CN107446930A - The coded sequence of 3C of the Festuca Arundinacea gene of resistance to Low nitrogen stress Fa14 3 a kind of and its application - Google Patents

The coded sequence of 3C of the Festuca Arundinacea gene of resistance to Low nitrogen stress Fa14 3 a kind of and its application Download PDF

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CN107446930A
CN107446930A CN201710117593.3A CN201710117593A CN107446930A CN 107446930 A CN107446930 A CN 107446930A CN 201710117593 A CN201710117593 A CN 201710117593A CN 107446930 A CN107446930 A CN 107446930A
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low nitrogen
festuca arundinacea
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李小冬
蔡璐
王小利
吴佳海
蔡鸣
蔡一鸣
孙方
陈光吉
舒健虹
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GUIZHOU INSTITUTE OF PRATACULTURE
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Abstract

The invention discloses 3C of the Festuca Arundinacea gene of resistance to Low nitrogen stress Fa14 3 coded sequence and its reference.The invention also discloses application of its coded sequence in the resistance to Low nitrogen stress of plant is adjusted, repellence of the plant to low nitrogen environment can be adjusted, biological yield increase and root elongation can be promoted, guarantee is provided to improve crop anti-adversity under low nitrogen obigotrophic condition, for use in the resistance to low nitrogen breed breeding of crops and vegetables, thus reduce nitrogenous fertilizer to use, alleviate a large amount of applied nitrogen increase production costs and the pollution to environment.

Description

The coded sequence of the Festuca Arundinacea gene of resistance to Low nitrogen stress Fa14-3-3C a kind of and its application
Technical field
The present invention relates to plant genetic engineering field, and in particular to a kind of Festuca Arundinacea gene of resistance to Low nitrogen stress Fa14-3-3C Coded sequence and its application.
Background technology
Nitrogen is one of main nutrient elements for influenceing growth and development of plants, and it participates in the structure group of plant organism Into also assisting in plant physiology metabolic response.Therefore, Rational Application nitrogenous fertilizer plays the effect of greatly facilitating to improving crop yield, But excessive apply of nitrogenous fertilizer causes the environment such as exceeded, the earth's surface water eutrophication of Nitrate content in ground water in agricultural production in recent years Problem.China is nitrogenous fertilizer consumption big country, and nitrogenous fertilizer usage amount is still in incremental trend, but crop is to artificial application nitrogenous fertilizer Utilization rate only has 50% or so, therefore the New Crop Varieties for cultivating resistance to low nitrogenous fertilizer stress have to efficiently production and environmental protection Extremely important meaning.
Festuca Arundinacea is main Cool-Season Forage Species and turfgrass, in water and soil conservation, environmental protection, urban afforestation and sports ground Played an important role in planting.But as a kind of graminous pasture, to nitrogenous fertilizer in its cultivation and seed production process Dependence is very strong, therefore, cultivates economic and environment-friendly resistance to low nitrogen Festuca Arundinacea new varieties, to effectively reducing grass-land cultivation management cost, Reduce environmental pollution, promote ley farming production sustainable development to have important facilitation.
Because nitrogen is most important to plant growth and agricultural production etc., forefathers are from the absorption of nitrogen, transhipment, same Change, transfer, regeneration and carbon nitrogen balance etc. have carried out systematic research.It was found that and elaborate the work(of multiple family genes Can, including the family genes such as NRT, NAXT, NAR (response NO3 -), AMT family genes (response NH4 +), ProT, LHT, The family genes such as AAP (response organic nitrogen), and function of these genes in multiple species is guarded.It is first except nitrogen is directly participated in Outside the gene of element metabolism, receive the regulation of nitrogen element content between many genes, 14-3-3 family genes are one of them.In high sheep Mao Zhong, 4 14-3-3 family genes can be significantly by high temperature, high salt, arid and low nitrogen induced expression, but it is specifically responded Mechanism and Land use systems are not studied also, understand fully the family gene to form of the plant under Low nitrogen stress, Physiology and biochemistry with And the influence of gene expression, the germ plasm resource of low nitrogen resisting stress is further cultivated using technique for gene engineering, it is dirty to reducing environment Dye, cultivation nitrogen efficiently play the role of important using new varieties.
