CN110184278A - A kind of Chinese tamarisk salt stress responsive genes TcNAC2 and its miRNA resistance target rTcNAC2 and application - Google Patents

Abstract

The invention discloses a kind of Chinese tamarisk salt stress responsive genes TcNAC2 and its miRNA resistance target rTcNAC2 and applications.The nucleotide sequence of the Chinese tamarisk salt stress responsive genes TcNAC2 expresses the amino acid sequence of albumen as shown in SEQ ID No.2 as shown in SEQ ID No.1.The present invention, by the miR164 response element same sense mutation of TcNAC2 gene, obtains miR164 resistance target rTcNAC2, nucleotide sequence is as shown in SEQ ID No.3 by big primer mutagenesis technology.Under salt stress, TcNAC2 is by the post-transcriptional control of miR164, and rTcNAC2 is not regulated and controled then by it.Chinese tamarisk salt stress responsive genes TcNAC2 and its miR164 resistance target rTcNAC2 has important value in the research and application in plant salt endurance or forest resistance breeding field.

Description

A kind of Chinese tamarisk salt stress responsive genes TcNAC2 and its miRNA resistance target rTcNAC2 And application

Technical field

The invention belongs to field of plant genetic project technology, and in particular to a kind of Chinese tamarisk salt stress responsive genes TcNAC2 and The application of its miRNA resistance target rTcNAC2.

Background technique

China cultivates Salinity tolerance germplasm there are the salt-soda soil that large area can not efficiently use, by breeding for stress tolerance method, can be with Increase cultivable ground area, improve grain yield work.Although having illustrated part in arabidopsis, rice isotype species Plant salt tolerance mechanism excavates related resistant gene of salt, but effect is not significant in resistance breeding application, genetically modified plants salt tolerance The effect is unsatisfactory for raising, is not able to satisfy demand (Zhu, 2002) in actual production.Compared to herbaceous plant and grass family Crop, salt tolerant tree species can on salinization soil, coastal tidal normal growth, have unique high salt-tolerant trait, salt tolerant Mechanism is the theoretical basis of breeding for stress tolerance, the directive significance that relevant resistant gene of salt resource cultivates the crop varieties of high-salt tolerance It is huge.About 90 kinds of Tamarix (Tamarix) plant, main distributed area concentrates on Asia and north African, northwest China and North China 18 kinds are shared, there is 1 mutation athel tree in Taiwan.Chinese Chinese tamarisk (Tamarix chinensis Lour.) is China's Gui Distributed area is most wide in sallow, is grown thickly in the form of shrub in inland Desert Regions and coastal tidal, the Liao Dynasty, Ji, Henan, Shandong, northern Suzhou, The ground such as Wan Bei, Xinjiang have wild population to be distributed (Zhang Pengyun, 1990).Chinese seed, the anatomic form of leaf and cauline leaf surface Salt glandular structure be adaptable to the characteristic feature of salt marsh, drought environment, can on 2% salt-soda soil normal growth, be salt tolerant One of strongest tree species of property.The Chinese unique resistance to characteristic with high salt of Chinese tamarisk, has only parsed related mechanism in physiology level at present, has such as secreted The mechanism such as salt, ion compartment, but the understanding of its molecular level is less, has cloned in other species of Tamarix at present To tens degeneration-resistant relevant transcription factors, but there has been no the separation of resistant gene of salt identifications to report for Chinese Chinese tamarisk, is badly in need of carrying out Correlative study.It excavates Chinese salt stress to respond relevant transcription factor gene and study its expression regulation mode, can not only add Understand Chinese tamarisk salt tolerant molecule mechanism deeply, while molecular tool can be provided for salt lick desalination, adversity resistant plant breeding,

NAC transcription factor family is distributed widely in higher plant, and number of members is huge, there is 138 members in arabidopsis. The N-terminal of NAC transcription factor is more conservative, and containing conservative NAC structural domain, C-terminal alterable height plays transcriptional activation and widow Dimerization effect, determines that NAC is functioned in the form of homologous or heterodimer.NAC family function is extensive, participates in the non-of plant The bioprocess such as biotic response, orga- nogenesis, secondary tissue development, fruit maturation, leaf senile, due to enormous amount, (Olsen et al., 2005) is still not clear in many member's functions.In the relevant NAC of abiotic stress, many NAC are reported as resistance to The positive regulatory factor of salt, in Tamarix, two Tamarix hispida (Tamarix hispida) NAC genes have carried out function and have ground Study carefully, wherein Tamarix hispida ThNAC23 heterogenous expression improves arabidopsis salt stress and osmotic stress tolerance ability (Wang, Li, Lu And Wang, 2017), the similar ThNAC23 of the overexpression of Tamarix hispida ThNAC7 improve plant salt tolerance (Zhang Mingyi, 2015).It is salt tolerance negative regulatory factor that part research, which reports NAC, such as the target gene NAC negative regulation of a miR164 in rice Paddy rice anti contravariance, three NAC, which coerce lower expression patterns, in diversiform-leaved poplar inconsistent or even opposite situation, and one of NAC is in root Middle negative regulation salt tolerance.Recent study shows the NAC gene containing miR164 response element, more show with it is abiotic Coerce closely related relationship, as arabidopsis AtNAC4 (ANAC080) be initially reported for influence lateral root occur and leaf senile into Journey, the nutrients such as research in recent years discovery and N, P coerce closely related and contain conservative miR164 response element (Lee et Al., 2016).

The above results show that NAC plays an important role in terms of plant salt stress response, and by the transcription of miR164 after The influence of regulation.Currently, there has been no the NAC transcription factor relevant report of Chinese Chinese tamarisk, clone and the Chinese Chinese tamarisk NAC of development and utilization Transcription factor, not only facilitates the molecular mechanism for illustrating its height salt tolerant, and can be to screen important resistant gene of salt and degeneration-resistant Genetic breeding is provided fundamental basis and molecular tool, utilizes to salt-soda soil and overall productivity in agriculture is improved with important valence Value.

