CN106834315B

CN106834315B - It is a kind of than white thorn NbCIPK25 gene and its expression albumen and application of undercuting

Info

Publication number: CN106834315B
Application number: CN201710184070.0A
Authority: CN
Inventors: 陈金慧; 鲁路; 张文军; 成铁龙; 施季森; 张景波; 杨秀艳; 史胜青
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2017-03-24
Filing date: 2017-03-24
Publication date: 2019-10-18
Anticipated expiration: 2037-03-24
Also published as: CN106834315A

Abstract

The invention discloses a kind of than the white thorn that undercutsNbCIPK25Gene and its expression albumen and application, it is described than the white thorn that undercutsNbCIPK25The nucleotide sequence of gene is as shown in SEQ ID NO.1, and amino acid sequence is as shown in SEQ ID NO.2.The present invention is according to the adverse-resistant characteristic than the white thorn that undercuts, and using the leaf tissue than the white thorn that undercuts, compares design primer according to homologous sequence, has cloned degeneration-resistant more relevant than the white thorn that undercutsCIPKFull length gene, and be named as according to the homologous gene in arabidopsisNbCIPK25.TurnNbCIPK25The salt-tolerant drought-resistant of the Arabidopsis plant of gene is analysis shows than the white thorn that undercutsNbCIPK25Function of the gene in terms of plant stress-resistance, increases the resource of plant stress-resistance gene pool.

Description

It is a kind of than white thorn NbCIPK25 gene and its expression albumen and application of undercuting

Technical field

The invention belongs to field of plant genetic project technology, and in particular to it is a kind of than undercut white thorn NbCIPK25 gene and its Express albumen and application.

Background technique

Than undercuting, white thorn (Nitraria billardieri) belongs to Sapindales (Sapindales) Bai Cike (Nitrariaceae) Nitraria (Nitraria) is distributed mainly on Australian southern aridity grass land or desert, is Ao Zhoute There is white thorn type.Nitraria is strong xerophytic deciduous shrubs, has extremely strong salt-tolerant drought-resistant.Studies have shown that than undercuting white thorn not only Salt content can be survived in reach in the environment of 900mM, but also salt can be improved, improve the fertility of soil, it is flat for ecology The maintenance of weighing apparatus plays a significant role.In addition, the classes active material such as alkaloid, flavones and phenolic acid rich in berry of nitraria tangutorum bobr, With blood pressure lowering, reducing blood lipid is anti-oxidant, the medical values such as antitumor and anti-inflammatory.Therefore, white thorn is as a kind of ecology and economy Plant gradually attracts attention.Currently, the research than the white thorn that undercuts mainly includes kind of a source distribution, alkaloids active material and drought resisting Salt tolerant Physiology and biochemistry property etc., for the breeding than the white thorn that undercuts, active material development and utilization and degeneration-resistant molecule mechanism are ground Study carefully and data support is provided, also lays the foundation for the excavation of resistant gene and application.

CIPKs (CBL-interacting protein kinase) forms CBL-CIPK complex with CBL, participates in planting Object Ca in stressful environmental²⁺Signal transduction, be the distinctive Ser/Thr class Phospoprotein kinase gene of plant.CIPKs is in N-terminal There is conservative kinase domain, is responsible for catalysis destination protein；C-terminal contains NAF structural domain, is responsible for and CBL protein binding.Number at present According to showing there are 26 CIPK class pa-ncreatic and duodenal homeobox1s in model plant arabidopsis, there are 30 CIPK classes homologous different in rice Flask gene has 27 CIPK class homeotic genes in poplar.It is numerous studies have shown that CIPK genoid is in plant salt tolerance, it is resistance to It plays a significant role during low potassium, cold-resistant and drought resisting etc. are degeneration-resistant.

At present studies have reported that be chick-pea CIPK25 gene, result of study show chick-pea in NaCl, PEG, Under the processing environments such as low temperature, ABA, IAA, MeJA and SA, the equal up-regulated expression of CIPK25 gene is to respond stressful environmental； CaCIPK25 can promote seed sprouting and the root growth of transgene tobacco, improve transgene tobacco under condition of salt stress Salt tolerance.In addition, the PeCIPK25 gene of diversiform-leaved poplar and PeCBL1, which form complex, adjusts Na⁺/K⁺Balance.

Summary of the invention

Goal of the invention: the object of the present invention is to provide a kind of than the white thorn NbCIPK25 gene that undercuts.Another mesh of the invention Be to provide than undercut it is white thorn NbCIPK25 gene expression albumen.Further object of the present invention is to provide than the white thorn that undercuts Application of the NbCIPK25 gene in plant drought salt tolerant breeding.