The content of the invention
It is an object of the invention to：Provide a kind of Festuca Arundinacea Fa14-3-3C nucleotide sequence and amino acid sequence with And its application in the resistance to Low nitrogen stress of plant, tolerance of the plant to Low nitrogen stress is changed by overexpression Fa14-3-3C Property, guarantee is provided to improve crop anti-adversity under the conditions of Low nitrogen stress, to cultivate resistance to low nitrogen New Crop Varieties, reduces production During nitrogenous fertilizer usage amount, reduce production cost while preserve the ecological environment.
What the present invention was realized in：
A kind of Festuca Arundinacea Fa14-3-3C coded sequence, it has such as SEQ ID NO in sequence table：Nucleotides shown in 1 Sequence, or with SEQ ID NO：The nucleotide sequence of DNA sequence dna at least 70% homology shown in 1；Or there is coded sequence SEQ ID NO in table：The polynucleotides of amino acid sequence shown in 2；Either by SEQ ID NO：2 amino acid residue sequence Row have and SEQ ID NO by substitution, missing or the addition of more than one amino acid residue：2 amino acid sequence is at least The sequence of 70% homology.
Festuca Arundinacea Fa14-3-3C coded sequence is SEQ ID NO in sequence table：Protein shown in 2 belongs to 14-3-3 Protein family.
Festuca Arundinacea Fa14-3-3C is positioned at cytoplasm.
Festuca Arundinacea Fa14-3-3C can be by Low nitrogen stress induced expression.
Application of the Festuca Arundinacea Fa14-3-3C coded sequence in the resistance to Low nitrogen stress of plant is adjusted.
By in Festuca Arundinacea Fa14-3-3C gene transferred plants, to improve the resistance to Low nitrogen stress ability of plant.
Described Low nitrogen stress refers to that nitrogen content in soil is 0~25mMol/L.
Described plant be rice, corn, sorghum, hybrid giant napier, Chinese pennisetum, rye grass, oat, orchardgrass, vegetables, fruit tree or Tobacco.
The overexpression Fa14-3-3C in wildtype Arabidopsis thaliana, bio of the plant under the conditions of low nitrogen can be dramatically increased Amount, and increasing degree and Fa14-3-3C expression quantity positive correlation.
Quantitative observation Festuca Arundinacea Fa14-3-3C is overexpressed plant root growth, finds genetically modified plants under normal operation There is no significant difference with wild type, and under Low nitrogen stress treatment conditions, 14-3-3C is overexpressed plant root long and significantly compares wild type Grow, illustrate that Fa14-3-3C positive can adjust tolerance of the plant to Low nitrogen stress.
Beneficial effect
The present invention is domestic and international first public play-by-play Festuca Arundinacea Fa14-3-3C sequence, and its overexpression can at present Enhancing plant does not have relevant report also to the function of Low nitrogen stress tolerance.The present invention is test result indicates that in wildtype Arabidopsis thaliana Middle overexpression Festuca Arundinacea Fa14-3-3C can remarkably promote plant biological yield, and biological yield increase degree with Fa14-3-3C expression quantity is proportionate.Under the conditions of Low nitrogen stress, Festuca Arundinacea Fa14-3-3C genetically modified plants root longs are than wild Type dramatically increases, and it is resistant to the enhancing of Low nitrogen stress ability.Therefore the present invention proposes that overexpression Festuca Arundinacea Fa14-3-3C can be utilized To change plant to low nitrogen tolerance, and the new varieties of the resistance to Low nitrogen stress of seed selection, the usage amount of nitrogenous fertilizer in production process is reduced, it is real Existing economic and environment-friendly agriculture purpose.
