CN103045639B - Application of AtTGA 4gene in improving plant adverse resistance - Google Patents
Application of AtTGA 4gene in improving plant adverse resistance Download PDFInfo
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Abstract
The invention discloses a novel application of an AtTGA 4 protein and an encoding gene thereof. The novel application of the AtTGA 4 protein and the encoding gene thereof provided by the invention concretely is the application of the AtTGA 4 protein and the encoding gene thereof in regulating and controlling the plant drought resistance and/or regulating and controlling uptake of nitrogen. The AtTGA 4 protein is the protein of the following (1) or (2): (1) the protein consisting of an amino acid sequence shown by a sequence 2 in a sequence table; and (2) the protein which consists of the amino acid sequence shown by the sequence 2 in the sequence table being replaced and/or deleted and/or added by one or several amino acid residues, is derived by the protein in (1) and has the same function with the protein in (1). Experiments prove that compared with the wild type plates, the drought resistance and the uptake capability of nitrogen of the plant with the encoding gene of the AtTGA 4 protein cultured by the invention are greatly increased.
Description
Technical field
The present invention relates to AtTGA4 albumen and encoding gene thereof in regulating plant drought resistance and/or regulation and control to the application in Nitrogen Absorption, particularly AtTGA4 albumen and encoding gene thereof improve plant drought resistance and improve plant nitrogen absorb in application.
Background technology
Nitrogen is the necessary nutritive substance of growth and development of plants.Nitrogen is not only the nutritive element of plant, plant degeneration-resistant in also play an important role.Under drought condition, plant not only can by " root-hat " the information transmission between root and over-ground part, make plant can adjust overall water relation (Davies WJ rapidly, Zhang J.Root signalsand the regulation of growth and development of plants in drying soil.Annu ReV PlantPhysiol Plant Mol Biol, 1991, 42, 55-76), and plant also accumulates if the Soluble osmoticums such as proline(Pro) are as osmotic protection material (Delauney AJ, Hu C AA, Kishor P B K, et al.cloning ofornithine-aminotransferase cDNA from Vigna aconitifolia by trans-complementation inEscherichia coli and regulation of proline biosynthesis [J] .J Biol Chem, 1993, 268:18673-18678), the intermediate product of Proline Metabolism has inducible gene expression (Iyer S simultaneously, CaplanA.Prodcts of proline catabolism can induce osmotically regulated genes in rice [J] .PlantPhysiol, 1998, 116:203-211) and reduce oxygen injury effect (the Hong ZL that causes of osmotic stress, LakkineniK, Zheng ZH, et al.Removal of feedback inhibition of pyrroline-5-carboxylate synthetaseresults in increased proline accumulation and protection of plants from osmotic stress [J] .Plant Physiol, 2000, 122:1129-1136).These drought stress response mechanisms of plant all have close relationship with nitrogen.Large quantity research shows that ABA is core substance (the Zhang J that mediation " root-hat " information is transmitted, Davies WJ.Antitranspirant activty in the xylem sap of maize plants.J Exp Bot, 1991, 42, 317-321), nitrogen nutrition simultaneously and ABA accumulation have close relationship (Wilkinson S, Daies WJ.ABA-based chemicalsignaling:the co-ordination of responses to stress in plant.Plant Cell Environ.2002, 25, 195-210), the increase of nitrogen nutrition can significantly improve susceptibility (the Liu J of pore to root signal, Wei KF, Gao ZH, Li BB, Ren HB, Hu JF, Jia WS.Nitrate as an enhancer of root signal in theregulation of stomatal movement in plants under drought stress.Chin Bull Bot, 2008, 25, 34-40).Under drought condition, the increase of plant proline(Pro) also has close relationship (Voetberg G S with the absorption of nitrogen and metabolism, Sharp R E, Growth at the maize primary root at low water potential.3.Roleof increased proline deposition in osmotic adjustment [J] .Plant Physiol, 1991,96:1125-1130).Excavation can strengthen the gene of plant drought resistance under drought condition by improving the absorption of plant to nitrogen, for the drought resistance improving plant, increase yield is significant simultaneously.
Not yet have at present and whether AtTGA4 transcription factor is played a role the report studied in plant drought.
Summary of the invention
The object of this invention is to provide the novelty teabag of a kind of AtTGA4 albumen and encoding gene thereof.AtTGA4 albumen is obtained by nucleoprotein screening system (NTT) screening.
Novelty teabag provided by the present invention is specially AtTGA4 albumen or its encoding gene in regulating plant drought resistance and/or regulation and control to the application in Nitrogen Absorption;
Described AtTGA4 albumen is following 1) or 2) protein:
1) protein be made up of the aminoacid sequence shown in sequence in sequence table 2;
2) by the aminoacid sequence shown in sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation, and have and 1) identical function by 1) derivative protein.
In the present invention, described regulating plant drought resistance and/or to Nitrogen Absorption be embodied in improve plant drought resistance and/or improve plant to Nitrogen Absorption ability.
Another object of the present invention is to provide AtTGA4 albumen or the application of its encoding gene in following (a1)-(a6) at least one of regulating plant;
(a1) plant proline content;
(a2) Plant Leaf chlorophyll contents;
(a3) plant peroxidase activity;
(a4) plant total nitrogen content;
(a5) plant nitrate reductase activity;
(a6) expression amount of plant nitrogen pathway key gene, described nitrogen pathway key gene is specially at least one in NRT2.1, NRT2.2, NIA1 and MIA2;
Described AtTGA4 albumen is following 1) or 2) protein:
1) protein be made up of the aminoacid sequence shown in sequence in sequence table 2;
2) by the aminoacid sequence shown in sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation, and have and 1) identical function by 1) derivative protein.
In the present invention, described regulating plant above-mentioned (a1)-(a6) proterties is embodied in: can improve plant proline content after drought-induced, and/or improves Plant Leaf chlorophyll contents, and/or reduces plant peroxidase activity; At low nitrogen, (as ammonium nitrogen or nitric nitrogen, nitrogen content is lower than MS
0nitrogen element content in substratum) plant total nitrogen content can be improved under condition, and/or improve plant nitrate reductase activity, and/or improve the expression amount of plant nitrogen pathway key gene (as NRT2.1, NRT2.2, NIA1 or NIA2).