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

It is a kind of than the white thorn NbCIPK25 gene that undercuts, nucleotide sequence is as shown in SEQ ID NO.1.

Above-mentioned ratio undercut it is white thorn NbCIPK25 gene expression albumen, amino acid sequence is as shown in SEQ ID NO.2.

Above-mentioned ratio undercuts white application of the thorn NbCIPK25 gene in plant drought salt tolerant breeding.

Contain the carrier than the white thorn NbCIPK25 gene that undercuts.

Contain the host cell than the white thorn NbCIPK25 gene that undercuts.

The utility model has the advantages that compared with prior art, the present invention combines the drought resistance and salt tolerance characteristic than the white thorn that undercuts, using than undercuting The blade of white thorn extracts tissue RNA and has been cloned CIPK more relevant than the white thorn drought resistance and salt tolerance that undercuts by homologous comparison design primer Genetic fragment, then full length gene is obtained by 5 ' RACE and 3 ' RACE technologies, and name according to the homologous gene in arabidopsis For NbCIPK25.Transgenic Arabidopsis plants are obtained by agrobacterium-mediated transformation and arabidopsis titbit infestation method again.Normally training Support environment in, than undercut it is white thorn NbCIPK25 gene overexpression arabidopsis T3 for homozygous plants growth and development with it is wild Type arabidopsis no significant difference.In arid and salt stress treatment process, the salt tolerance of drought of homozygous transgenic Arabidopsis plant It is significantly higher than wildtype Arabidopsis thaliana, thus demonstrates than function of the white thorn NbCIPK25 gene in terms of plant drought salt tolerant that undercuts Can, resource is increased for plant stress-resistance gene pool, the degeneration-resistant Journal of Sex Research for improving plant is of great significance.

Detailed description of the invention

Fig. 1 is than 1% agarose gel electrophoresis figure of the white thorn total serum IgE that undercuts；

Fig. 2 is the PCR result figure of NbCIPK25 gene entire open reading frame clone；

Fig. 3 is the expression vector figure of NbCIPK25 gene；

Fig. 4 is NbCIPK25 transgenic arabidopsis on NaCl containing 0mM, 100mM NaCl and 150mM NaCl culture medium Sprout state diagram in 4 days and 10 days；

Fig. 5 is NbCIPK25 transgenic arabidopsis on NaCl containing 0mM, 100mM NaCl and 150mM NaCl culture medium Sprout 4 days germination rate statistical charts；

Fig. 6 is that NbCIPK25 transgenic arabidopsis is adding 0mM NaCl respectively, 100mM NaCl and 150mM NaCl's 14 days growth conditions figures are handled on 1/2MS culture medium；

Fig. 7 is that NbCIPK25 transgenic arabidopsis is adding 0mM NaCl respectively, 100mM NaCl and 150mM NaCl's 14 days root long statistical charts are handled on 1/2MS culture medium；

Fig. 8 is that NbCIPK25 transgenic arabidopsis is adding 0mM, 100mM, 150mM, the 1/2MS of 200mM mannitol respectively State diagram in 3 days is sprouted on culture medium；

Fig. 9 is that NbCIPK25 transgenic arabidopsis is adding 0mM, 100mM, 150mM, the 1/2MS of 200mM mannitol respectively 3 days germination rate statistical charts are sprouted on culture medium；

Figure 10 is NbCIPK25 transgenic arabidopsis on the 1/2MS culture medium for adding 0mM and 150mM mannitol respectively Reason 4 days growth conditions and radical, the number of sheets and root long statistical results chart.

Specific embodiment

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

Embodiment 1

Using the blade than the white thorn that undercuts as material, total serum IgE is extracted, and be inverted to cDNA, designs corresponding primer and carry out PCR, After agarose gel electrophoresis, purpose band is recycled, is connect with pMD19-T carrier, is transferred to Escherichia coli, be sequenced and analyze.It determines After purpose sequence, according to sequence design RACE primer, PCR obtains 5 ' and 3 ' sequences, and after sequencing analysis, splicing is obtained NbCIPK25 full length sequence.According to full length sequence design primer, PCR obtains full length fragment, connect, be transferred to pMD19-T carrier Escherichia coli are sequenced again and analyze, are determined as overall length target fragment.Restriction enzyme site is added at target fragment both ends.Expression carries After body pBI121 double digestion, it is connected under the action of T4 ligase with target fragment.Recombinant vector be transferred to Agrobacterium EHA105 and It in GV3101, after waiting arabidopsis of the right age, is converted using titbit infusion method, and passes through antibiotic-screening and PCR detection mirror Surely T1 generation is obtained, then T2 generation and T3 carry out drought resisting to homozygous transgenic plant and WT lines for homozygous transgenic plant Salt tolerance comparative analysis.It is specific as follows:

(1) extraction of total serum IgE

Using the blade than the white thorn that undercuts as material, according to NORGEN kit (Norgen Biotek) operating procedure into Row RNA extraction, used reagent and consumptive material, which are handled, makes it without RNA enzyme.Than 1% agarose of the Nitraria leaf piece total serum IgE that undercuts Gel electrophoresis result is as shown in Figure 1, band is more visible；Measure the absorbance of total serum IgE, OD₂₆₀/OD₂₈₀Value is 2.02, OD₂₆₀/ OD₂₃₀It is 1.91, can be used as gene cloning.