Brief description of the drawings
Fig. 1 clones for Festuca Arundinacea Fa14-3-3C full-length genes；
Using the careless No. 1 Festuca Arundinacea leaf cDNA in Guizhou Province as template, Festuca Arundinacea Fa14- is obtained using 5 ' RACE and 3 ' RACE technologies 3-3C full length sequences；
Fig. 2 is Festuca Arundinacea Fa14-3-3C Overexpression vector schematic diagrames；
Clone obtains Festuca Arundinacea Fa14-3-3C gene orders and will be connected to p1300-GFP expression vectors；
Fig. 3 is screening p1300-Fa14-3-3C-GFP positive transgenic plant；
The transgenic seed disinfected is seeded in 1/2MS+ Ticarcillin/Clavulanate Acid 300mg/L+ hygromycin 25mg/L culture basal growths 20 days (A)；Using NPT primer detection Festuca Arundinacea Fa14-3-3C overexpressing plants (B)；Using 35S+Fa14-3-3C R primers Detect overexpressing plants (C)；
Fig. 4 is Festuca Arundinacea Fa14-3-3C Subcellular Localizations；
It will turn have the Agrobacterium conversion tobacco epidermal cells of pCAMBIA1300-Fa14-3-3C-GFP carriers, swash ultraviolet Luminous lower observe observe signal is distributed in plant cell to indicate that Fa14-3-3C protein subcellulars position.A is ultraviolet Fluorescence signal is distributed under exciting light, and B is the form of Tobacco Epidermis under light field.
Fig. 5 is that Festuca Arundinacea Fa14-3-3C Expressed in Transgenic Plant is analyzed and low nitrogen induced expression is analyzed；
A, the RNA of 10 -day-old of Festuca Arundinacea Fa14-3-3C overexpressions and WT lines is extracted, reverse transcription, which is lived, to be obtained CDNA, Fa14-3-3C expression quantity is then analyzed using qRT-PCR；B, analyze in Low nitrogen stress 15d Festuca Arundinacea seedling leaves Fa14-3-3C expression change；
Fig. 6 is Festuca Arundinacea Fa14-3-3C overexpressions compared with biological yield after wildtype Arabidopsis thaliana Low nitrogen stress；
The seed disinfected is seeded in 1/2MS cultures basal growth 3 days, consistent plant is transferred to normal (control, A) With being handled 15 days under the conditions of nitrogen-free agar (processing, B), weigh, count biological yield；
Fig. 7 is Festuca Arundinacea Fa14-3-3C overexpressions and WT lines root long under normal and nitrogen-free treatment conditions Growth change schematic diagram；
(B, D) Fa14-3-3C overexpressions and WT lines growthform under the conditions of normal (A, C) with Low nitrogen stress (A, B) and statistical analysis (C, D).
Embodiment
Embodiments of the invention 1：Festuca Arundinacea Fa14-3-3C gene clonings and expression vector establishment.
According to transcript profile design data RACE primers, 5 ' RACE：FaGF14-C-REV15’ -GTTCTTGTAGGCGACG- 3 ', FaGF14-C-REV 25 '-CTCCTCGACGGTGAGCTC3 ', FaGF14-C-REV 35 '- CGACCATCTCCTCGTACC-3’,3’RACE：FaGF14-C -FWD15’-GCAGAGCAGGCTGAGAGTTATGAAGAGA- 3’,FaGF14-C-FWD2
5 '-TTCATGGAGAAGGTGGCAAAGACAGTT-3 ' obtain according to RACE kits (Clontech) specification Festuca Arundinacea Fa14-3-3C full length mRNA sequences.
Design FaGF14-C-FWD4：[5’-CGGGATCCATGTCGGCACCAGCGGAGCTTTC -3’]；FaGF14-C- REV4：[5 '-GCTCTAGACTCGGCGGCTCCCTTTCG -3 '], for cloning Festuca Arundinacea Fa14-3-3C sequences, and connected PEASY-Blunt cloning vectors (pEASY-Blunt Cloning Kit, Transgene) are connected to, carry out sequencing analysis, are utilized Correct clone is connected to p1300-GFP carriers by BamH I and Xba I.Concrete outcome reference picture 1.
1st, the acquisition of Festuca Arundinacea 14-3-3C sequences.
(1) RNA extraction refers to (TRIZOLTMKit RNA extracts reagents specification)
(2) reverse transcription of the chains of cDNA first uses RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas), operation are carried out with reference to used kit explanation.
(3) RACE reactions use Clontech SMART RACE kit, and operating method is with reference to kit specification.