Summary of the invention

Goal of the invention: being directed to the deficiencies in the prior art, and the object of the present invention is to provide a kind of Chinese tamarisk salt stress to ring Gene TcNAC2 is answered, the application demand of plant salt endurance or forest resistance breeding is met.It is a further object of the present invention to provide one The above-mentioned improved miRNA resistance target rTcNAC2 of Chinese tamarisk salt stress responsive genes TcNAC2 of kind.Further object of the present invention is The application of above-mentioned Chinese tamarisk salt stress responsive genes TcNAC2 or miRNA resistance target rTcNAC2 a kind of is provided.

Technical solution: to solve the above-mentioned problems, the technical solution adopted in the present invention is as follows:

A kind of Chinese tamarisk salt stress responsive genes TcNAC2, nucleotide sequence is as shown in SEQ ID No.1.

The expression albumen of the Chinese tamarisk salt stress responsive genes TcNAC2, amino acid sequence is as shown in SEQ ID No.2.

A kind of miR164 resistance target rTcNAC2 is rung by the Chinese tamarisk salt stress responsive genes TcNAC2 by miR164 Element same sense mutation is answered to obtain, nucleotide sequence is as shown in SEQ ID No.3.

Carrier containing the Chinese tamarisk salt stress responsive genes TcNAC2.

Carrier containing the miR164 resistance target rTcNAC2.

Preferably, the Chinese tamarisk salt stress responsive genes TcNAC2 carrier is Dual-Luciferase report carrier 35sGLO: TcNAC2 is equipped with composing type strongly expressed promoter P35S and firefly in 5 ' end upstream groups of the miR164 response element of TcNAC2 Luciferase reporter gene, 3 ' end downstream groups are equipped with Renilla luciferase reporter gene.

Preferably, the carrier of the miR164 resistance target rTcNAC2 is Dual-Luciferase report carrier 35sGLO: RTcNAC2 is equipped with composing type strongly expressed promoter in 5 ' end upstream groups of the miR164 response element same sense mutation of rTcNAC2 P35S and firefly luciferase reporter gene, 3 ' end downstream groups are equipped with Renilla luciferase reporter gene.

Preferably, the carrier of the miR164 resistance target rTcNAC2 is the over-express vector PBI121GW of rTcNAC2: RTcNAC2 assembles 35S promoter；NPTII expression casette is assembled, LB and RB sequence is assembled, is between LB and RB sequence RTcNAC2 gene expression construct and gene NPTII.

The expression egg of the Chinese tamarisk salt stress responsive genes TcNAC2 or the Chinese tamarisk salt stress responsive genes TcNAC2 Application of the carrier of the white or described Chinese tamarisk salt stress responsive genes TcNAC2 in plant salt endurance or forest resistance breeding.

The carrier of the miR164 resistance target rTcNAC2 or the miR164 resistance target rTcNAC2 are in plant Application in salt tolerance or forest resistance breeding.

The utility model has the advantages that compared with prior art, the present invention for material, is led to Chinese Chinese tamarisk (Tamarix chinenses) It crosses RACE technology and has cloned TcNAC2 gene；The miR164 response element of TcNAC2 is identified by Dual-luciferase reportor systerm Part；Spatial and temporal expression profile of the TcNAC2 gene under salt stress is had detected by Real-Time Fluorescent Quantitative PCR Technique；Pass through big primer MiR164 response element same sense mutation is obtained miR164 resistance target rTcNAC2 by mutating technology.Under salt stress, The miR164 response element of TcNAC2 is regulated and controled by miR164, and TcNAC2 is specifically lowered rapidly in Chinese tamarisk root, and mutation should RTcNAC2 after element is not by the post-transcriptional control of miR164, Chinese tamarisk salt stress responsive genes TcNAC2 and its miR164 resistance Target rTcNAC2 has significant application value in plant salt endurance or forest resistance breeding field.

Detailed description of the invention

Fig. 1 is the expression pattern figure under TcNAC2 gene salt stress；

Fig. 2 is luciferase reporter gene carrier 35sGLO structure chart；

Fig. 3 is the miR164 response element fluorescence detection figure of TcNAC2 gene；

Fig. 4 is over-express vector PBI121GW structure chart.

Specific embodiment

The present invention is further described below combined with specific embodiments below.Do not make point illustrated in following embodiment Sub- biological experimental method can refer to side listed in one book of " Molecular Cloning:A Laboratory guide " (third edition) J. Pehanorm Brooker Method or the conventional method of this field carry out, or carry out according to kit and product description.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Embodiment 1: TcNAC2 gene is cloned by RACE technology

Based on the TcNAC2 transcript of Chinese tamarisk RNA-seq data, the RACE primer of design 5 ' and 3 ' is expanded by nest-type PRC Increase two sections of specific products, be sequenced by T- carrier cloning, sequencing result is spliced by overlay region, obtains cNDA overall length.Specifically It is as follows:

I. design of primers

3 ' RACE forward primers:

Outer Primer:5 '-GAGGTGATGAAGAACCAATAGAG-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 '-TATGGCCTTTTGGAGCCCGCCCCTT-3 '；

Inner Primer:5 '-TTACGCTCACCACTTGCTC-3 '.

II.3 ' RACE reaction process:

(1) following component is added in the small centrifuge tube for the RNase-free that one is placed on ice by reverse transcription: 1 μ g Total RNA (vegetable material be Chinese tamarisk blade), 4 μ L dNTP Mix, 2 μ L 3 ' RACE Adapter, 2 μ L 10X RT Buffer, 1 μ L RNase Inhibitor, 1 μ L M-MLV Reverse Transcriptase, Nuclease-free Water is mended to 20 μ L.