The detailed process that RNA is extracted are as follows: liquid nitrogen is added in the mortar of 0.1%DEPC solutions overnight processing and is sufficiently ground Sample is transferred to 2mL after then taking 800 μ L Lysis Solution to be homogenized in sample and newly manages.12000rpm centrifugation 2min takes supernatant to move in new pipe.70% isometric ethyl alcohol is added, is vortexed and mixes.Mixed liquor is moved in pillar (under connect 2mL collecting pipe), it is centrifuged 1min, filtrate is outwelled, puts back to collecting pipe.400 μ L Wash Solution are added, are centrifuged 1min, abandon filter Liquid puts back to collecting pipe.DNaseI working solution is added, 12000rpm is centrifuged 1min, filtrate is sucked back on pillar, 25-30 DEG C of standing 15min.400 μ L Wash Solution, 12000rpm are added and are centrifuged 1min, abandon filtrate.400 μ L Wash are added again Solution, 12000rpm are centrifuged 1min, abandon filtrate.Pillar is put back into collecting pipe, 12000rpm is centrifuged 2min, abandons collecting pipe. Pillar is put into 1.5mL pipe, 50 μ LElution Solution are added.First 200~2000rpm is centrifuged 2min, then 12000rpm is centrifuged 1min, and volume is centrifuged 1min less than 50 μ L, then with 14000rpm.

(2) acquisition of cDNA

Using the RNA of extraction as template, reverse transcription obtains cDNA, and used is Invitrogen companyIII First-Strand Synthesis Kit.RNA usage amount is 1 μ g, detailed process are as follows: configuration reaction Liquid (1 μ g RNA, 1 μ L Primer (OligodT), 1 μ L 10mM dNTP mix, DEPC-TreatedWater, up to, 10 μ L), after of short duration low-speed centrifugal, 65 DEG C of 5min are immediately placed on 1~2min on ice.Reagent (2 μ L 10 are added in pipe one step up × RT Buffer, 4 μ L 25mM MgCl₂, 2 μ L 0.1M DTT, 1 μ LRNaseOUT (40U/ μ L), 1 μ LSuperScript III RT), it is placed in PCR instrument, response procedures are 50 DEG C of 50min, 85 DEG C of 5min.The reaction solution of previous step is centrifuged, in every pipe The RNase H, 37 DEG C of 20min of 1 μ L is added.

(3) homologous clone of target gene

It according to the part transcript profile data than the white thorn that undercuts, is compared, is designed using Oligo7 special by NCBIblast Specific primer, clone obtain NbCIPK25 genetic fragment, are then attached carrier, conversion Escherichia coli, aim sequence sequencing It is analyzed with sequence, is determined as target gene.Cloning primer, PCR system and PCR program are as follows.

NbCIPK25 segment cloning primer are as follows:

CIPK25 forward primer:5'-AACAAGGATAAAGTGAAGAAGGAAGT-3',

CIPK25 reverse primer:5'-CCTGACAAAACTTAACATACTCCAAG-3'.

PCR reaction system (20 μ L) are as follows: 2 μ L 10 × PCR Buffer, 1.2 μ L Mg²⁺(25mM/L)、0.4μL 10× dNTP、0.1μL Tag(5.0U/ul)、1μL Forward primer(10uM/L)、1μL Reverse primer(10uM/ L)、1μL Template cDNA(100ng/ul)、13.3μL ddH₂O。

PCR response procedures are as follows: 95 DEG C of denaturation 5min, 55 DEG C of annealing 30s, 72 DEG C of extension 1min20s, 35 recycle.

(4) target gene 5 ' end and 3 ' terminal sequences clone

RACE primer is designed using Oligo7, carries out the clone of CIPK25 gene 3 ' end and 5 ' ends, gel extraction obtains Target fragment is obtained, is then connect with carrier pMD19-T, Escherichia coli, picking monoclonal, sequencing and sequence analysis are converted, it is determining After two end sequences for obtaining NbCIPK25 gene, splicing obtains NbCIPK25 full length gene sequence.RACE primer, PCR are anti- Answer system and program as follows.