(4) splice the result of sequence according to RACE, design full-length clone primer, using the cDNA of synthesis in (2) as template gram Grand total length 14-3-3C CDS sequences, kit PhusionTMHigh-Fidelity DNA Polymerase, reaction system It is as follows：5 × buffer, 4 μ l；DNTP mix, 0.5 μ l；MgCl2,1.5μl；FaGF14-C-FWD4,1 μ l； FaGF14-C- REV4,1 μ l；CDNA templates, 2 μ l；DNA Polymerase,0.2μ l；dd H2O, 10 μ l.Program is as follows, 95 DEG C of 1min, and 95 DEG C 10s,
58 DEG C of 10s, 72 DEG C of 1min, 30cycles, 72 DEG C of 5min, 25 DEG C of 5min.
(5) using Shanghai life work glue reclaim kit (SK8132, Shanghai life work) recovery Festuca Arundinacea Fa14-3-3C genes.
(6) by the fragment of recovery be connected to pEASY-Blunt cloning vectors (pEASY-Blunt Cloning Kit, Transgene), Festuca Arundinacea 14-3-3C genes are obtained using sequencing analysis, its base sequence is SEQ ID NO：Core shown in 1 Nucleotide sequence.Its corresponding protein sequence is SEQ ID NO：Amino acid sequence shown in 2.
2nd, Overexpression vector is built
Using BamH I and digestion pEASY-Blunt-Fa14-3-3C and the p1300-GFP empty carriers of Xba I, reaction system It is as follows：dd H2O, 33 μ l；1×Tango buffer, 5μl；BamHⅠ,1μl；XbaⅠ,1μl；Plasmid, 10 μ l.37 DEG C, digestion 2h.Using Shanghai life work glue reclaim kit (SK8132, Shanghai life work) recovery Festuca Arundinacea Fa14-3-3C genes and P1300-GFP empty carriers.
Purpose fragment is connected with T4DNA ligases, reaction system is as follows, Fa14-3-3C fragments, 4 μ l；P1300-GFP is empty Carrier segments, 4 μ l；T4DNA connections buffer, 1 μ l；T4DNA ligases, 1 μ l；4 DEG C, connect 16h.Converted using heat shock method DH5 α competent cells, are screened using kanamycins, and through vector primer (35S starts word aligning primer) and gene primer (FaGF14-C-REV4) performing PCR identification is entered, response procedures are cloned with Fa14-3-3C, finally obtain p1300-Fa14-3-3C- GFP carriers, as shown in Figure 1.
Embodiments of the invention 2：P1300-Fa14-3-3C-GFP transfer-gen plants obtain and its expression analysis.
1st, Agrobacterium-mediated Transformation
P1300-Fa14-3-3C-GFP carriers are transformed into by GV3101 competent cells using electric robin, celebrated using sulfuric acid Big mycin and kanamycins are screened, and enter performing PCR identification, the same Fa14-3- of response procedures using 35S and FaGF14-C-REV4 3C is cloned, and obtains conversion p1300-Fa14-3-3C-GFP positive Agrobacterium colonies.
2nd, transformation of Arabidopsis thaliana
(1) the Agrobacterium single bacterium containing target plasmid activated from picking on resistance plate falls on 5mL LB+25mg/L celebratings The LB culture mediums of big mycin+50mg/L kanamycins activate 24 h, 28 DEG C, 220rpm.
(2) according to 1:The bacterium solution of activation is inoculated into 200mL LB+ gentamicin 25mg/L+ kanamycins by 50 ratio 28 DEG C of 50mg/L culture medium, 220rpm culture 8-12h,.
(3) room temperature 5000rpm centrifuges 15min.Supernatant is abandoned, Agrobacterium precipitation is suspended in the inducing culture of fresh configuration In (10g sucrose is completely dissolved in 200ml distilled water, and 20 μ l Silwet (Shanghai life work) are added before conversion).
(4) wild-type Arabidopsis plants for removing flower and silique are directly dipped to agrobacterium suspension 30s.
(5) lucifuge overnight incubation, then normal culture to seed maturity.
3rd, transfer-gen plant screens
(1) T0 is distributed into 200mg for the arabidopsis seed of transgenosis often to manage, with 75% ethanol disinfection 1min, with 50% 84 thimerosals (clean auntie, commercially available) sterilize 3min.