(2) it mixes gently, of short duration centrifugation, 42 DEG C incubate 1 hour, into PCR step；

(3) 3 ' RACE nest-type PRCs；

3 ' RACE Outer PCR reaction systems (50 μ L) composition: 5.0 μ L 10 × LAPCR Buffer (Mg²⁺Free), 5.0μL MgCl₂(25mM), 8.0 μ L dNTP Mixture (each 2.5mM), 2.0 μ L, 3 ' RACE Outer PrimerF (10 μM), 2.0 μ L, 3 ' Outer PrimerR (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.

3 ' RACE Inner PCR reaction systems (50 μ L) composition: 5.0 μ L 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 Inner PrimerF (10 μM), 2.0 μ L 3 ' RACE Inner PrimerR (10 μM), 1 μ L Outer 3 ' RACE PCR product, 0.5 μ L TakaRa LA Taq (5U/ μ L), 26.5 μ L Nuclease-free Water.

Response procedures: 94 DEG C 3 minutes；94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 1 minute, 35 cycles；72 DEG C 7 points Clock.

(4) the connection reaction of purified fragments

Target DNA molecule, reaction system (5 μ L): 2.2 are cloned using the pMD19-T simple Vetor of TaKaRa company The PCR product of μ L purification and recovery, 0.3 μ L pMD-19 Simple Vector, 2.5 μ L Solution I.Reaction condition: 16 DEG C 30 minutes；4 DEG C overnight.

(5) Escherichia coli convert: on ice by fresh preparation or -70 DEG C of Escherichia coli TOP10 competent cells frozen Melt；The connection product for taking 5 μ L purified fragments and cloning vector is added in 100 μ L competent cells, and mixes gently, ice Bath 30 minutes；Thermal shock 90 seconds in 42 DEG C of water-baths is immediately placed in 3-5 minutes on ice；It is added 800 μ LLB fluid nutrient mediums, 37 DEG C 100 Rev/min shake bacterium 1 hour；4000 revs/min are centrifuged 3 minutes, sop up 800 μ L culture medium of upper layer, mix remaining bacterium solution；By bacterium solution It is applied on the LB sifting motion cultivation plate containing Amp, 37 DEG C of inversion overnight incubations.

(6) positive colony screening and sequencing analysis

It is inoculated in LB liquid medium from picking individual colonies on sifting motion cultivation plate, 37 DEG C 250 revs/min are shaken bacterium and stayed overnight； Directly detected using the PCR that the bacterium solution of overnight incubation carries out recombinant conversion as template.

Reaction system (20 μ L): 2.0 μ L 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 solution, 1.0 μ L rTaq, 12.1 μ L Milli-Q Water.Response procedures: 94 DEG C 3 minutes；94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 1 minute, 28 cycles；72 DEG C 7 minutes.

Positive colony is sequenced to obtain base sequence by Sanger PCR sequencing PCR.

III.5 ' RACE reaction process

(1) RNA is handled: following ingredient is added in the small centrifuge tube of RNase-free: 10 μ g Total by CIP reaction RNA, 2 μ L 10X CIP buffer, 2 μ L Calf Intestine Alkaline Phosphatase (CIP), Nuclease- Free Water to 20 μ L.

(2) it mixes gently, of short duration centrifugation；37 DEG C incubate 1 hour；

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

(4) sufficient vortex, room temperature high speed centrifugation (>=10000g) 5 minutes；

(5) 150 μ L chloroforms, sufficient vortex, room temperature high speed centrifugation is added into a new centrifuge tube in transfer upper strata aqueous phase (>=10000g) 5 minutes；

(6) 150 μ L isopropanols, sufficient vortex are added into a new centrifuge tube in transfer upper strata aqueous phase, and ice bath 10 divides Clock；

(7) maximum (top) speed is centrifuged 20 minutes, rinses precipitating with 70% ethyl alcohol of 0.5ml pre-cooling, maximum (top) speed is centrifuged 5 points Clock carefully abandons ethyl alcohol, the dry precipitating of gas；

(8) precipitating is resuspended to get CIP ' RNA with 11 μ L Nuclease-free Water, places be further used on ice TAP reaction, or -20 DEG C of preservations；

(9) TAP reacts, and following component is added in the small centrifuge tube of a RNase-free: 5 μ LCIP ' d RNA, 1 μ L 10X TAP buffer, 2 μ L Tobacco Acid Pyrophosphatase (TAP), 2 μ L Nuclease-free Water；

(10) it mixes gently, of short duration centrifugation, 37 DEG C incubate 1 hour to get CIP/TAP-treated RNA；Into connector Connection Step, or -20 DEG C of preservation reactants；

The connection of (11) 5 ' RACE connectors, is added to following component in the small centrifuge tube of one RNase-free: 2 μ L CIP/TAP-treated RNA, 1 μ L 5 ' RACE Adapter, 1 μ L 10 × RNA Ligase Buffer, 2 μ L T4 RNA Ligase (2.5U/ μ L), 4 μ L Nuclease-free Water.

(12) it mixes gently, of short duration centrifugation, 37 DEG C incubate 1 hour to get Ligated RNA；Into reverse transcription step, Or -20 DEG C of preservation reactants.

(13) following component is added in the small centrifuge tube for the RNase-free that one is placed on ice: 2 μ L 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.

(14) it mixes gently, of short duration centrifugation；42 DEG C incubate 1 hour to get RT reaction；Into PCR step, or -20 DEG C save reactant.

(15) 5 ' RACE nest-type PRCs: reaction system, reaction condition are consistent with the nest-type PRC of 3 ' RACE.

(16) PCR product cloning and sequencing, operation are consistent with 3 ' RACE clone.