Another object of the present invention is to provide a kind of method of cultivating transgenic plant, comprises the steps: the encoding gene of AtTGA4 albumen to import in object plant, obtains the transgenic plant with at least one proterties in following (b1)-(b8):
(b1) drought resistance improves compared with described object plant;
(b2) receptivity comparing nitrogen with described object plant improves;
(b3) with described object plant all after drought-induced, its plant proline content is higher than described object plant;
(b4) with described object plant all after drought-induced, its Plant Leaf chlorophyll contents is higher than described object plant;
(b5) with described object plant all after drought-induced, its plant peroxidase activity is lower than described object plant;
(b6) with described object plant all after low nitrogen process, its plant total nitrogen content is higher than described object plant;
(b7) with described object plant all after low nitrogen process, its plant nitrate reductase activity is higher than described object plant;
(b8) with described object plant all after low nitrogen process, in its plant, the expression amount of nitrogen pathway key gene is higher than described object plant, and described nitrogen pathway key gene is specially at least one in NRT2.1, NRT2.2, NIA1 and NIA2;
Low nitrogen described in step (b6)-(b8) is that nitrogen element content is lower than the normal required nitrogen element content of described object plant.In the present invention, described low nitrogen is that nitrogen element content is lower than MS
0nitrogen element content or lower than the nitrogen element content in Hoagland water planting liquid (formula is shown in embodiment) in solid medium (formula is shown in embodiment); In the present invention, described low nitrogen is specially 0.2mM NO
3 -or 2mM NH
4 +or 1mM NH
4 +.
Described AtTGA4 albumen is following 1) or 2) protein:
1) protein be made up of the aminoacid sequence shown in sequence in sequence table 2;
2) by the aminoacid sequence shown in sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation, and have and 1) identical function by 1) derivative protein.
In above-mentioned application or method, the encoding gene (AtTGA4 gene) of described AtTGA4 albumen specifically can be following a)-c) in arbitrary described gene:
A) gene of encoding sequence as shown in sequence in sequence table 1;
B) gene of nucleotide sequence as shown in sequence in sequence table 1;
C) under strict conditions with gene recombination a) or b), and the gene of protein of the composition of aminoacid sequence shown in encoding sequence 2.
Wherein, sequence 1 is made up of 1095 Nucleotide, and whole sequence 1 is the encoding sequence of AtTGA4 gene, and the albumen (AtTGA4 albumen) in polynucleotide shown in sequence 2, sequence 2 is made up of 364 amino acid altogether.
In the above-mentioned methods, the encoding gene of described AtTGA4 albumen is imported in described object plant by the recombinant expression vector of the encoding gene containing described AtTGA4 albumen.
The promotor that the encoding gene starting described AtTGA4 albumen in described recombinant expression vector is transcribed can be 35s promotor.
In one embodiment of the invention, described recombinant expression vector is specially the multiple clone site encoding gene of described AtTGA4 albumen being inserted into pBI121 plasmid (35s promotor) downstream, the recombinant plasmid of the encoding gene of the described AtTGA4 albumen of the expression obtained.Described multiple clone site is specially Sma I and Spe I.
In above-mentioned application or method, described plant can be dicotyledons, also can be monocotyledons.
In one embodiment of the invention, described plant is dicotyledons, and described dicotyledons is specially Arabidopis thaliana, as Arabidopis thaliana kind Columbia0.
In the present invention, above-mentioned application or the drought resisting involved by method and/or Nitrogen Absorption are embodied at least one in following index:
(c1) plant survival rate;
(c2) plant phenotype, as root length, diameter, surface-area, stem length and leaf area etc.;
(c3) proline content of plant;
(c4) chlorophyll content of plant;
(c5) peroxidase activity of plant;
(c6) total nitrogen content of plant;
(c7) nitrate reductase activity of plant;
(c8) expression amount of nitrogen pathway key gene in plant, as NRT2.1, NRT2.2, NIA1 or NIA2.
In general, drought resistance raising is embodied in compared with not genetically modified wild type control, the plant of the encoding gene of AtTGA4 albumen is proceeded to after Osmotic treatment, its survival rate is relatively high, root length, diameter and stem long relatively length, root system surface area and leaf area is relatively large, the proline content of plant and chlorophyll content are relatively high, and the peroxidase activity of plant is relatively low; The raising of Nitrogen Absorption ability is embodied in compared with not genetically modified wild type control, the plant of the encoding gene of AtTGA4 albumen is proceeded to after low nitrogen (as ammonium nitrogen or nitric nitrogen) process, its survival rate is relatively high, root length, diameter and stem long relatively length, root system surface area and leaf area is relatively large, the total nitrogen content of plant is relatively high, the nitrate reductase activity of plant is relatively high, and in plant, the expression amount of nitrogen pathway key gene NRT2.1, NRT2.2, NIA1 and NIA2 is relatively high.
Experiment proves, the plant proceeding to the encoding gene of AtTGA4 albumen utilizing method provided by the present invention to cultivate to obtain is compared with WT lines, and its drought resistance and the receptivity to nitrogen are all greatly improved.
Accompanying drawing explanation
Fig. 1 is the expression of AtTGA4 gene in Osmotic treatment different time sections.
Fig. 2 is AtTGA4 gene (0.2mM NO under low nitrogen condition
3 -with 2mM NH
4 +) expression of different time sections.Wherein, A is that AtTGA4 gene is at 0.2mM NO
3 -the expression of process different time sections; B is that AtTGA4 gene is at 2mM NH
4 +the expression of process different time sections.In A and B, " D " expression in X-coordinate " my god ".
Fig. 3 is the structure schema of recombinant expression vector pBI121-TGA4.
Fig. 4 is that 50mg/mL Kan screens the T obtained
0in generation, turns AtTGA4 gene Arabidopsis plant seedling (being positioned at arrow indication circle).