(2) sterile water wash seed is used 3-4 times, even suspension is spread in 1/2MS+ Ticarcillin/Clavulanate Acids in 0.1% agarose 300mg/L+ kanamycins 50mg/L culture mediums, 4 DEG C of vernalization 3 days.
(3) in temperature：22 DEG C, humidity：80%, intensity of illumination：80-200μmol/m2/ s, photoperiod：16h illumination, 8h are black Cultivated 15-30 days in dark phjytotron, the plantlet of transplant for selecting green stalwartness is standby into Nutrition Soil.
4th, transfer-gen plant is identified
(1) with Tiangeng plant DNA extraction kit (DP305) extraction transgenosis and the fresh blade group of wildtype Arabidopsis thaliana The DNA knitted, program technic is with reference to kit specification.
(2) using 35S and FaGF14-C-REV4 primers combine carry out PCR identifications, PCR reaction using Shanghai life work 2 × Taq PCR reaction kits, reaction system are as follows：2 × Taq PCR Mixture, 10 μ l；35S forward primers, 1 μ l； FaGF14-C-REV4 reverse primers, 1 μ l；DNA, 2 μ l；dd H2O, 6 μ l.Laggard performing PCR amplified reaction is mixed, program is as follows： 94 DEG C of 3min, 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 30s, 30cycles, 72 DEG C of 5min, 25 DEG C of 5min.Take 8 μ l PCR primers electric Swimming detection, selects positive plant and is used for expression analysis, as a result as (Fig. 3 C) shows.
5th, transfer-gen plant expression analysis
(1) wild type and transgenic arabidopsis total serum IgE are extracted with TRIZOL RNA kits (Invitrigen), with reference to examination Agent specification
(2) RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) are used The chains of reverse transcription cDNA first, operating method is with reference to kit explanation.
(3) using GoTaq Real-Time PCR Systems (Promega, A6001) detection genetically modified plants and wild 14-3-3C expression, primer are combined as in type：Fa14-3-3C-FWD5,5 '-TTGCCTACCCTGGATAAGATCTAAG-3 ' With Fa14-3-3C-REV5,5 '-TAATAAACCCAGTCGTATCGCTTAG-3 ', reference gene uses Ubiquitin, primer It is combined as UBI-FWD1,5 '-CACCTCGATCACCCACCTCT-3 ', UBI-REV1
5 '-AGGGTCTCCGATAACCTCCA-3 ', for operating method according to kit specification, response procedures are 94 DEG C 2min, 94 DEG C of 15s, 58 DEG C of 15s, 72 DEG C of 30s, read plate, 45 circulations, 72 DEG C of 5min, 25 DEG C of 5min.Analyze 14-3- 3C expression change, wherein 14-3-3C OE-1 significantly raise with -3 expression quantity, and OE-2 up-regulation unobvious.
Embodiments of the invention 3：Fa14-3-3C-GFP Subcellular Localizations.
1st, tobacco is planted
The leaflet tobacco seed newly harvested is uniformly sowed in peat soil：Vermiculite=1:In 1 matrix, 22~24 DEG C, 16h Illumination, 8h dark-growns 30-60 days, water 1 time within every 3~7 days.
2nd, agrobacterium strains are activated
It is mould that Agrobacterium containing p1300-Fa14-3-3C-GFP plasmids in LB+25mg/L gentamicins+50mg/L is blocked that 28 DEG C of activation culture 2-4d of solid medium of element, picking monoclonal block that to 50mL LB+25mg/L gentamicins+50mg/L Fluid nutrient medium concussion and cultivate 16~24h of mycin, 28 DEG C, 220rpm.5000rpm room temperatures centrifuge 10min, and supernatant is outwelled, With 50mL dilution buffers (10mM MgCl2, 10mM MES, pH=5.7,0.5% glucose, 200mM acetosyringones) it is outstanding Floating, 5000rpm room temperatures centrifuge 10min again, and supernatant is outwelled, and are suspended with 50mL dilution buffers, standby.
3rd, convert and observe fluorescence signal
The Agrobacterium activated is squeezed into tobacco leaf following table chrotoplast, half-light culture with 1~3mL disposable syringe 3~5d, tear the fluorescence signal that tobacco lower epidermis observes 508nm under uv excitation light.
