CN1137265C - Method for raising plant nitrogen assimilation efficiency - Google Patents

Method for raising plant nitrogen assimilation efficiency Download PDF

Info

Publication number
CN1137265C
CN1137265C CNB011044322A CN01104432A CN1137265C CN 1137265 C CN1137265 C CN 1137265C CN B011044322 A CNB011044322 A CN B011044322A CN 01104432 A CN01104432 A CN 01104432A CN 1137265 C CN1137265 C CN 1137265C
Authority
CN
China
Prior art keywords
plant
gdh
gene
mould
arteries
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CNB011044322A
Other languages
Chinese (zh)
Other versions
CN1333349A (en
Inventor
波 田
田波
王芳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Microbiology of CAS
Original Assignee
Institute of Microbiology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Microbiology of CAS filed Critical Institute of Microbiology of CAS
Priority to CNB011044322A priority Critical patent/CN1137265C/en
Priority to AU2001246330A priority patent/AU2001246330A1/en
Priority to PCT/CN2001/000294 priority patent/WO2002002776A1/en
Publication of CN1333349A publication Critical patent/CN1333349A/en
Application granted granted Critical
Publication of CN1137265C publication Critical patent/CN1137265C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The present invention provides a method for raising the assimilatory efficiency of plant nitrogen, which comprises: (a). a fungus glutamate dehydrogenase (GDH) gene is connected with a promotor which can guide the expression of an exogenous gene in plants to construct mosaic genes; (b). the constructed mosaic genes are guide into plant cells and are screened, and transformed plants are cultured.