Fig. 5 is 4 and turns AtTGA4 gene strain and wildtype Arabidopsis thaliana germination period drought resistance detected result.Wherein, grow the statistics of the seedling number of 2 greenery when A is 8%PEG process to 10 day, concrete, A-1 is normal MS
0the growthhabit of each Arabidopis thaliana in solid medium treatment group, A-2 is the MS containing 8%PEG
0the growthhabit of each Arabidopis thaliana in solid medium treatment group, A-3 is the sample distribution schematic diagram of A-1 and A-2 treatment group, and A-4 is the MS containing 8%PEG
0the seedling number statistics of 2 greenery is grown in solid medium treatment group, when B is 8%PEG process to 17 day, Phenotypic Observation, root system scans, stem length and Leaf area determination result, the phenotype (its distribution is as shown in A-3) that B-1 is each Arabidopis thaliana during 8%PEG process to 17 day, when B-2 is 8%PEG process to 17 day, each Arabidopis thaliana Root morphology compares, each Arabidopis thaliana root long statistics when B-3 is 8%PEG process to 17 day, each Arabidopis thaliana root surface area statistics when B-4 is 8%PEG process to 17 day, each Arabidopis thaliana average root diameter statistics when B-5 is 8%PEG process to 17 day, each Arabidopis thaliana stem long statistics when B-6 is 8%PEG process to 17 day, each Arabidopis thaliana leaf area statistics when B-7 is 8%PEG process to 17 day.
Fig. 6 is 4 and turns AtTGA4 gene strain, wildtype Arabidopsis thaliana and AtTGA4 Arabidopsis Mutants drought resistance of seedling detected result---phenotype.Wherein, A is before Osmotic treatment; B is that Osmotic treatment is after 18 days; C is Osmotic treatment 18 days and rehydration after 5 days.Mutant represents AtTGA4 Arabidopsis Mutants.
Fig. 7 is 4 and turns AtTGA4 gene strain, wildtype Arabidopsis thaliana and AtTGA4 Arabidopsis Mutants drought resistance of seedling detected result---survival results.Mutant represents AtTGA4 Arabidopsis Mutants.
Fig. 8 is 4 and turns AtTGA4 gene strain, wildtype Arabidopsis thaliana and AtTGA4 Arabidopsis Mutants drought resistance of seedling detected result---the mensuration of Physiological Indices of Drought Resistance.Wherein, A is proline content; B is that peroxidase (POD) is active; C is chlorophyll content.Mutant represents AtTGA4 Arabidopsis Mutants.
Fig. 9 is 4 detected results turning AtTGA4 gene strain and wildtype Arabidopsis thaliana germination period Nitrogen Absorption ability.Wherein, A is each Arabidopsis plant phenotype, and A-1 is normal MS
0the each Arabidopsis plant form of solid medium treatment group, A-2 is containing 0.2mM NO
3 -mS
0the growing state of each Arabidopis thaliana on solid medium, A-3 is containing 0.2mM NO
3 -mS
0on solid medium, each Arabidopis thaliana Root morphology compares, and A-4 is the sample distribution schematic diagram of A-1, A-2 and A-5, and A-5 is containing 2mM NH
4 +mS
0the growing state of each Arabidopis thaliana on solid medium, A-6 is containing 2mM NH
4 +mS
0on solid medium, each Arabidopis thaliana Root morphology compares; B is that the root system of each Arabidopsis plant scans, stem is long and Leaf area determination result, and B-1 is for containing 0.2mM NO
3 -at MS
0the root long data statistics of each Arabidopis thaliana on solid medium, B-2 is containing 0.2mM NO
3 -mS
0the root surface area data statistics of each Arabidopis thaliana on solid medium, B-3 is containing 0.2mM NO
3 -mS
0the average root diameter data statistics of each Arabidopis thaliana on solid medium, B-4 is containing 0.2mM NO
3 -mS
0the stem long data statistics of each Arabidopis thaliana on solid medium, B-5 is containing 0.2mM NO
3 -mS
0the leaf area data statistics of each Arabidopis thaliana on solid medium, B-6 is containing 2mM NH
4 +mS
0the root long data statistics of each Arabidopis thaliana on solid medium, B-7 is containing 2mM NH
4 +mS
0the root surface area data statistics of each Arabidopis thaliana on solid medium, B-8 is containing 2mMNH
4 +mS
0the average root diameter data statistics of each Arabidopis thaliana on solid medium, B-9 is containing 2mM NH
4 +mS
0the stem long data statistics of each Arabidopis thaliana on solid medium, B-10 is containing 2mM NH
4 +mS
0the leaf area data statistics of each Arabidopis thaliana on solid medium.
Figure 10 is 4 detected result---phenotypes turning AtTGA4 gene strain and wildtype Arabidopsis thaliana seedling stage (under water planting condition) Nitrogen Absorption ability.Wherein, A is for containing 0.2mM NO
3 -the form of Hoagland water planting liquid process each Arabidopsis plant after 10 days; B is for containing 1mM NH
4 +the form of Hoagland water planting liquid process each Arabidopsis plant after 10 days.
Figure 11 is 4 detected result---total nitrogens turning AtTGA4 gene strain and wildtype Arabidopsis thaliana Nitrogen Absorption in seedling stage ability.Wherein, A is for containing 0.2mM NO
3 -the total nitrogen of Hoagland water planting liquid process each Arabidopsis plant after 10 days; B is for containing 1mM NH
4 +the total nitrogen of Hoagland water planting liquid process each Arabidopsis plant after 10 days.
Figure 12 is 4 and turns nitrogen approach relevant physiological reaction detection result under AtTGA4 gene strain and wildtype Arabidopsis thaliana Drought at seedling stage condition.Wherein, A is total nitrogen; B is that nitrate reductase (NR) is active; C is that glutamine synthetase (GS) is active.
Figure 13 is 4 RT-PCR analytical resultss turning 8 key genes participating in nitrogen approach in AtTGA4 gene strain, wildtype Arabidopsis thaliana and AtTGA4 Arabidopsis Mutants.For the detected result of each gene, be from left to right all followed successively by AtTGA4 Arabidopsis Mutants, wildtype Arabidopsis thaliana, TGA4-1, TGA4-2, TGA4-3, TGA4-4.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Arabidopis thaliana (Arabidopsis thaliana L.) environmental Columbia0: order from Arabidopis thaliana database, http://www.arabidopsis.org/; AtTGA4 Arabidopsis Mutants seed: order from Arabidopis thaliana database, http://www.arabidopsis.org/.Arabidopis thaliana is planted in Institute of Crop Science, Chinese Academy of Agricultural Science's phytotron, and growth room's temperature is set to 21 DEG C, and the long day is that 16h illumination/8h is dark, and short day is that 10h illumination/14h is dark.