Embodiments of the invention 4：Fa14-3-3C-GFP induced expressions are analyzed.
1st, Festuca Arundinacea Low nitrogen stress is handled
It is sterile with 100mL from tall fescue seed (the Guizhou Province grass No. 1) min of 75% alcohol surface sterilizations of 50mL 1 newly harvested Water rinses 3~5 times.Germinateed culture Festuca Arundinacea seedling in the culture dish for being lined with sterilizing filter paper, 22 DEG C, germinate 7d, add 3 daily~ 4mL sterilized waters.The seedling for selecting neat and consistent carries out water planting processing, and control group is placed in Hoagland nutrient solutions and cultivates 15d, locates Reason group is by seedling in nitrogen-free water planting liquid (NO in Hoagland nutrient solutions3- substituted by Cl-) Low nitrogen stress 15d, changed 1 time per 3d Nutrient solution, illumination 16h, dark 8h, 22 DEG C of growth temperature.
2nd, induced expression is analyzed
5th part in RNA preparations, reverse transcription and quantitative fluorescence analysis reference case study on implementation 2, wherein, Fa14-3-3C energy Significantly induced by Low nitrogen stress.
Embodiments of the invention 5：The resistance to Low nitrogen stress analysis of p1300-Fa14-3-3C-GFP transfer-gen plants.
1st, Fa14-3-3C promotes plant biological yield increase under the conditions of Low nitrogen stress
By wild type and turn Fa14-3-3C genes arabidopsis sterilization after be seeded in 1/2MS solid mediums, 22 DEG C, after 16h/8h (light dark) is cultivated 4 days, the seedling for selecting neat and consistent is transplanted to the full nutrient mediums of 1/2MS and 1/ respectively Grown 15 days in 2MS nitrogen stress culture mediums, separately sampled weighing fresh weight, under normal operation, 14-3-3C transfer-gen plants with it is wild Type arabidopsis fresh weight does not have marked difference, and under the conditions of Low nitrogen stress, 14-3-3C transfer-gen plants OE-1 and OE-3 fresh weights Significantly than wildtype Arabidopsis thaliana increase, and OE-2 increases unobvious, and change and the changes in gene expression of phenotype are coincide.
2nd, Fa14-3-3C promotes the root system of plant under the conditions of Low nitrogen stress to extend
It will be seeded in after wild type and Fa14-3-3COE-1 arabidopsis seed disinfections in 1/2MS solid mediums, 22 DEG C, 16h/8h (light dark) cultivate 4 days after, select neat and consistent seedling be transplanted to respectively containing the full nutrient mediums of 1/2MS with Grown 10 days in the square culture dish of 1/2MS nitrogen stress culture mediums, measure root length daily, count lower turn of different growth conditions The change of 14-3-3C genes and wildtype Arabidopsis thaliana root length.Under normal operation, Fa14-3-3COE-1 root systems and open country Raw type is not significantly different, and under the conditions of Low nitrogen stress, since Fa14-3-3COE-1 root systems handling the 2nd day significantly than wild Type arabidopsis is grown, and is maintained to processing and terminates, and illustrates that by overexpression Fa14-3-3C genes plant pair can be remarkably promoted The tolerance of Low nitrogen stress.Daily data analysis use Excel 2010, graphics software use Photoshop CS6 and PowPoint 2010 is handled.