IV.ORF amplification

3 ' RACE and 5 ' RACE sequences are spliced, and predict its reading frame using NCBI-ORF finder tool.Root According to full length gene primers (amplicon includes initiation codon and terminator codon), the overall length of TcNAC2 gene is carried out Clone.Wherein, TcNAC2 ORF forward primer: 5 '-ATGACCATGATTACGCCAAG-3 ', TcNAC2 ORF reverse primer: 5 '-TCAGTTACAATAACTCCAAAAC-3 ', High fidelity PCR reaction system are as follows: 10 × LA PCR Buffer, 5.0 μ L； 2.5mM dNTP Mixture 8.0μL；25mM Mg²⁺5.0μL；LA Taq DNA Polymerase(5U/μL)0.5μL；Just To 2 μ L of primer (10 μM)；2 μ L of reverse primer (10 μM)；1 μ L of template (Chinese tamarisk cDNA)；Sterile ddH2O is added to supply 50 μ L.Reaction Program: 94 DEG C of initial denaturation 3 minutes-(94 DEG C 40 seconds -55 DEG C 30 seconds -72 DEG C 30 seconds) -72 DEG C of × 35 circulations 10 minutes.

TcNAC2 full length cDNA sequence is 1579bp, and sequence is complete comprising 1362bp as shown in SEQ ID No.1 The amino acid sequence of whole reading frame (CDS), corresponding TcNAC2 albumen is 453aa, and sequence is as shown in SEQ ID No.2.

Embodiment 2: TcNAC2 gene expression pattern is analyzed by quantitative fluorescent PCR

The salt stress response of TcNAC2 gene, the region CDS based on TcNAC2 gene are verified by fluorescent quantitative PCR technique Fluorescence quantification PCR primer is designed, the TIF gene based on Chinese tamarisk designs internal control primer, and sequence is as follows:

TcNAC2 forward primer: 5 '-GATCCAAGCTCGCGGAAGCC-3 '；

TcNAC2 reverse primer: 5 '-CACGAATGTCGGTGGCCGAT-3 '；

TIF forward primer: 5 '-ACCACAGGAGTGTCCACCACA-3 '；

TIF reverse primer: 5 '-TGATGCTTTGCGTGCCAGTG-3 '.

Using saturable dye evagreen (Biotium company) and fluorescence quantitative PCR instrument Viia7 (ABI company), examine in real time Survey the fluorescence intensity of PCR process, specific PCR system is referring to evagreen specification, by comparing TcNAC2 gene and internal reference Reach the recurring number of fluorescence threshold to calculate the relative expression quantity of determining TcNAC2 gene.Wherein, pcr template is different tissues The cDNA that mRNA reverse transcription obtains, sampling come from Chinese tamarisk cuttage seeding, salt treatment 0.5 hour, 1 hour root, stem, leaf, with no side of body The root for compeling processing is control, calculates relative quantification, as a result as shown in Figure 1, TcNAC2 gene significantly lowers (the salt side of body in Chinese tamarisk root Compel expression quantity after lowering about 0.5,1 hour 0.5 hour to lower significantly, abundance is difficult to detect close to zero), the table in leaf and stem Up to measuring lower and being constitutive expression (expression quantity faint variation between 0.05-0.2), illustrate TcNAC2 specifically in Chinese tamarisk Salt stress is responded rapidly in root.It separately measures Chinese tamarisk miR164 significantly to be induced by salt stress, TcNAC2 and miR164 show as the salt side of body Compel lower expression negative correlativing relation, shows that TcNAC2 gene may be by the regulation of Chinese tamarisk miR164.

Embodiment 3

Multipoint mutation is carried out using large primer PCR method, after the miR164 response element same sense mutation for obtaining TcNAC2 ORF, the specific steps are as follows:

According to Codon degeneracy, same sense mutation primer is designed, introduces mutational site as much as possible, but not have more than The poly structure of 3 bases, while flank retains at least complementary base of 10nt, TcNAC2 mutant primer is 5 '-ACAGAATCAT CTCATGTAAGCTGTTTCAGTAATCCCATGATC-3 ' passes through TcNAC2 mutant primer and implementation in a wheel PCR reaction The TcNAC2 ORF reverse primer of example 1, the wheel PCR product segment expanded.It is anti-using a wheel in two wheel PCR reactions Answer product as big primer, the TcNAC2 ORF forward primer of embodiment 1 carries out PCR and obtains the resistance for introducing multisite mutation It is as follows that target rTcNAC2 segment, the first round and the second wheel PCR are all made of High fidelity PCR reaction system: 10 × LA PCR Buffer5.0μL；2.5mM dNTP Mixture 8.0μL；25mM Mg²⁺5.0μL；LA Taq DNA Polymerase(5U/ μL)0.5μL；2 μ L of forward primer (10 μM)；2 μ L of reverse primer (10 μM)；1 μ L of template (Chinese tamarisk cDNA)；Add sterile ddH₂O is mended 50 μ L of foot.Response procedures: 94 DEG C of initial denaturation 3 minutes-(94 DEG C 40 seconds -55 DEG C 30 seconds -72 DEG C 30 seconds) × 35 circulations -72 DEG C 10 minutes.Second wheel PCR product is connected into carrier T, carries out cloning and sequencing after converting Escherichia coli, recycling includes positive piece The carrier T of section.

Sequencing obtains the miR164 resistance target rTcNAC2 of TcNAC2, and nucleotide sequence is as shown in SEQ ID No.3.

The miRNA response element of embodiment 4:TcNAC2 gene is identified

Embodiment 2 the result shows that TcNAC2 gene may be by the regulation of Chinese tamarisk miR164, binding sequence is complementary, in advance It measures TcNAC2 gene and contains a miR164 response element (5 '-TCACGTGTCCTGCTTCTCCA-3 '), embodiment 3 uses Large primer PCR method carries out multipoint mutation, ORF, i.e. rTcNAC2 after obtaining the miR164 response element same sense mutation of TcNAC2. The present embodiment measures fluorescence intensity change, passes through quantitative miRNA164 by building luciferase reporter gene carrier (Fig. 2) Interaction intensity between response element verifies TcNAC2 response element.