RACE primer are as follows:

CIPK25:3 ' race primer A:5 '-CAATTCCGGCCATCATGCGTCTCCCCT-3',

CIPK25:3 ' race primer B:5'-AGGTTACAGGGGAAATTCGAGGGGAGG-3',

CIPK25:3 ' race primer C:5'-GAAGTTGCGGTGGTGGAATTCTCGAAGT-3',

CIPK25:5 ' race primer A:5 '-GTATCGGCTTTGGCTCCGTCGTATCC-3 ',

CIPK25:5 ' race primer B:5 '-ACTGTGTGTGGAGGAGCCCGTCGTTG-3 ',

CIPK25:5 ' race primer C:5 '-ATCGACGGCGCTGATCAACTGCTGG-3 ',

The PCR reaction system (20 μ L) of RACE A item are as follows: 2 μ L 10 × PCR Buffer, 1.2 μ L Mg²⁺(25mM/ L)、0.4μL 10×dNTP、0.1μL Tag(5.0U/ul)、1μLCIPK25:3’/5’race primer A(10uM/L)、1μ L10×Universal Primer A Mix、1μL Template cDNA(100ng/ul)、13.3μL ddH₂O。

The PCR response procedures of RACE A item: 95 DEG C of denaturation 5min, 69 DEG C/66 DEG C annealing (3 ' end/5 ' end) 30s, 72 DEG C of extension (3 ' end/5 ' end) 50s/45s, 35 circulations.

The PCR reaction system (20 μ L) of RACE B and C item are as follows: 2 μ L 10 × PCR Buffer, 1.2 μ L Mg²⁺ (25mM/L)、2μL dNTP、0.4μL Kode、0.6μL Universal Primer A Mix、0.6μLCIPK25:3’/5’ race primer B/C、1μLDiluent of race A product、12.2μL ddH₂O。

The PCR response procedures of RACE B and C item: 94 DEG C of initial denaturation 2min, 98 DEG C of denaturation 10s, 67 DEG C/66 DEG C/69 DEG C/68 DEG C of (3 ' race B/3 ' raceC/5 ' raceB/5 ' raceC) annealing 30s, 68 DEG C of extension 30s/30s/40s/30s (3 ' Race B/3 ' race C/5 ' race B/5 ' race C), 35 circulations.

(5) full length gene obtains

According to CIPK25 full length gene sequence, full-length gene cloning primer is designed using Oligo7, PCR and gel extraction obtain NbCIPK25 full-length gene is obtained, is transferred to Escherichia coli after connecting with pMD19-T, by sequencing analysis, is determined as again complete CIPK25 gene.CIPK25 full length gene than the white thorn that undercuts is 1925bp, is named as NbCIPK25, particular sequence such as SEQ ID Shown in NO.1, open reading frame (ORF) sequence length is 1356bp, and expressed protein sequence includes 452 amino acid, such as Shown in SEQ ID NO.2.Fig. 2 is clone result of the NbCIPK25 gene containing complete ORF.The primer of full-length gene clone, PCR are anti- Answer system and program specific as follows:

Full-length gene cloning primer are as follows:

CIPK25:wl forward primer:5'-ATGGCGGAGGAGGATAAAC-3',

CIPK25:wl reverse primer:5'-AACTTCCACAGTCATCTCATCTC-3',

PCR reaction system (20 μ L) are as follows: 2 μ L 10 × PCR Buffer, 1.2 μ L Mg²⁺(25mM/L)、0.4μL 10× dNTP、0.1μL Tag(5.0U/ul)、1μLForward primer(10uM/L)、1μLReverse primer(10uM/L)、1 μL Template cDNA(100ng/ul)、13.3μL ddH₂O。

PCR response procedures: 95 DEG C of denaturation 5min, 57 DEG C of annealing 30s, 72 DEG C of extension 2mins, 35 recycle.

The verifying of 2 gene function of embodiment

35S:CIPK25 expression vector is constructed, is transferred in wild type Colombia arabidopsis, is observed than the white thorn that undercuts Whether NbCIPK25 gene can enhance the salt tolerance of drought of arabidopsis, compare the phenotype of transgenic arabidopsis and wildtype Arabidopsis thaliana Difference analyzes the function than the white thorn CIPK25 gene that undercuts.

1) building of carrier

Coli strain used in the present invention is E.coli JM109 (precious bioengineering (Dalian) Co., Ltd buys)；Table It is pBI 121 up to carrier (Biovector Co., LTD company buys).Restriction enzyme is purchased from New England Biolabs(NEB).T4 ligase is purchased from Takara.