SEQUENCE LISTING
Sequence table
<110>Guizhou Province Grass Industry Research Institute
<120>The coded sequence of the gene of resistance to Low nitrogen stress Fa14-3-3C a kind of and application
<130> nm:
<160> 2
<170> PatentIn version
<210> 1
<211> 783
<212> DNA
<213>Tall fescue (Festuca Arundinacea)
<400>
ATGTC GGCAC CAGCG GAGCT TTCCC GGGAG GAGAA CGTGT ACATG GCCAA 50
GCTCG CCGAG CAGGC CGAGC GCTAC GAGGA GATGG TCGAC TTCAT GGAGA 100
AGGTG GCCAA GACGG TCGAC TCGGA GGAGC TCACC GTCGA GGAGC GCAAC 150
CTCCT CTCCG TCGCC TACAA GAACG TCATC GGGGC GCGCC GCGCC TCCTG 200
GCGCA TCATC TCCTC CATCG AGCAG AAGGA GGAGA GCCGC GGCAA CGAGG 250
ACCGC GTCAC CCTCA TCAAG GACTA CCGCG GCAAG ATCGT GACTG AGCTC 300
ACCAA GATCT GCGAC GGCAT CCTCA AGCTG CTCGA GACCC ACCTC GTCCA 350
ATCTT CCACC GCACC CGAGT CCAAG GTCTT CTACC TCAAG ATGAA GGGTG 400
ACTAC TACAG GTATC TTGCG GAATT CAAGA GTGGG GCTGA GAGGA AGGAT 450
GCTGC TGAGA ATACC ATGGT GGCAT ACAAG GCTGC TCAGG ATATT GCTTT 500
GGCTG AGCTG GCTAC AACCC ATCCC ATTAG GCTTG GGCTG GCACT CAACT 550
TCTCG GTGTT CTACT ATGAG ATCCT CAACT CGCCT GATCG TGCTT GCAAC 600
CTCGC AAAGC AGGCT TTCGA TGAGG CCATC TCTGA GTTGG ACACC CTGAA 650
CGAGG AATCC TACAA GGACA GCACT TTGAT CATGC AACTC CTACG TGACA 700
ACTTG ACCCT GTGGA CTTCA GACAT CACGG AGGAC ACTGC GGAGG AGGAG 750
ATCAG GGAGG CCCCG AAGGG AGCCG CCGAG TAA 783
<210> SEQ ID NO. 2
<211> 1
<212> 87
<213>Tall fescue (Festuca Arundinacea)
<400>
MSAPA ELSRE ENVYM AKLAE QAERY EEMVD FMEKV AKTVD SEELT VEERN 50
LLSVA YKNVI GARRA SWRII SSIEQ KEESR GNEDR VTLIK DYRGK IVTEL 100
TKICD GILKL LETHL VQSST APESK VFYLK MKGDY YRYLA EFKSG AERKD 150
AAENT MVAYK AAQDI ALAEL ATTHP IRLGL ALNFS VFYYE ILNSP DRACN 200
LAKQA FDEAI SELDT LNEES YKDST LIMQL LRDNL TLWTS DITED TAEEE 250
IREAP KGAAE - 260
<210> 3
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>According to transcript profile sequence information, reversely drawing for the RACE of Fa14-3-3C 5 ' is designed with Primer Premier 5.0 Thing is used to clone the terminal sequences of Fa14-3-3C 5 '.
<400> 3
GTTCT TGTAG GCGAC G 16
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>According to transcript profile sequence information, reversely drawing for the RACE of Fa14-3-3C 5 ' is designed with Primer Premier 5.0 Thing is used to clone the terminal sequences of Fa14-3-3C 5 '.
<400> 4
CTCCT CGACG GTGAG CTC 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>According to transcript profile sequence information, reversely drawing for the RACE of Fa14-3-3C 5 ' is designed with Primer Premier 5.0 Thing is used to clone the terminal sequences of Fa14-3-3C 5 '.
<400> 5
CGACC ATCTC CTCGT ACC 18
<210> 6
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>According to transcript profile sequence information, the forward direction that the RACE of Fa14-3-3C 3 ' are designed with Primer Premier 5.0 is drawn Thing is used to clone the terminal sequences of Fa14-3-3C 3 '.
<400> 6
GCAGA GCAGG CTGAG AGTTA TGAAG AGA 28
<210> 7
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>According to transcript profile sequence information, the forward direction that the RACE of Fa14-3-3C 3 ' are designed with Primer Premier 5.0 is drawn Thing is used to clone the terminal sequences of Fa14-3-3C 3 '.
<400> 7
TTCAT GGAGA AGGTG GCAAA GACAG TT 27
<210> 8
<211> 31
<212> DNA
<213>Artificial sequence
<220>
<223>According to RACE results, then clone's Fa14-3-3C total lengths are designed with the Software for Design of Primer Premier 5.0 CDS forward primer, add the joints of BamH I.