It is specific as follows:

I.35sGLO:TcNAC2 construction of recombinant vector

1) oligonucleotides designs

TcNAC2 response element flank adds restriction enzyme site, according to two pairs of oligonucleotides of design claimed below:

TcNAC2 positive-sense strand: restriction enzyme site A+Notl restriction enzyme site+TcNAC2 response element+restriction enzyme site B residue； TcNAC2 antisense strand: restriction enzyme site B+ antisense TcNAC2 response element+Notl restriction enzyme site+restriction enzyme site A residue；

RTcNAC2 positive-sense strand: restriction enzyme site A+Notl restriction enzyme site+rTcNAC2 mutant nucleotide sequence+restriction enzyme site B residue； RTcNAC2 antisense strand: restriction enzyme site B+ antisense rTcNAC2 mutant nucleotide sequence+Notl restriction enzyme site+restriction enzyme site A residue.

2) double digestion combination selection

There are a variety of enzymes combinations to select for the multiple cloning sites of 35sGLO, avoids isocaudarner and isoschizomers in principle, such as SalI and XhoI, XbaI and NheI, and two restriction enzyme sites are apart from not too near, the protection base of flank at least 6nt.NheI (restriction enzyme site A) and SalI (restriction enzyme site B) meet the above rule, can carry out efficient double digestion.Utilize NheI (restriction enzyme site A) and SalI (restriction enzyme site B) is restriction enzyme site, and design oligonucleotides is as follows, utilizes PAGE synthetic oligonucleotide

TcNAC2+:5 '-CTAGATAGCGGCCGCTATTCACGTGTCCTGCTTCTCCAG-3 '；

TcNAC2-:5 '-TCGACTGGAGAAGCAGGACACGTGAATAGCGGCCGCTAT-3 '；

RTcNAC2+:5 '-CTAGATAGCGGCCGCTATCATGTAAGCTGTTTCAGTAG-3 '；

RTcNAC2-:5 '-TCGACTACTGAAACAGCTTACATGATAGCGGCCGCTAT-3 '.

3) 35sGLO vector linearization

Theoretical best digestion system (50 μ L): 1 μ is obtained by NEBcloner (https: //nebcloner.neb.com) L 35sGLO (1 μ g/ μ L), 5 μ L 10X CutSmart Buffer, 0.5 μ L NheI (20U/ μ L), 0.5 μ L SalI (20U/ μ L), 43 μ L Nuclease-free Water.

It prepares reaction system (50 μ L), piping and druming mixes, 37 DEG C of incubation 5-15min, electrophoresis detection digestion products, and recycling is linear Change plasmid.

4) Insert Fragment is obtained

In 2 0.2ml centrifuge tubes, according to following system (50 μ L), two pairs of oligonucleotide hybridizations are carried out respectively:

2 μ L TcNAC2+/rTcNAC2+ (1 μ g/ μ L), 2 μ L TcNAC2-/rTcNAC2- (1 μ g/ μ L), 46 μ L Oligo Annealing Bufier。

TcNAC2 the and rTcNAC2 hybrid product obtained after 90 DEG C of reactions 3min, 37 DEG C of annealing 15min, as insertion piece Section.

5) carrier recombinates

It prepares T4 (NEB) linked system (10 μ L): 1 μ L TcNAC2 or rTcNAC2 hybrid product (4ng/ μ L), 0.5 μ L T4 ligase (400U/ μ L), 1 μ L T4 10X bufier, X μ L linearize 35sGLO (50ng), 7.5-X μ L ddH₂O。

25 DEG C of incubation 10min (or 16 DEG C of connections overnight)

Plasmid is extracted after conversion, the segment that NotI digestion obtains 140bp or so is positive plasmid.

6) after positive plasmid sequence verification is correct, 35sGLO:TcNAC2 and 35sGLO:rTcNAC2 recombinant vector is obtained.

The building of II.miR164 transient expression vector

By gateway system, Chinese tamarisk miR164 (5 '-UGACAGAAGAGAGUGAGCAC-3 ') are inserted into and are opened with 35S The P2GW7 carrier of mover constructs miRNA transient expression vector.

It is reacted first with BP, miR164 segment is inserted into PCR8/GW/TOPO carrier, sequence verification Insert Fragment obtains MiR164 entry vector.Then it is reacted, is passed through using LRLR Clonase^TMII Enzyme Mix kit, into The recombining reaction of row miR164 entry vector and the P2GW7 overexpression vector suitable for plant protoplast, sequence verification insertion Segment extracts positive plasmid, obtains P2GW7:miR164a recombinant vector.

III. it transfects protoplast and measures fluorescence intensity

By control group: the 35sGLO:TcNAC2 carrier of 9 μ L；

TcNAC2 group: 35sGLO:TcNAC2 carrier (the 1 μ g/ μ of mi164:P2FGW7 carrier (the 1 μ g/ μ L) and 3 μ L of 6 μ L L)；

RTcNAC2 group: 35sGLO:rTcNAC2 carrier (the 1 μ g/ of mi164:P2FGW7 carrier (the 1 μ g/ μ L) and 3 μ L of 6 μ L μL)；

Cotransfection is distinguished to 100 μ L poplar protoplasts (6 × 10⁵/ ml) in, it is repeated 4 times, room temperature dark culturing 16~ After sufficiently being transfected for 24 hours, fluorescence detection is carried out.

1) luciferase reaction substrate (Promega E1910 kit) is configured, on ice for use；

LAR is prepared: 10mL Luciferase Assay Buffer II dissolves Luciferase Assay Substrate is mixed, current or -70 freeze；

Stop&Glo is prepared: Stop&Glo Buffer and Substrate is prepared according to 50: 1, is mixed, ready-to-use, or Person -20 freezes；

2) protoplast after transfection is collected by centrifugation in 4000rpm, and 100 μ L 1X Passive Lysis Buffer are added 100ml cracking precipitating；

3) transfection liquid after cracking is transferred to 96 hole elisa Plates holes (100 μ L), control group, TcNAC2 group and rTcNAC2 group It is each to repeat 4 holes；

4) Glomax-96 Chemiluminescence Apparatus power supply is connected, GloMax software is opened, according to software document, is established GloMax automatic assay program measures fluorescence intensity；

The fluorescence intensity of control group is set as 100%, TcNAC2 group and rTcNAC2 group relative intensity of fluorescence is calculated, It is plotted as histogram, such as Fig. 3, it can be seen that TcNAC2 group fluorescence is significantly suppressed, and illustrates that TcNAC2 is pressed down by Chinese tamarisk miR164 System, and rTcNAC2 group fluorescence reverts to 34%, the rTcNAC2 after illustrating mutation is released to a certain degree by miR164 inhibition, The miR164 response element for demonstrating TcNAC2 is inhibited by miR164.