Detailed process is as follows:

1. add XbaI and SmaI restriction enzyme site respectively to CIPK25 genetic fragment upstream and downstream by PCR, PCR system and anti- Condition is answered to expand with overall length, primer is respectively:

NbCIPK25F+XbaI:5'-GCTCTAGAATGGCGGAGGAGGATAAAC-3',

NbCIPK25R+SmaI:5'-TCCCCCGGGAACTTCCACAGTCATCTCATCTC-3',

Forward restricted site of XbaI:5'-T ∧ CTAGA-3',

Reverse restricted site ofSmaI:5'-CCC ∧ GGG-3',

After the sequencing of 2.PCR product is correct, under the action of T4 ligase, the building of carrier can be carried out.Using XbaI and SmaI restriction endonuclease carries out endonuclease reaction and handles 121 expression vector of pBI.

Double enzyme digestion reaction (20 μ L) system and step: 2 μ L CutSmart buffer, 0.5 μ LXbaI, 1 μ gpBI121 matter Grain, ddH₂O supplies 20 μ L；4h is reacted in 37 DEG C of water-baths；0.5 μ LSmaI, endonuclease reaction 4h is added；1% agarose gel electrophoresis into Row separation；Digestion products are recycled and purified with AxyPrep DNA Gel Extraction Kit (AXYGEN), are dissolved in 20 μ In the TE buffer of L.Same operation obtains target gene from cloning vector.

3. detecting recycled digestion products concentration, each reagent (target fragment molecular number: carrier point is added by linked system Subnumber=3:1~5:1), 16 DEG C of connections overnight.25 μ L coupled reaction systems are as follows: 2.5 μ L T4 DNA ligase buffer (10 ×), the expression vector of 5 μ L digestions, the PCR product of 15.5 μ L digestions, 2 μ L T4DNA ligase, ddH₂O supplies 25 μ L.

4. connection product converts e. coli jm109 telegraphy cell, picking single colonie is inoculated into LB liquid medium, 37 DEG C shake culture is stayed overnight；Bacterium solution PCR is carried out using overall length primer, with screening positive clone, uses AxyPrep Plasmid later MiniprepKit (AXYGEN) extracts plasmid and carries out digestion verification.It is sequenced whether detection vector construction dashes forward in the process simultaneously Change or deficient phenomena.Construction of expression vector is as shown in Figure 3.

2) conversion of Agrobacterium

1. the agrobacterium strains that the present invention uses are GV3101 (Biovector Co., LTD company buys).Using The pBI121+NbCIPK25 expression vector built is transferred to Agrobacterium by frozen-thawed method.Detailed process are as follows: 1) ice bath melted GV3101 competent cell is added the expression vector plasmid of at least 100ng recovery purifying, mixes gently, 20~30min of ice bath； 2) liquid nitrogen flash freezer l min, 37 DEG C of thermal shock 3min, is immediately placed in 1~2min on ice；3) the LB culture of 800 μ L antibiotic-frees is added Base, 28 DEG C, 200rpm recovery 3.5h；4) 4000rpm is centrifuged 3min, sops up 700 μ L culture mediums；5) remaining bacterium solution is mixed, is smeared In addition 50mg.L^-1Card receive mycin solid LB training base on；6) 30~48h is cultivated in 28 DEG C of inversions；7) PCR detection positive colony, 4 DEG C save positive colony it is spare.

It blooms 2. the arabidopsis of health status to be planted is grown to.The positive colony that PCR is detected, shakes bacterium to OD₆₀₀For When 0.8, carries out thaliana flower organ and impregnate conversion.Detailed process is as follows: 1) being centrifuged bacterium solution 5000rpm, 5min, collect bacterium Body is suspended with 5% sucrose solution；2) before immersion, SilwetL-77 is added, concentration is 0.05% (500 μ L/L), shakes blebbing Foam；3) aerial part of arabidopsis is impregnated into Agrobacterium aaerosol solution 30sec, during which shaked gently, 4) dipped is intended Southern mustard lies low in pallet, covers moisturizing with preservative film, masking foil sealing is protected from light for 24 hours；5) masking foil is opened, is trained under normal condition It supports, stops watering when seed maturation.

3. harvest seed is dried, with kanamycin screening T1 for seed after sterilizing, screening and culturing medium: 1/2MS+ card that Mycin 50mg/L+ cephalo 200mg/L.

Continue to cultivate in soil 4. the positive plant that screening obtains moves to, after collecting T1 for arabidopsis seed, continues to sieve Choosing obtains T2 for plant.Then, after collecting T2 for the seed of Arabidopsis plant, continue to screen, identify homozygosis T3 for strain.