PBI121 carrier: purchased from Clontech company, catalog number 6081-1.
MS
0solid medium:
Hoagland water planting liquid, i.e. Hoagland ' s(Gram suddenly) nutrient solution prescription: nitrocalcite 945mg/L, saltpetre 607mg/L, ammonium phosphate 115mg/L, magnesium sulfate 493mg/L, iron salt solutions 2.5ml/L, liquid microelement 5ml/L, pH=6.0.
Wherein, iron salt solutions: iron vitriol 2.78g, disodium ethylene diamine tetraacetate (EDTA.Na) 3.73g, distilled water 500ml, pH=5.5.Liquid microelement: potassiumiodide 0.83mg/L, boric acid 6.2mg/L, manganous sulfate 22.3mg/L, zinc sulfate 8.6mg/L, Sodium orthomolybdate 0.25mg/L, copper sulfate 0.025mg/L, cobalt chloride 0.025mg/L.
The present invention mainly studies the New function of AtTGA4 albumen and encoding gene thereof, described AtTGA4 albumen is that the present inventor (is documented in Liu Yangna by nucleoprotein screening system (NTT) in a large amount of candidate albumen, Chen Ming, Li Liancheng etc. the foundation of nucleoprotein screening system (NTT) and qualification. wheat crops journal, 3 phases in 2007) screening obtains.
The change of embodiment 1, AtTGA4 gene expression amount under arid and low nitrogen condition
One, the expression analysis of AtTGA4 gene under drought condition
1, the extraction of Arabidopis thaliana total serum IgE
By the seed of wildtype Arabidopsis thaliana Columbia0 70%(volume fraction in Bechtop) ethanol postincubation 3min, then with aseptic washing 2 times, wash 1min at every turn, after carrying out sterilising treatment 10min with the clorox of 5 ‰ (mass volume ratios), again with aseptic washing twice, wash one minute at every turn.With toothpick, aseptic Seed Points is sowed at MS
0on solid medium flat board, tissue culture room growth 1 week is transferred in 4 DEG C of vernalization after 3 days, select seedling of the same size and be forwarded to containing 8%PEG(mass volume ratio) MS
0solid medium flat board starts process.Treatment time is set to 0h, 0.25h, 0.5h, 1h, 3h, 6h, 12h, 24h respectively.
The wildtype Arabidopsis thaliana TRNzol method processed is extracted total serum IgE, and the RNA extracted is after 2% agarose gel electrophoresis detection is qualified, and carrying out reverse transcription by M-MLV ThermoScript II (TaKaRa company) to total serum IgE is cDNA, and-20 DEG C save backup.
2, RT-PCR detects the expression of AtTGA4 gene
According to AtTGA4 gene (At5G10030, arabidopsis gene group seat numbering; Http:// www.arabidopsis.org/; The 256-1350 position of Genbank:NM_121041) sequences Design Auele Specific Primer TAG4-F1 and TGA4-R1, object fragment is 200bp.With Arabidopis thaliana Actin gene for internal reference designs primer AraAct-F and AraAct-R, object fragment is 227bp.
The cDNA of the different time sections process obtained in step 1 is diluted to suitable concentration and carries out real-time quantitative PCR mensuration as template.Real-time quantitative PCR is carried out according to Real Master Mix (SYBR Green) PCR kit (TIANGEN company) specification sheets.Reaction system comprises 2.5 × Real Master Mix 4 μ l, upstream and downstream primer each 0.5 μ l, ddH
2o 4.5 μ l, template 1 μ l.Pcr amplification program is: 95 DEG C of denaturation 3min, 95 DEG C of sex change 20s, 53 DEG C of annealing 20s, and 72 DEG C extend 20s, 40 circulations, and each circulation the 3rd step carries out fluorescent collecting, is finally annealed to 65 DEG C, to rise 0.5 DEG C to 95 DEG C sex change 1min every 30s.Then detect its fluorescent value, and carry out solubility curve analysis, each sample arranges 3 repetitions.
TAG4-F1:5 '-GCTTCCACATCTAGACATCCTG-3 ' (At5G10030:214-235bp, i.e. the 214-235 position of sequence 1 in sequence table)
TGA4-R1:5 '-AAGAGCATTGGTATCTACTCCG-3 ' (At5G10030:396-417bp, the i.e. reverse complementary sequence of the 396-417 position of sequence 1 in sequence table)
AraAct-F：5’-CCCCTGCTATGTATGTGGCTAT-3’
AraAct-R：5’-TGCTGTGGTGGTGAAAGAGTAA-3’
RT-PCR analytical results shows, and solubility curve is unimodal, and amplified production specificity is good, and fluorescence curve can react amplification well.Carry out statistical study to AtTGA4 gene at the expression of Osmotic treatment different time sections, as shown in Figure 1, under drought condition, the expression of this gene has instantaneous sharply decline to result, and As time goes on expression amount rises again gradually.Illustrate that this gene may be the gene that response responses of drought stress compares downstream, under drought condition, the background of this gene is expressed and is reduced, but along with stress time increase some directly respond drought stress gene expression amount increase, and then act on this gene, make the expression amount of this gene increase gradually again.
Two, the express spectra of AtTGA4 gene under low nitrogen condition
1, the extraction of Arabidopis thaliana total serum IgE
In Bechtop by wildtype Arabidopsis thaliana Columbia0 after sterilising treatment, with toothpick program request and MS
0on solid medium flat board, 4 DEG C of vernalization moves to after tissue culture room grows 1 week after 3 days, selects seedling of the same size and transfers in containing 2mM NH respectively
4 +with 0.2mM NO
3 -mS
0solid medium starts process, the treatment time is set to 0 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days.The wildtype Arabidopsis thaliana TRNzol method processed is extracted total serum IgE.The Reverse Transcription box reverse transcription detecting qualified RNA TakaRa company is cDNA, and-20 DEG C save backup.