Embodiment 5:rTcNAC2 gene plant expression vector establishment

Utilize the Overexpression vector of gateway cloning technology building rTcNAC2 gene.Use specific PCR primers (embodiment 1 TcNAC2 ORF primer), using in embodiment 3 include rTcNAC2 segment carrier T as template, carry out PCR amplification, will RTcNAC2 gene ORF is building up to entry vector.Entry vector is pCR8/GW/TOPOTM vector (Invitrogen).Reaction System are as follows: Fresh PCR product (purified) 10-20ng；Salt solution 1μL；pCR8/GW/TOPOTM vector 1μL；Add sterile ddH₂O supplies 6 μ L.Response procedures are as follows: be stored at room temperature 30 minutes.

Picking positive colony carries out sequence verification, positive entry vector and plant expression vector from sifting motion cultivation plate PBI121GW carries out LR reaction.Vector plasmid is as shown in Figure 4.Reaction system are as follows: entry vector 100ng；PBI121GW 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 1 hour.After LR reacts, in rTcNAC2 gene transfered plant expression vector PBI121GW.PBI121GW is assembled with access AttR1 and attR2 element is cloned, rTcNAC2 gene expression frame is quickly assembled and ensures accurate translation；It is assembled with LB and RB sequence Column, the rTcNAC2 gene and riddled basins NPTII for promoting to assemble therebetween are integrated into the arabidopsis thaliana chromosome infected. In addition, it can make TcNAC2 high efficient expression in 5 ' end assembling composing type strongly expressed promoter P35S of rTcNAC2 gene.Pass through PCR detection and sequence verification, confirmation overexpression vector construction success, are named as PBI121GW:rTcNAC2, which is located at After promoter P35S, under the driving of promoter P35S, rTcNAC2 can in plant high efficient expression.

Since rTcNAC2 does not contain miR164 response element, by the carrier, the gene is in the intracorporal expression water of plant It is flat not by miR164 post-transcriptional control, it is forest transgenic salt-tolerant wheat research that the salt stress response effectiveness after overexpression is more significant Important molecule tool.

Described above to be merely exemplary for the purpose of the present invention, and not restrictive, those of ordinary skill in the art understand, In the case where not departing from spirit and scope as defined in the appended claims, many modifications, variation or equivalent can be made, but all Protection scope of the present invention will be fallen into.

Sequence table

<110>Nanjing Forestry University

<120>a kind of Chinese tamarisk salt stress responsive genes TcNAC2 and its miRNA resistance target rTcNAC2 and application