3) turn the test of NbCIPK25 gene arabidopsis thaliana salt-tolerance

4 homozygous transgenic arabidopsis strains and each 50 seeds of wildtype Arabidopsis thaliana are taken to be individually placed to containing 0mM, It is sprouted on the 1/2MS culture medium of 100mM and 150mM NaCl, is put under light and sprouts after seed vernalization 2-3 days, statistics is sprouted after 4 days Hair rate.4 Transgenic wheat lines and wildtype Arabidopsis thaliana strain are respectively designated as OX-1, OX-2, OX-3, OX-4 and WT.Six It is secondary to repeat to test while carrying out.4 days and 10 days phenotypes are sprouted as shown in figure 4, sprouting 4 days germination rate statistical result such as Fig. 5 It is shown.

Statistical result showed in 0mM NaCl environment, turns 4 overexpression strains of NbCIPK25 gene after sprouting four days The seed germination rate of system and the seed germination rate difference of WT are more apparent, and transgenic line is higher than the germination rate of WT by 3.53% respectively, 4.80%, 4.90% and 5.39%.In 100mMNaCl environment, transgenic line is higher than the germination rate of WT by 53.00% respectively, 50.06%, 53.10% and 54.62%, be in extremely significant sex differernce (P < 0.01).Under the conditions of 150mM salt treatment, transgenic line System is respectively than WT high 38.92%, 53.96%, 55.50% and 51.40%, and wherein difference is extremely significant between OX-2 and OX-3 and WT (P<0.01).Result is sprouted it is found that germination rate of the arabidopsis seed in salt stress environment can be improved in NbCIPK25 from salt.

In 1/2MS culture after germination and growth 1 week, each strain takes 30 plants to be transferred to difference for transgenic plant and WT seed 0mM is added, on the 1/2MS culture medium of 100mM and 150mM NaCl, observes the growth table of transgenic plant and WT lines Type.It repeats to test while carrying out three times.Growth phenotype is as shown in Figure 6.Observe the life of transgenic arabidopsis and wildtype Arabidopsis thaliana Long status, it is known that NbCIPK25 can promote the root growth of plant under normal operation.Under 100mM and 150mMNaCl environment, NbCIPK25 can promote the growth of Arabidopsis leaf and root with plant, show growth conditions more better than WT.Fig. 7 is to turn base Because of the root long statistical result of arabidopsis and wildtype Arabidopsis thaliana.

4) the drought resistance test of NbCIPK25 transgenic arabidopsis

Three homozygous transgenic arabidopsis strain seeds and WT arabidopsis seed kind are being added to 0mM respectively, 100mM, On the 1/2MS culture medium of 150mM and 200mM mannitol, after sprouting 3 days, germination rate is counted.Statistical result is as shown in Figure 8,9, sprouts When sending out the 3rd day, high by 49%, 43% He is distinguished in the germination rate of the culture medium of the mannitol containing 0mM, three transgenic line ratio WT 45%, and difference is extremely significant.For mannitol concentration in 100mM and 150mM, the germination rate of transgenic line ratio WT strain is high, but The difference of OX-2 and WT is unobvious.When mannitol concentration is 200mM, the germination rate of transgenic arabidopsis is distinguished high than wild type 78%, 73% and 72%, it is in the extremely significant property (P < 0.01) of difference.

OX-1 transgenic arabidopsis and WT seed are sprouted after a week on 1/2MS culture medium, each strain respectively takes 60 plants It is transferred on the 1/2MS culture medium for adding 0mM and 150mM mannitol respectively, after growth 4 days, observes arabidopsis growth conditions, system Count radical amount, blade quantity and root long data.

The results are shown in Figure 10, after being handled 4 days under 0mM mannitol environment, the root long ratio WT of transgenic arabidopsis OX-1 Length 19%, difference is extremely significant (P < 0.01), blade quantity and radical and WT no significant difference；Under 150mM mannitol environment After processing 4 days, the root long ratio WT's of transgenic arabidopsis OX-1 is long by 29%, and difference is extremely significant (P < 0.01), transgenic arabidopsis For the radical and blade quantity of OX-1 respectively 42% and 20% more than WT, difference is extremely significant (P < 0.01).