<400> 8
CGGGA TCCAT GTCGG CACCA GCGGA GCTTT C 31
<210> 9
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>According to RACE results, then clone's Fa14-3-3C total lengths are designed with the Software for Design of Primer Premier 5.0 CDS reverse primer, add the joints of Sal I, while can be used for Molecular Detection genetically modified plants.
<400> 9
GCTCT AGACT CGGCG GCTCC CTTTC G 26
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>35S promoter sequence is obtained from ncbi database retrieval, then with the Software for Design of Primer Premier 5.0 The forward primer 35S held close to promoter 3 ', for detecting transfer-gen plant.
<400> 10
GGCGC GCCAG CCGTC TTCGT CGCG 24
<210> 11
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>Determined according to total length Fa14-3-3C sequences with the Software for Design of Primer Premier 5.0 design 14-3-3C fluorescence Expression analysis forward primer is measured, for detecting 14-3-3C changes in gene expression.
<400> 11
TTGCC TACCC TGGAT AAGAT CTAAG 25
<210> 12
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>Determined according to total length Fa14-3-3C sequences with the Software for Design of Primer Premier 5.0 design 14-3-3C fluorescence Expression analysis reverse primer is measured, for detecting 14-3-3C changes in gene expression.
<400> 12
TAATA AACCC AGTCG TATCG CTTAG 25
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>BLAST obtains Festuca Arundinacea Ubiquitin gene orders, then with the Software for Design bases of Primer Premier 5.0 The forward primer of cause, to the internal reference reacted as quantitative fluorescent PCR.
<400> 13
CACCT CGATC ACCCA CCTCT 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>BLAST obtains Festuca Arundinacea Ubiquitin gene orders, then with the Software for Design bases of Primer Premier 5.0 The reverse primer of cause, to the internal reference reacted as quantitative fluorescent PCR.
<400> 14
AGGGT CTCCG ATAAC CTCCA 20

Claims (5)

  1. A kind of 1. Festuca Arundinacea gene of resistance to Low nitrogen stress Fa14-3-3C coded sequence, it is characterised in that：It has as in sequence table SEQ ID NO：Nucleotide sequence shown in 1, or with SEQ ID NO：The core of DNA sequence dna at least 70% homology shown in 1 Nucleotide sequence；Or with SEQ ID NO in polynucleotide：The polynucleotides of amino acid sequence shown in 2；Either by SEQ ID NO：2 amino acid residue sequence passes through substitution, missing or the addition of more than one amino acid residue, has and SEQ ID NO：The sequence of 2 amino acid sequence at least 70% homology.
  2. 2. a kind of Festuca Arundinacea Fa14-3-3C as claimed in claim 1 coded sequence is in the resistance to Low nitrogen stress of plant is adjusted Using.
  3. 3. Festuca Arundinacea Fa14-3-3C according to claim 2 coded sequence answering in the resistance to Low nitrogen stress of plant is adjusted With, it is characterised in that：By in Festuca Arundinacea Fa14-3-3C gene transferred plants, to improve the resistance to Low nitrogen stress ability of plant.
  4. 4. Festuca Arundinacea Fa14-3-3C according to claim 2 coded sequence answering in the resistance to Low nitrogen stress of plant is adjusted With, it is characterised in that：Described Low nitrogen stress refers to that nitrogen content in soil is 0~25mMol/L.
  5. 5. Festuca Arundinacea Fa14-3-3C according to claim 2 coded sequence answering in the resistance to Low nitrogen stress of plant is adjusted With, it is characterised in that：Described plant be rice, corn, sorghum, hybrid giant napier, Chinese pennisetum, rye grass, oat, orchardgrass, vegetables, Fruit tree or tobacco.
CN201710117593.3A 2017-03-01 2017-03-01 The coded sequence of 3C of the Festuca Arundinacea gene of resistance to Low nitrogen stress Fa14 3 a kind of and its application Pending CN107446930A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592106A (en) * 2019-10-31 2019-12-20 宁夏大学 Molecular marker Lb14-3-3c gene and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110592106A (en) * 2019-10-31 2019-12-20 宁夏大学 Molecular marker Lb14-3-3c gene and application thereof

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