2, RT-PCR detects the expression of AtTGA4 gene
According to the method in above-mentioned steps 1 respectively with the cDNA of 8 of two different treatment time periods for template carries out real-time quantitative PCR mensuration, each sample arranges 3 repetitions.
RT-PCR result is presented at 0.2mM NO
3 -under treatment condition, As time goes on the expression amount of AtTGA4 gene has obvious rising, and when processing 5 days, expression amount is the highest, reduces (in Fig. 2 A) gradually subsequently; At 2mM NH
4 +under treatment condition, the expression amount of AtTGA4 gene is As time goes on stable rises, and again rises (in Fig. 2 B) after having decline a little when processing 6 days.The result shows that AtTGA4 gene the ammonium nitrogen of lower concentration and nitric nitrogen may have response to external world, and strong to the response ratio ammonium nitrogen of nitric nitrogen.
The clone of embodiment 2, AtTGA4 gene and the acquisition of process LAN strain
One, the clone of AtTGA4 gene and the structure of recombinant expression vector pBI121-TGA4
With the wildtype Arabidopsis thaliana Columbia0 of Adult plant for experiment material, extract total serum IgE by TRNzol method, the meal transcript reagent box reverse transcription detecting qualified RNA TaKaRa company is cDNA, and-20 DEG C save backup.
According to CDS sequences Design Auele Specific Primer TGA4-F2 and TGA4-R2 of the AtTGA4 gene that TAIR website provides, amplification AtTGA4 full length gene.Reaction system comprises 2 × GC Buffer 25 μ l, dNTP Mix 4 μ l, upstream and downstream primer each 1 μ l, primerstar 0.3 μ l, ddH
2the above PCR reaction reagent of O 16.7 μ l, template cDNA 2 μ l(all comes from TaKaRa company pcr amplification test kit).Pcr amplification program is 94 DEG C of denaturation 10min, 94 DEG C of sex change 30s, 49 DEG C of annealing 45s, and 72 DEG C extend 1min, 40 circulations, last 72 DEG C of annealing 10min, 4 DEG C of preservations.
TGA4-F2:5 '-TCC
cCCGGGaTGAATACAACCTCGACAC-3 ' (underscore part is the recognition site of SmaI, and sequence is thereafter the 1-19 position of sequence 1)
TGA4-R2:5 '-GG
aCTAGTtTACGTTGGTTCACGTTG-3 ' (underscore part is the recognition site of Spe I, and sequence is thereafter the reverse complementary sequence of the 1078-1095 position of sequence 1)
PCR primer is carried out agarose gel electrophoresis, reclaims test kit with the DNA gel of TaKaRa company and reclaim purifying target DNA.Utilize the P Easy-Blunt cloning Vector system of TIANGEN company, the PCR primer of acquisition is inserted on cloning vector.Reaction system 10 μ l:8 μ l PCR primer, 2 μ l vector, after room temperature places 15min, be added to connection product in 100 μ l TOP10 competent cells, and mixing ice bath 30s, puts 2min on ice after 42 DEG C of heat shock 90s immediately.Add the LB nutrient solution of 500 μ l balances to room temperature afterwards, in 37 DEG C of shaking tables, 200rpm cultivates 1h.After being smoothened on the LB substratum containing Amp resistance by the bacterium liquid glass stick of muddiness, put overnight incubation in 37 DEG C of constant incubators.Picking white mono-clonal, utilizes primer TGA4-F2 and TGA4-R2 to carry out bacterium liquid PCR and detects (object clip size is about 1095bp).
PCR is identified correct bacterial strain shakes bacterium upgrading grain, with restriction endonuclease sma I and Spe I double digestion plasmid, size is about the object band of 1095bp, be connected with the pBI121 carrier large fragment through same double digestion, obtain recombinant plasmid.Recombinant plasmid called after pBI121-TGA4(Fig. 3 of DNA fragmentation (AtTGA4 gene) shown in sequence 1 in the restriction enzyme site Sma I and Spe I insertion sequence table of pBI121 will be shown) through order-checking.In recombinant expression vector pBI121-TGA4, the promotor starting AtTGA4 genetic expression is 35s promotor.Wherein, the albumen (AtTGA4 albumen) in sequence 1 polynucleotide shown in sequence 2.
Two, acquisition and the qualification of AtTGA4 gene Arabidopis thaliana is turned
The recombinant expression vector pBI121-TGA4(that step one is obtained or pBI121 carrier) use freeze-thaw method transformation Agrobacterium C58C1(purchased from Biovector CO., LTD company, article No. article No. Biovector009) (the method for freeze-thaw method transformation Agrobacterium, with reference to Holsters M, de Waele D, Depicker A.Transfection and transformation ofAgrobacterium tumefaciens.Mol Gen Genet, 1978,183:181-187).Carry out bacterium liquid PCR with primer TGA4-F2 and TGA4-R2 to identify, will show through qualification the recombinational agrobacterium called after C58C1/pBI121-TGA4 containing DNA fragmentation (about 1095bp) shown in sequence 1 in ordered list; The recombinational agrobacterium called after C58C1/pBI121 of pBI121 empty carrier will be proceeded to.
Adopt method (the Bechtold N etc. that Agrobacterium inflorescence infects, (1993) In plantaAgrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants.C.R.Acad.Sci.316:1194 – 1199) by recombinational agrobacterium C58C1/pBI121-TGA4(or C58C1/pBI121 of above-mentioned gained) the environmental Columbia0 of arabidopsis thaliana transformation, through the MS containing 50mg/mL Kan
0solid medium screens, and filters out 4 strain T altogether
0in generation, turns AtTGA4 gene plant, is denoted as TGA4-1, TGA4-2, TGA4-3 and TGA4-4(wherein three T
0in generation, turns AtTGA4 gene plant as the seedling in arrow indication circle in Fig. 4).By the positive plant filtered out transplant cultivate in greenhouse to Nutrition Soil, breed often for seed all at the MS containing 50mg/mL Kan
0the enterprising row filter of solid medium, breeding is to T
2for time start to carry out Function Identification.