SEQUENCE LISTING

<110>Nanjing Forestry University

<120>a kind of than white thorn NbCIPK25 gene and its expression albumen and the application of undercuting

<130> 100

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 1925

<212> DNA

<213> Nitraria billardieri

<400> 1

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ggagtccaat agagtcttac gaagttcccc cctttcttca accaaaatct taaaaccctt 240

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atgagatggg gagattgctt gggaagggca ctttcgccaa agtctatcac gggaagaaca 420

tcgtttctgg ggaaagtgtc gccattaaag tgatcaacaa ggataaagtg aagaaggaag 480

tgatgatgga gcaaatcaag agagaaatct ccgttatgcg gctcgctcgt catccgaacg 540

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accgagatct gaaaccggag aatctgctct tggacgaaaa cgaggatctc aaaatctccg 780

atttcggtct ctcggctctg ccggaacatt tacgcaacga cgggctcctc cacacacagt 840

gcgggacgcc tgcttacgtg gcgccggagg ttttaaggaa aaaaggatac gacggagcca 900

aagccgatac atggtcctgt ggagtgattt tatatgtgct cctcgccgga ttcttaccgt 960

ttcaggatga gaatctgatg aagatgtacc ggaaaatttt caaagcagaa tttgaatttc 1020

cgccgtggtt ttcgacggat gcgaagcgtt tgatctccag acttctaatc cctgatccag 1080

aacggagaat tacaattccg gccatcatgc gtctcccctg gttccggaag gatttaacgc 1140

agacgacgtc gtttgcgatg gaagaacctg ctgaaattga aaaaacagag gaagatataa 1200

acgactcagc ccgtaagagt accaaaacga cgtcgccgaa gttctttaac gcgttcgaat 1260

ttatctcatc gatgtcgtcg ggatttgatt tctctagcct gttcgagaac acgagaaaat 1320

caggatcgat gttcacgtcg aagtgctcgg cgagtggtat tatggagaaa atcgaaggag 1380

tcggaaacgc gatgaatttc aaagtgtcga aactgaagga tttcaaagtg aggttacagg 1440

ggaaattcga ggggaggaaa gggcggctgg ccgtgaccgc ggaagtatac gaggtggcgg 1500

cggaagttgc ggtggtggaa ttctcgaagt cggctggaga taccttggag tatgttaagt 1560

tttgtcagga agatgttagg ccggcgttga aagacattgt ttggacatgg caaggtgaca 1620

atgttgtcgg taactgtaac ggtaataaaa ctgagggaga tgagatgact gtggaagttt 1680

gagtggtaat ttcgcagtgg tgaaaacatg acatgaacct ctttaggatt tttgtcttaa 1740

gaaaaagtat tttgtgattc gtgcttttag ttttggtttt tggggaaact tggtttaatg 1800

agccctttgt aaatagtttg tgtttactgc atcagttgga acttggaagc aatgtggttg 1860

tagaaaacgt gaacttccaa tttaaactaa tattttgaaa gtaaaaaaaa aaaaaaaaaa 1920

aaaaa 1925

<210> 2

<211> 452

<212> PRT

<213> Nitraria billardieri

<400> 2

Met Ala Glu Glu Asp Lys His Met Leu Phe Gly Lys Tyr Glu Met Gly

1 5 10 15

Arg Leu Leu Gly Lys Gly Thr Phe Ala Lys Val Tyr His Gly Lys Asn

20 25 30

Ile Val Ser Gly Glu Ser Val Ala Ile Lys Val Ile Asn Lys Asp Lys

35 40 45

Val Lys Lys Glu Val Met Met Glu Gln Ile Lys Arg Glu Ile Ser Val

50 55 60

Met Arg Leu Ala Arg His Pro Asn Val Val Glu Leu Lys Glu Val Met

65 70 75 80

Ala Thr Lys Thr Lys Ile Phe Phe Met Met Glu Tyr Val Lys Gly Gly

85 90 95

Glu Leu Phe Ala Lys Val Ala Lys Gly Lys Met Ser Glu Asp Leu Ala

100 105 110

Arg Lys Tyr Phe Gln Gln Leu Ile Ser Ala Val Asp Phe Cys His Ser

115 120 125

Arg Gly Val Ser His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp

130 135 140

Glu Asn Glu Asp Leu Lys Ile Ser Asp Phe Gly Leu Ser Ala Leu Pro

145 150 155 160

Glu His Leu Arg Asn Asp Gly Leu Leu His Thr Gln Cys Gly Thr Pro

165 170 175

Ala Tyr Val Ala Pro Glu Val Leu Arg Lys Lys Gly Tyr Asp Gly Ala

180 185 190

Lys Ala Asp Thr Trp Ser Cys Gly Val Ile Leu Tyr Val Leu Leu Ala

195 200 205

Gly Phe Leu Pro Phe Gln Asp Glu Asn Leu Met Lys Met Tyr Arg Lys

210 215 220

Ile Phe Lys Ala Glu Phe Glu Phe Pro Pro Trp Phe Ser Thr Asp Ala

225 230 235 240

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