<130> 100

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1579

<212> DNA

<213> Tamarix chinenses

<400> 1

tcacacagga aacagctatg accatgatta cgccaagctc agaattaacc ctcactaaag 60

ggactagtcc tgcaggttta aacgaattgg cccttctaat acgactcact atagggcaag 120

cagtggtatc aacgcagagt acatggggat tttactcttt ttttttctct gacaaaagaa 180

gaagaagaag aagaaggatt agtattctta ttattagaag gaaagaagaa atggtaaaca 240

ttaatcctgg atccgttgtt ggaggtgatg aagaaccaat agagttacct cctggattcc 300

gattccatcc gacggacgaa gagctgatca ctcactatct ttcaccaaaa gtagctgata 360

acagcttctc tgctatagct gttggtgaag ttgatttgaa caactgtgaa ccctgggact 420

tgcctaagca ttcaaagatg ggagaaaagc agtggtattt cttctgtgtt agaggcagga 480

aatacccaac tggttcaagg ataaatagag ctactgatgc tggttattgg aaggcaaccg 540

gtatggacaa ggaaatttac agaggaaaac agctggctgg tatgaagaaa actttggttt 600

tctacaaggg gcgggctcca aaaggccata aatctaattg ggtcatacat gaatatagat 660

tggaggaaaa attctccttc caaaacctct ctgattcatc taagaaggat gagtgggtct 720

tatgccgggc cttcgagaag agtgcaggag agaagaagat accttaccca ggaccgattc 780

catccaattc tatccatttt caaaatccat ttgcaagctt acctcccttg atagaatcct 840

cgtcacgcat cactgaccat ctcaaaccaa caagtataac agaatcatct cacgtgtcct 900

gcttctccaa tcccatgatc aaaacccctc aacaaaccat catcaatgac aattcctttg 960

attcttaccg taagcataac atccacagcc ctttcatgcc cggctactat gctgaattac 1020

aatcatcgac ggaggactca tactgtggat ctcaaattag ttcaagcttc gcctatggca 1080

ctttgccgta tccaggtgga gaatacatgt cggatcagtc aatcttgagg tctatcctta 1140

agaacaatgg aacagaggga gcaatgaaaa cagagagtaa tcattgtaat atggttactg 1200

ctattgaatc tcgggatact ggtttgtcta gtgatatgta tgctgaaata tcatctgtgg 1260

tcgataaata taagacgagg ggtactagag atgcatttga tgatcatcag ggtcaccagg 1320

gctgttctat ttctggcgga cctcaagata ttgattggtt ttggagttat tgtaactgaa 1380

tcagtcagaa gaagatgccg atggcccatg gtgcttgctg ttatggcgat gagataatga 1440

ctcgctaggg tcattagatt caacagaacc ggaaaacagg aaagaaagga agaaaacact 1500

atccttcact gtatgcaaaa tatatatgaa aactcaggtc atgtggatac agaaaaaaaa 1560

aaaaaaaaaa aaaaaaaaa 1579

<210> 2

<211> 453

<212> PRT

<213> Tamarix chinenses

<400> 2

Met Thr Met Ile Thr Pro Ser Ser Glu Leu Thr Leu Thr Lys Gly Thr

1 5 10 15

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

20 25 30

Gly Gln Ala Val Val Ser Thr Gln Ser Thr Trp Gly Phe Tyr Ser Phe

35 40 45

Phe Phe Ser Asp Lys Arg Arg Arg Arg Arg Arg Arg Ile Ser Ile Leu

50 55 60

Ile Ile Arg Arg Lys Glu Glu Met Val Asn Ile Asn Pro Gly Ser Val

65 70 75 80

Val Gly Gly Asp Glu Glu Pro Ile Glu Leu Pro Pro Gly Phe Arg Phe

85 90 95

His Pro Thr Asp Glu Glu Leu Ile Thr His Tyr Leu Ser Pro Lys Val

100 105 110

Ala Asp Asn Ser Phe Ser Ala Ile Ala Val Gly Glu Val Asp Leu Asn

115 120 125

Asn Cys Glu Pro Trp Asp Leu Pro Lys His Ser Lys Met Gly Glu Lys

130 135 140

Gln Trp Tyr Phe Phe Cys Val Arg Gly Arg Lys Tyr Pro Thr Gly Ser

145 150 155 160

Arg Ile Asn Arg Ala Thr Asp Ala Gly Tyr Trp Lys Ala Thr Gly Met

165 170 175

Asp Lys Glu Ile Tyr Arg Gly Lys Gln Leu Ala Gly Met Lys Lys Thr

180 185 190

Leu Val Phe Tyr Lys Gly Arg Ala Pro Lys Gly His Lys Ser Asn Trp

195 200 205

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

210 215 220

Ser Asp Ser Ser Lys Lys Asp Glu Trp Val Leu Cys Arg Ala Phe Glu

225 230 235 240

Lys Ser Ala Gly Glu Lys Lys Ile Pro Tyr Pro Gly Pro Ile Pro Ser

245 250 255

Asn Ser Ile His Phe Gln Asn Pro Phe Ala Ser Leu Pro Pro Leu Ile

260 265 270

Glu Ser Ser Ser Arg Ile Thr Asp His Leu Lys Pro Thr Ser Ile Thr

275 280 285

Glu Ser Ser His Val Ser Cys Phe Ser Asn Pro Met Ile Lys Thr Pro

290 295 300

Gln Gln Thr Ile Ile Asn Asp Asn Ser Phe Asp Ser Tyr Arg Lys His

305 310 315 320

Asn Ile His Ser Pro Phe Met Pro Gly Tyr Tyr Ala Glu Leu Gln Ser

325 330 335

Ser Thr Glu Asp Ser Tyr Cys Gly Ser Gln Ile Ser Ser Ser Phe Ala

340 345 350

Tyr Gly Thr Leu Pro Tyr Pro Gly Gly Glu Tyr Met Ser Asp Gln Ser

355 360 365

Ile Leu Arg Ser Ile Leu Lys Asn Asn Gly Thr Glu Gly Ala Met Lys

370 375 380

Thr Glu Ser Asn His Cys Asn Met Val Thr Ala Ile Glu Ser Arg Asp

385 390 395 400

Thr Gly Leu Ser Ser Asp Met Tyr Ala Glu Ile Ser Ser Val Val Asp

405 410 415

Lys Tyr Lys Thr Arg Gly Thr Arg Asp Ala Phe Asp Asp His Gln Gly

420 425 430

His Gln Gly Cys Ser Ile Ser Gly Gly Pro Gln Asp Ile Asp Trp Phe

435 440 445

Trp Ser Tyr Cys Asn

450

<210> 3

<211> 1362

<212> DNA

<213> rTcNAC2(Artificial)

<400> 3

atgaccatga ttacgccaag ctcagaatta accctcacta aagggactag tcctgcaggt 60

ttaaacgaat tggcccttct aatacgactc actatagggc aagcagtggt atcaacgcag 120

agtacatggg gattttactc tttttttttc tctgacaaaa gaagaagaag aagaagaagg 180

attagtattc ttattattag aaggaaagaa gaaatggtaa acattaatcc tggatccgtt 240

gttggaggtg atgaagaacc aatagagtta cctcctggat tccgattcca tccgacggac 300

gaagagctga tcactcacta tctttcacca aaagtagctg ataacagctt ctctgctata 360

gctgttggtg aagttgattt gaacaactgt gaaccctggg acttgcctaa gcattcaaag 420

atgggagaaa agcagtggta tttcttctgt gttagaggca ggaaataccc aactggttca 480

aggataaata gagctactga tgctggttat tggaaggcaa ccggtatgga caaggaaatt 540

tacagaggaa aacagctggc tggtatgaag aaaactttgg ttttctacaa ggggcgggct 600

ccaaaaggcc ataaatctaa ttgggtcata catgaatata gattggagga aaaattctcc 660

ttccaaaacc tctctgattc atctaagaag gatgagtggg tcttatgccg ggccttcgag 720

aagagtgcag gagagaagaa gataccttac ccaggaccga ttccatccaa ttctatccat 780

tttcaaaatc catttgcaag cttacctccc ttgatagaat cctcgtcacg catcactgac 840

catctcaaac caacaagtat aacagaatca tctcatgtaa gctgtttcag taatcccatg 900

atcaaaaccc ctcaacaaac catcatcaat gacaattcct ttgattctta ccgtaagcat 960

aacatccaca gccctttcat gcccggctac tatgctgaat tacaatcatc gacggaggac 1020

tcatactgtg gatctcaaat tagttcaagc ttcgcctatg gcactttgcc gtatccaggt 1080

ggagaataca tgtcggatca gtcaatcttg aggtctatcc ttaagaacaa tggaacagag 1140

ggagcaatga aaacagagag taatcattgt aatatggtta ctgctattga atctcgggat 1200

actggtttgt ctagtgatat gtatgctgaa atatcatctg tggtcgataa atataagacg 1260

aggggtacta gagatgcatt tgatgatcat cagggtcacc agggctgttc tatttctggc 1320

ggacctcaag atattgattg gttttggagt tattgtaact ga 1362

<210> 4

<211> 23

<212> DNA

<213>3'RACE forward primer Outer Primer (Artificial)

