtgaatcca g 21

<210> 14

<211> 20

<212> DNA

<213> Artificial

<220>

<223>TcARF6 forward primers

<400> 14

tctgggcatt cggcgagcta 20

<210> 15

<211> 20

<212> DNA

<213> Artificial

<220>

<223>TcARF6 reverse primers

<400> 15

gcggctattt gtcgctgctg 20

<210> 16

<211> 24

<212> DNA

<213> Artificial

<220>

<223>TIFY forward primers

<400> 16

tggagtaact gaaccaggga ggag 24

<210> 17

<211> 21

<212> DNA

<213> Artificial

<220>

<223>TIFY forward primers

<400> 17

ggctgtaggt gcctgaactg g 21

Claims (8)

1. a kind of Chinese tamarisk salt stress responds key gene TcARF6, its nucleotide sequence is as shown in SEQ ID NO.1.

2. the expressing protein of the Chinese tamarisk salt stress response key gene TcARF6 described in claim 1, its amino acid sequence such as SEQ Shown in ID NO.2.

3. the carrier containing the Chinese tamarisk salt stress response key gene TcARF6 described in claim 1.

4. carrier according to claim 2, it is characterised in that：5 ' end assembling composing types of the carrier in TcARF6 genes Strongly expressed promoter P35S.

5. carrier according to claim 2, it is characterised in that：The carrier assembles strong end at 3 ' ends of TcARF6 genes Only sub- NOS.

6. carrier according to claim 2, it is characterised in that：The carrier assembles HPT expression casettes, is used as transgenosis The selection markers of plant, the screening of genetically modified plants is carried out with hygromycin.

7. carrier according to claim 2, it is characterised in that：The carrier assembles LB and RB sequences, promotes to be assembled in it Between TcARF6 gene expression constructs and riddled basins HPT be integrated into dicotyledon recipient cell chromosome.

8. applications of the salt stress response key gene TcARF6 in plant resistance to environment stress breeding described in claim 1.