245 250 255

Thr Ile Pro Ala Ile Met Arg Leu Pro Trp Phe Arg Lys Asp Leu Thr

260 265 270

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

275 280 285

Glu Glu Asp Ile Asn Asp Ser Ala Arg Lys Ser Thr Lys Thr Thr Ser

290 295 300

Pro Lys Phe Phe Asn Ala Phe Glu Phe Ile Ser Ser Met Ser Ser Gly

305 310 315 320

Phe Asp Phe Ser Ser Leu Phe Glu Asn Thr Arg Lys Ser Gly Ser Met

325 330 335

Phe Thr Ser Lys Cys Ser Ala Ser Gly Ile Met Glu Lys Ile Glu Gly

340 345 350

Val Gly Asn Ala Met Asn Phe Lys Val Ser Lys Leu Lys Asp Phe Lys

355 360 365

Val Arg Leu Gln Gly Lys Phe Glu Gly Arg Lys Gly Arg Leu Ala Val

370 375 380

Thr Ala Glu Val Tyr Glu Val Ala Ala Glu Val Ala Val Val Glu Phe

385 390 395 400

Ser Lys Ser Ala Gly Asp Thr Leu Glu Tyr Val Lys Phe Cys Gln Glu

405 410 415

Asp Val Arg Pro Ala Leu Lys Asp Ile Val Trp Thr Trp Gln Gly Asp

420 425 430

Asn Val Val Gly Asn Cys Asn Gly Asn Lys Thr Glu Gly Asp Glu Met

435 440 445

Thr Val Glu Val

450

<210> 3

<211> 26

<212> DNA

<213> Artificial

<220>

<223>CIPK25 forward primer sequence

<400> 3

aacaaggata aagtgaagaa ggaagt 26

<210> 4

<211> 26

<212> DNA

<213> Artificial

<220>

<223>CIPK25 reverse primer sequence

<400> 4

cctgacaaaa cttaacatac tccaag 26

<210> 5

<211> 27

<212> DNA

<213> Artificial

<220>

<223>CIPK25:3'race primer A sequence

<400> 5

caattccggc catcatgcgt ctcccct 27

<210> 6

<211> 27

<212> DNA

<213> Artificial

<220>

<223>CIPK25:3'race primer B sequence

<400> 6

aggttacagg ggaaattcga ggggagg 27

<210> 7

<211> 28

<212> DNA

<213> Artificial

<220>

<223>CIPK25:3'race primer C sequence

<400> 7

gaagttgcgg tggtggaatt ctcgaagt 28

<210> 8

<211> 26

<212> DNA

<213> Artificial

<220>

<223>CIPK25:5'race primer A sequence

<400> 8

gtatcggctt tggctccgtc gtatcc 26

<210> 9

<211> 26

<212> DNA

<213> Artificial

<220>

<223>CIPK25:5'race primer B sequence

<400> 9

actgtgtgtg gaggagcccg tcgttg 26

<210> 10

<211> 25

<212> DNA

<213> Artificial

<220>

<223>CIPK25:5'race primer C sequence

<400> 10

atcgacggcg ctgatcaact gctgg 25

<210> 11

<211> 19

<212> DNA

<213> Artificial

<220>

<223>CIPK25:wl forward primer sequence

<400> 11

atggcggagg aggataaac 19

<210> 12

<211> 23

<212> DNA

<213> Artificial

<220>

<223>CIPK25:wl reverse primer sequence

<400> 12

aacttccaca gtcatctcat ctc 23

<210> 13

<211> 27

<212> DNA

<213> Artificial

<220>

<223>I sequence of NbCIPK25F+Xba

<400> 13

gctctagaat ggcggaggag gataaac 27

<210> 14

<211> 32

<212> DNA

<213> Artificial

<220>

<223>I sequence of NbCIPK25R+Sma

<400> 14

tcccccggga acttccacag tcatctcatc tc 32

<210> 15

<211> 6

<212> DNA

<213> Artificial

<220>

<223>I sequence of Forward restricted site of Xba

<400> 15

tctaga 6

<210> 16

<211> 6

<212> DNA

<213> Artificial

<220>

<223>I sequence of Reverse restricted site ofSma

<400> 16

cccggg 6

Claims

1. a kind of than the white thorn NbCIPK25 gene that undercuts, nucleotide sequence is as shown in SEQ ID NO.1.

2. the expression albumen described in claim 1 than the white thorn NbCIPK25 gene that undercuts, amino acid sequence such as SEQ ID Shown in NO.2.

3. described in claim 1 improving the application in plant salt tolerance drought resistance than the white thorn NbCIPK25 gene that undercuts.

4. containing the carrier described in claim 1 than the white thorn NbCIPK25 gene that undercuts.

5. containing the host cell described in claim 1 than the white thorn NbCIPK25 gene that undercuts.