Further to T
2the transgenic arabidopsis seedling in generation carries out PCR detection, and with the genomic dna of transgenic arabidopsis seedling for template, the primer that PCR detects is TGA4-F2 and TGA4-R2.PCR detected result shows, and 4 different turns AtTGA4 gene strain and all increase and obtain the fragment that length is about 1200bp.And carry out PCR detection with same primer pair wildtype Arabidopsis thaliana Columbia0 plant and the Arabidopsis plant that proceeds to pBI121 empty carrier, all do not obtain above-mentioned amplified fragments.Through qualification, the Arabidopis thaliana called after Col/pBI121-TGA4 of recombinant expression vector pBI121-TGA4 will be proceeded to, proceeds to the Arabidopis thaliana called after Col/pBI121 of pBI121 empty carrier.
Embodiment 3, turn the Identification of Drought of AtTGA4 gene Arabidopis thaliana
One, germination period drought resistance detects
Wildtype Arabidopsis thaliana Columbia0(WT), 4 T with following three kinds of Arabidopsis plant for experiment material:
2in generation, turns AtTGA4 gene strain (TGA4-1, TGA4-2, TGA4-3 and TGA4-4), proceeds to the Arabidopis thaliana of pBI121 empty carrier, after carrying out aseptically process, is sowed at MS respectively
0solid medium and containing 8%(mass volume ratio) MS of PEG
0on solid medium, tissue culture room growth is put in 4 DEG C of vernalization after 3 days, when processing to adding up the seedling number growing 2 greenery when 10 days, when continuing process to 17 days, observing phenotypic difference and carrying out root system scanning, stem length and Leaf area determination.Experiment repetition 3 times, results averaged.
Result shows, and process 10 days and add up afterwards to grow the seedling number of two panels greenery, and 4 turn between AtTGA4 gene strain and wildtype Arabidopsis thaliana and have obvious difference, and green seedling number is obviously more than wild-type (as A in Fig. 5).When processing to 17 days, the growth tendency turning AtTGA4 gene strain is obviously better than wild-type, root system scanning (relates to root length, diameter, and root system surface area), the long and leaf area measuring result display of stem: under 8%PEG treatment condition, AtTGA4 transfer-gen plant has stronger drought resistance (in as Fig. 5 B) than also wildtype Arabidopsis thaliana, be in particular in: compared with wildtype Arabidopsis thaliana, the root length turning AtTGA4 gene strain is relatively long, roots oxidizing is relatively large, and root system surface area is relatively large; Stem is long relatively long; Leaf area is relatively large.When being no matter process 10 days, or during process 17 days, the Arabidopis thaliana drought resistance detected result proceeding to pBI121 empty carrier compared with wildtype Arabidopsis thaliana without significant difference.
Two, drought resistance of seedling detects
With following four kinds of Arabidopsis plant for experiment material: AtTGA4 Arabidopsis Mutants, wildtype Arabidopsis thaliana Columbia0(WT), 4 T
2in generation, turns AtTGA4 gene strain (TGA4-1, TGA4-2, TGA4-3 and TGA4-4), proceeds to the Arabidopis thaliana of pBI121 empty carrier, at MS
0solid medium is sent out after seedling, is transferred in soil and plants, start to carry out Osmotic treatment (not watering) when being cultured to 10 days and before Arabidopis thaliana bolting.Observe its phenotypic difference during Osmotic treatment to 18 day and add up surviving rate, then carrying out rehydration process, rehydration adds up its surviving rate after 5 days again.And respectively before Osmotic treatment and rehydration 5 days time sampling drought resisting relative physiologic index is measured: proline content, chlorophyll content, peroxidase (POD) are active.Experiment repetition 3 times, results averaged.
Proline content mensuration concrete grammar reference Zhang Dianzhong etc. (Zhang Dianzhong etc. free proline content measuring method [J]. Plant Physiology Communications, 1990 (4): 62-65.);
Measuring chlorophyll content concrete grammar reference Su Zhengshu etc. (Su Zhengshu etc. the Measures compare [J] of several mensuration chlorophyll content of plant. Plant Physiology Communications, 1989 (5): 77-78.)
Peroxidase Activity Determination concrete grammar reference Yuan Qinghua etc. (Yuan Qinghua etc. the comparison [J] of superoxide-dismutase, peroxidase and polyphenol oxidase activity in clover anti-sense brown spot kind. blade of grass journal .2001(11): 100-104.)
Result shows, Osmotic treatment is after 18 days, and rehydration is after 5 days, 4 green plant numbers turning AtTGA4 gene strain are all obvious more than wild-type and mutant (Fig. 6), Osmotic treatment carried out rehydration process after 18 days, add up its surviving rate during rehydration 5 days and find that the surviving rate of process LAN strain is about 1.5 times of wild-type, and the surviving rate of wildtype Arabidopsis thaliana is about mutant 4 times (Fig. 7).
Respectively by before Osmotic treatment and rehydration after 5 days plant sampling carry out the mensuration of Physiological Indices of Drought Resistance, result as shown in Figure 8: the proline content of whole plant and peroxidase activity increase after Osmotic treatment, but the decline of chlorophyllous content.Turn the proline(Pro) of AtTGA4 gene strain and chlorophyll content after process more than wild-type, and wild-type content is more than mutant; Peroxidase activity is then contrary, according to turning AtTGA4 gene strain, wild-type, mutant sequentially.
For the detection of above each side, the Arabidopis thaliana proceeding to pBI121 empty carrier compared with wildtype Arabidopsis thaliana without significant difference.
Embodiment 4, turn the qualification of the Nitrogen Absorption ability of AtTGA4 gene Arabidopis thaliana
Containing 0.2mM NO
3 -mS solid medium: Repone K 1.3866g, saltpetre 0.02g, vitamin (mother liquid concentration is 1mg/mL) 1mL, pyridoxine hydrochloride (mother liquid concentration is 0.5mg/mL) 1mL, inositol (final concentration is in the medium 50mg/L) 10mL, glycine (mother liquid concentration is 2mg/mL) 1mL, nicotinic acid (mother liquid concentration is 0.5mg/mL) 1mL, without MS substratum (SIGMA company) 0.78g of N, sucrose 30g, plant gel 2.4g, after being dissolved in appropriate distilled water, 1L is supplemented to, pH7.0 with distilled water.