<400> 4

gaggtgatga agaaccaata gag 23

<210> 5

<211> 22

<212> DNA

<213>3'RACE forward primer Inner Primer (Artificial)

<400> 5

tagacatggt tggtacagac tc 22

<210> 6

<211> 45

<212> DNA

<213>3'RACE reverse primer Outer Primer (Artificial)

<400> 6

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 7

<211> 22

<212> DNA

<213>3'RACE reverse primer Inner Primer (Artificial)

<400> 7

ctaatacgac tcactatagg gc 22

<210> 8

<211> 45

<212> DNA

<213>5'RACE forward primer Outer Primer (Artificial)

<400> 8

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 9

<211> 22

<212> DNA

<213>5'RACE forward primer Inner Primer (Artificial)

<400> 9

ctaatacgac tcactatagg gc 22

<210> 10

<211> 25

<212> DNA

<213>5'RACE reverse primer Outer Primer (Artificial)

<400> 10

tatggccttt tggagcccgc ccctt 25

<210> 11

<211> 19

<212> DNA

<213>5'RACE reverse primer Inner Primer (Artificial)

<400> 11

ttacgctcac cacttgctc 19

<210> 12

<211> 20

<212> DNA

<213>TcNAC2 ORF forward primer (Artificial)

<400> 12

atgaccatga ttacgccaag 20

<210> 13

<211> 22

<212> DNA

<213>TcNAC2 ORF reverse primer (Artificial)

<400> 13

tcagttacaa taactccaaa ac 22

<210> 14

<211> 20

<212> DNA

<213>TcNAC2 forward primer (Artificial)

<400> 14

gatccaagct cgcggaagcc 20

<210> 15

<211> 20

<212> DNA

<213>TcNAC2 reverse primer (Artificial)

<400> 15

cacgaatgtc ggtggccgat 20

<210> 16

<211> 21

<212> DNA

<213>TIF forward primer (Artificial)

<400> 16

accacaggag tgtccaccac a 21

<210> 17

<211> 20

<212> DNA

<213>TIF reverse primer (Artificial)

<400> 17

tgatgctttg cgtgccagtg 20

<210> 18

<211> 42

<212> DNA

<213>TcNAC2 mutant primer (Artificial)

<400> 18

acagaatcat ctcatgtaag ctgtttcagt aatcccatga tc 42

<210> 19

<211> 39

<212> DNA

<213>TcNAC2+ primer sequence (Artificial)

<400> 19

ctagatagcg gccgctattc acgtgtcctg cttctccag 39

<210> 20

<211> 39

<212> DNA

<213>TcNAC2- primer sequence (Artificial)

<400> 20

tcgactggag aagcaggaca cgtgaatagc ggccgctat 39

<210> 21

<211> 38

<212> DNA

<213>rTcNAC2+ primer sequence (Artificial)

<400> 21

ctagatagcg gccgctatca tgtaagctgt ttcagtag 38

<210> 22

<211> 38

<212> DNA

<213>rTcNAC2- primer sequence (Artificial)

<400> 22

tcgactactg aaacagctta catgatagcg gccgctat 38

Claims (10)

1. a kind of Chinese tamarisk salt stress responsive genes TcNAC2, nucleotide sequence is as shown in SEQ ID No.1.

2. the expression albumen of Chinese tamarisk salt stress responsive genes TcNAC2 described in claim 1, amino acid sequence such as SEQ ID Shown in No.2.

3. a kind of miR164 resistance target rTcNAC2, the Chinese tamarisk salt stress responsive genes TcNAC2 as described in claim 1 pass through MiR164 response element same sense mutation obtains, and nucleotide sequence is as shown in SEQ ID No.3.

4. the carrier containing Chinese tamarisk salt stress responsive genes TcNAC2 described in claim 1.

5. carrier according to claim 4, it is characterised in that: the carrier is Dual-Luciferase report carrier 35sGLO: TcNAC2 is equipped with composing type strongly expressed promoter P35S and firefly in 5 ' end upstream groups of the miR164 response element of TcNAC2 Luciferase reporter gene, 3 ' end downstream groups are equipped with Renilla luciferase reporter gene.

6. the carrier containing miR164 resistance target rTcNAC2 described in claim 3.

7. carrier according to claim 6, it is characterised in that: the carrier is Dual-Luciferase report carrier 35sGLO: RTcNAC2 is equipped with composing type strongly expressed promoter in 5 ' end upstream groups of the miR164 response element same sense mutation of rTcNAC2 P35S and firefly luciferase reporter gene, 3 ' end downstream groups are equipped with Renilla luciferase reporter gene.

8. carrier according to claim 6, it is characterised in that: the carrier is the over-express vector PBI121GW of rTcNAC2: RTcNAC2 assembles 35S promoter；NPTII expression casette is assembled, LB and RB sequence is assembled, is between LB and RB sequence RTcNAC2 gene expression construct and gene NPTII.

9. Chinese tamarisk salt stress described in Chinese tamarisk salt stress responsive genes TcNAC2 described in claim 1 or claim 2 responds base The carrier of Chinese tamarisk salt stress responsive genes TcNAC2 described in expression albumen or claim 4 because of TcNAC2 in plant salt endurance or Application in forest resistance breeding.

10. miR164 resistance target rTcNAC2 as claimed in claim 3 or miR164 resistance target as claimed in claim 6 Application of the carrier of rTcNAC2 in plant salt endurance or forest resistance breeding.