Containing 2mM NH
4 +mS solid medium: Repone K 1.4g, ammonium sulfate 0.132g, vitamin (mother liquid concentration is 1mg/mL) 1mL, pyridoxine hydrochloride (mother liquid concentration is 0.5mg/mL) 1mL, inositol (final concentration is in the medium 50mg/L) 10mL, glycine (mother liquid concentration is 2mg/mL) 1mL, nicotinic acid (mother liquid concentration is 0.5mg/mL) 1mL, without MS substratum (SIGMA company) 0.78g of N, sucrose 30g, plant gel 2.4g, after being dissolved in appropriate distilled water, 1L is supplemented to, pH7.0 with distilled water.
Containing 0.2mM NO
3 -hoagland water planting liquid: Ca (NO
3)
24H
2o 0.0236g, CaCl
20.435g, KH
2pO
40.123g, KCl 0.4498g, MgSO
47H
2o 0.49g, EDTA-Fe 0.0367g, MnCl
24H
2o 0.002g, CuSO
40.00024g, ZnSO
47H
2o 0.00029g, H
3bO
30.00186g, (NH
4)
6mo
7o
34H
2o 0.000035g, after being dissolved in appropriate distilled water, is supplemented to 1L with distilled water, pH=6.0.
Containing 1mMNH
4 +hoagland water planting liquid: CaCl
20.4465g, KCl 0.4498g, (NH
4)
2hPO
40.06603g, KH
2pO
40.05566g, MgSO
47H
2o 0.49g, EDTA-Fe 0.0367g, MnCl
24H
2o0.002g, CuSO
40.00024g, ZnSO
47H
2o 0.00029g, H
3bO
30.00186g, (NH
4)
6mo
7o
34H
2o 0.000035g, after being dissolved in appropriate distilled water, is supplemented to 1L with distilled water, pH=6.0.
One, the detection of germination period Nitrogen Absorption ability
Wildtype Arabidopsis thaliana Columbia0(WT), 4 T with following three kinds of Arabidopsis plant for experiment material:
2in generation, turns AtTGA4 gene strain (TGA4-1, TGA4-2, TGA4-3 and TGA4-4), proceeds to the Arabidopis thaliana of pBI121 empty carrier, is sowed at MS respectively after carrying out aseptically process
0solid medium, containing 0.2mM NO
3 -mS solid medium (formula as above), with containing 2mM NH
4 +mS solid medium (formula as above) in, 4 DEG C of vernalization are transferred to tissue culture room growth after 3 days, when process is to observing its phenotypic difference when 18 days and carrying out the long and Leaf area determination of root system scanning (relate to root length, diameter, and root system surface area), stem to it.
Result shows: no matter be the nitric nitrogen of lower concentration or the ammonium nitrogen of lower concentration, the growth tendency all better than wild-type (as A in Fig. 9) of 4 process LAN strains.Its root system, stem length, leaf area measuring result are also shown: when seed stage carries out NITROGEN IN LOW CONCENTRATION process to wildtype Arabidopsis thaliana and process LAN strain, relative to the utilising efficiency of wildtype Arabidopsis thaliana process LAN strain to nitrogen higher (as B in Fig. 9), be in particular in: compared with wildtype Arabidopsis thaliana, the root length turning AtTGA4 gene strain is relatively long, roots oxidizing is relatively large, and root system surface area is relatively large; Stem is long relatively long; Leaf area is relatively large.
Two, seedling stage Nitrogen Absorption ability detection
Wildtype Arabidopsis thaliana Columbia0(WT), 4 T with following three kinds of Arabidopsis plant for experiment material:
2in generation, turns AtTGA4 gene strain (TGA4-1, TGA4-2, TGA4-3 and TGA4-4), proceeds to the Arabidopis thaliana of pBI121 empty carrier, at MS
0cultured on solid medium, after one week, is selected the basically identical seedling of size and is transferred on 96 orifice plates at the end, allow it swim in black dish and carry out water planting process, has the Hoagland water planting liquid that two kinds different: containing 0.2mM NO
3 -hoagland water planting liquid (formula as above) and containing 1mMNH
4 +hoagland water planting liquid (formula as above).Each Arabidopsis plant phenotype is observed in water planting process after 10 days, and detects total nitrogen content.Test in triplicate, results averaged.
The detection concrete grammar reference Lv Weixian of total nitrogen content etc. (Lv Weixian etc. the comparative studies of nitric nitrogen, ammonia-state nitrogen, determination of total nitrogen content method in plant. spectroscopy and spectroscopic analysis .2004.24:204-206.)
Result shows, and no matter is, process 10 days in the nitric nitrogen of lower concentration or the nutrient solution of ammonium nitrogen after, turn AtTGA4 gene strain grows trend all than wild-type better (as Figure 10).Its mensuration of carrying out total nitrogen is compared to the nitrogen content more than wild-type (as Figure 11) finding to turn in AtTGA4 gene strain.For the detection of above each side, the Arabidopis thaliana proceeding to pBI121 empty carrier compared with wildtype Arabidopsis thaliana without significant difference.
Embodiment 4, AtTGA4 albumen and encoding gene thereof be nitrogen approach relevant physiological reaction detection under drought condition
With following four kinds of Arabidopsis plant for experiment material: AtTGA4 Arabidopsis Mutants (AtTGA4 gene expression amount is low), wildtype Arabidopsis thaliana Columbia0(WT, AtTGA4 gene normal expression), 4 T
2in generation, turns AtTGA4 gene strain (TGA4-1, TGA4-2, TGA4-3 and TGA4-4, AtTGA4 gene overexpression), proceeds to the Arabidopis thaliana of pBI121 empty carrier, at MS
0solid medium is sent out after seedling, is transferred in soil and plants, start to carry out Osmotic treatment (not watering) when being cultured to 10 days and before Arabidopis thaliana bolting.Rehydration process is carried out after Osmotic treatment to 18 day.Before Osmotic treatment and rehydration sample after 5 days, be determined as follows the physical signs relevant to nitrogen approach: nitrate reductase (NR) is active, glutamine synthetase (GS) active and total nitrogen.RNA is extracted in sampling simultaneously and reverse transcription is cDNA, take cDNA as Real-time PCR:NRT1.1, NRT2.1, NRT2.2, NRT2.5, NIA1, NIA2, GLN1.4, G-6-PD2 that template does some genes relevant to nitrogen approach, using Actin as internal reference, the primer is as shown in table 1, compares their expression amount difference under AtTGA4 gene three kinds is multi-form.Test in triplicate, results averaged.
The detection primer of table 1 nitrogen approach genes involved and amplified fragments size
Before Osmotic treatment and rehydration after 5 days, the nitrate reductase activity of each plant, activity of glutamine synthetase and total nitrogen measurement result are as shown in figure 12, result shows that nitrate reductase activity, glutamine activity and total nitrogen increase all to some extent under drought condition, but the change of total nitrogen and nitrate reductase is turning AtTGA4 gene strain, variant between wild-type and mutant, and the change of glutamine synthetase does not have notable difference between this three.Simultaneously to RT-PCR analytical results display (Figure 13) of 8 key genes participating in nitrogen approach: the expression amount of these four genes of NRT2.1, NRT2.2, NIA1, NIA2 is in mutant, wild-type and turn between AtTGA4 gene strain and have notable difference, the expression amount turning these 4 genes in AtTGA4 gene strain is about 2 times of wild-type, and the expression amount in wild-type is about 1.5 times of mutant; The expression amount of these four genes of NRT1.1, NRT2.5, GLN1.4, G-6-PD2 is in mutant, wild-type and turn between AtTGA4 gene strain and do not have obvious difference.This illustrates that NRT2.1, NRT2.2, NIA1, NIA2 may by the adjustments of AtTGA4 gene under drought condition.For the detection of above each side, the Arabidopis thaliana proceeding to pBI121 empty carrier compared with wildtype Arabidopsis thaliana without significant difference.
Claims (6)
1.AtTGA4 albumen or its encoding gene are in regulating plant drought resistance and/or to the application in Nitrogen Absorption; The protein that described AtTGA4 albumen is made up of the aminoacid sequence shown in sequence in sequence table 2; The encoding gene of described AtTGA4 albumen is the gene of nucleotide sequence as shown in sequence in sequence table 1; Described plant is Arabidopis thaliana; Described regulating plant drought resistance and/or to Nitrogen Absorption be embodied in improve plant drought resistance and/or improve plant to Nitrogen Absorption ability.
2.AtTGA4 albumen or the application of its encoding gene in following (a1)-(a6) of regulating plant;
(a1) plant proline content;
(a2) Plant Leaf chlorophyll contents;
(a3) plant peroxidase activity;
(a4) plant total nitrogen content;
(a5) plant nitrate reductase activity;
(a6) expression amount of plant nitrogen pathway key gene, described nitrogen pathway key gene is specially at least one in NRT2.1, NRT2.2, NIA1 and NIA2;
The protein that described AtTGA4 albumen is made up of the aminoacid sequence shown in sequence in sequence table 2; The encoding gene of described AtTGA4 albumen is the gene of nucleotide sequence as shown in sequence in sequence table 1; Described plant is Arabidopis thaliana.
3. cultivate a method for transgenic plant, comprise the steps: the encoding gene of AtTGA4 albumen to import in object plant, obtain the transgenic plant with at least one proterties in following (b1)-(b8):
(b1) drought resistance improves compared with described object plant;
(b2) receptivity comparing nitrogen with described object plant improves;
(b3) with described object plant all after drought-induced, its plant proline content is higher than described object plant;
(b4) with described object plant all after drought-induced, its Plant Leaf chlorophyll contents is higher than described object plant;
(b5) with described object plant all after drought-induced, its plant peroxidase activity is lower than described object plant;
(b6) with described object plant all after low nitrogen process, its plant total nitrogen content is higher than described object plant;
(b7) with described object plant all after low nitrogen process, its plant nitrate reductase activity is higher than described object plant;
(b8) with described object plant all after low nitrogen process, in its plant, the expression amount of nitrogen pathway key gene is higher than described object plant, and described nitrogen pathway key gene is at least one in NRT2.1, NRT2.2, NIA1 and NIA2;
The protein that described AtTGA4 albumen is made up of the aminoacid sequence shown in sequence in sequence table 2; The encoding gene of described AtTGA4 albumen is the gene of nucleotide sequence as shown in sequence in sequence table 1; Described plant is Arabidopis thaliana.
4. method according to claim 3, is characterized in that: the encoding gene of described AtTGA4 albumen is imported in described object plant by the recombinant expression vector of the encoding gene containing described AtTGA4 albumen.
5. method according to claim 4, is characterized in that: the promotor that the encoding gene starting described AtTGA4 albumen in described recombinant expression vector is transcribed is 35s promotor.
6. method according to claim 5, is characterized in that: described recombinant expression vector is the recombinant plasmid of the multiple clone site encoding gene of described AtTGA4 albumen being inserted into pBI121 plasmid, the encoding gene of the described AtTGA4 albumen of the expression obtained.
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Non-Patent Citations (5)
| Title |
|---|
| Analysis of the Arabidopsis nuclear proteome and its response to cold stress;Min Seok Bae et al;《The Plant Journal》;20031231;第36卷;第652-663页 * |
| NM_121041.4;Swarbreck D et al;《GenBank》;20110528;第1-2页 * |
| NP_196565.1;Swarbreck D et al;《GenBank》;20110528;第1-2页 * |
| Redox Regulation of the NPR1-TGA1 System of Arabidopsis thaliana by Nitric Oxide;Christian Lindermayr et al;《The Plant Cell》;20100831;第22卷;第2894-2907页 * |
| TGA1 and TGA4 Transcription Factor Control Nitrate response in Arabidopsis Thaliana Root Organs;Jose Alvarez et al;《21st International Conference on Arabidopsis Research》;20101231;第107页 * |
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