Description

A kind of method that improves plant nitrogen assimilation efficiency
Invention field
The present invention relates to a kind of method that improves plant nitrogen assimilation efficiency.
Background technology
Nitrogen is the required first nutritive element of plant-growth.The nitrogenous fertilizer of being used on the agricultural only has 30%-40% to be utilized by plant absorbing at present, and major part is all wasted.Some is converted into nitrate and is lost in the soil, causes environmental pollution.The crop that changes GDH can effectively increase the absorption of plant nitrogen, improves utilization rate of nitrogen fertilizer, thereby can save the amount of application of nitrogenous fertilizer, and it is huge playing economic benefit.Simultaneously, GDH extensively is present in animals and plants and the microorganism, and the plant that therefore changes GDH can not produce harm to people and animals and plants.
Mainly contain countries such as the U.S., Australia now and carry out research (the United States Patent (USP) NO.5955651 of glutamate dehydrogenase transgenic plant; 5985634; 5998700).Their gdh gene is mainly derived from chlorella and intestinal bacteria, and model plant is cash crop such as tobacco, corn.Find that after deliberation its nitrogen utilization efficiency of plant that changes gdh gene increases, show that plant leaf is big and quantity is more.The nitrogen content of changeing the plant different tissues of intestinal bacteria GDH Duos about 16% than control group after measured, and aminoacids content changes to some extent in the different tissues simultaneously.Be comparatively significantly, L-glutamic acid content is obviously lower, and L-Ala, Methionin, the increase of aspartic acid equal size are bigger.The capable amount of starch of changeing GDH gene potato is than the control group showed increased.But employed glutamate dehydrogenase is active much lower so far.
Summary of the invention
The purpose of this invention is to provide a kind of method that improves plant nitrogen assimilation efficiency.
The invention provides a kind of method that improves plant nitrogen assimilation efficiency, comprise: (a) can in plant, guide the promotor of exogenous gene expression to link to each other with a kind of fungi glutamate dehydrogenase (GDH) gene, chimeric genetic construct: (b) mosaic gene that makes up is imported in the vegetable cell, screen and turn out by plants transformed.
In the method for the invention, described gdh gene can be prothetic group NADP gdh gene or prothetic group NAD gdh gene.
In the method for the invention, described fungi gdh gene can be from the mould genus of the arteries and veins spore fungi of filamentous fungus, and the intercadence spore is mould in comprising, to eat mould, the coarse arteries and veins spore of arteries and veins spore well mould.Described gdh gene also can be from yeast fungus, for example cereuisiae fermentum (Saccharomycescerevisiae).Described gdh gene can also be from basidiomycetes, for example bisporous mushroom (Agaricus bisporus).
In the method for the invention, described plant can be tobacco, corn, cotton or paddy rice.
In the method for the invention; Described glutamte dehydrogenase can be from the mould Neurospora intermedia of the middle arteries and veins spore glutamte dehydrogenase of (being called for short Ni), and it has amino acid sequence as follows: MSNLPSEPEFEQAYKELAYTLENSSLFQKHPEYRTALTVASIPERVIQFRVVWEDD NGNVQVNRGYRVQFNSALGPYKGGLRLHPSVNLSILKFLGFEQIFKNALTGLSMGG GKGGADFDPKGKSDAEIRRFCCAFMAELHKHIGADTDVPAGDIGVGGREIGYMFGA YRKAANRFEGVLTGKGLSWGGSLIRPEATGYGLVYYVGHMLEYSGAGSYAGKRVAL SGSGNVAQYAALKLIELGATVVSLSDSKGALVATGESGITVEDINAVMAIKEARQS LTSFQHAGHLKWIEGARPWLHVGKVDIALPCATQNEVSKEEAEGLLAAGCKFVAEG SNMGCTLEAIEVFENHRKEKKGEAVWYAPGKAANCGGVAVSGLEMAQNSQRLNWTQ AEVDEKLKDIMKNAFFNGLNTAKIYVEAAEGELPSLVAGSNIAGFVKVAQAMHDQG DWWSKN
：ATGTCTAACCTTCCCTCTGAGCCCGAGTTTGAGCAGGCCTACAAGGAGTTGGCCTACACTCTCGAGAACTCCTCCCTCTTCCAGAAGCACCCCGAGTACCGCACTGCCCTCACCGTTGCCTCCATCCCCGAGCGTGTCATTCAGTTCCGTGTTGTCTGGGAGGACGACAACGGCAACGTCCAGGTCAATCGCGGTTACCGTGTCCAGTTCAACTCCGCCCTCGGTCCCTACAAGGGTGGTCTCCGTCTCCACCCCTCCGTCAACCTTTCCATTCTCAAGTTCCTCGGTTTCGAGCAGATCTTCAAGAATGCCCTCACTGGCTTGAGCATGGGTGGTGGCAAGGGTGGTGCCGACTTCGACCCCAAGGGCAAGAGCGACGCTGAGATCCGTCGCTTCTGCTGCGCTTTCATGGCTGAGCTTCACAAGCACATTGGTGCCGATACCGATGTTCCCGCTGGTGACATCGGTGTTGGTGGCCGTGAGATCGGCTACATGTTCGGTGCCTACCGCAAGGCCGCGAACCGTTTCGAGGGTGTCCTTACTGGCAAGGGCCTCTCCTGGGGTGGTTCGCTCATTCGCCCTGAGGCCACTGGTTACGGCCTCGTCTACTACGTCGGCCACATGCTCGAGTACTCTGGCGCCGGATCCTACGCTGGCAAGCGCGTTGCCCTCTCCGGTTCCGGCAACGTCGCCCAGTACGCCGCCCTCAAGCTCATTGAGCTAGGCGCCACCGTTGTCTCCCTCTCCGACTCCAAGGGTGCTCTTGTCGCCACTGGCGAGTCCGGCATCACCGTTGAGGACATCAACGCCGTTATGGCCATCAAGGAGGCCCGTCAGTCCCTTACCAGCTTCCAGCACGCTGGTCACCTGAAGTGGATCGAGGGCGCCCGCCCCTGGCTTCACGTTGGCAAGGTTGACATCGCTCTTCCTTGCGCTACCCAGAACGAGGTCTCCAAGGAGGAGGCTGAGGGTCTCCTTGCCGCCGGCTGCAAGTTCGTCGCTGAGGGTTCCAACATGGGCTGCACTCTCGAGGCCATTGAGGTCTTTGAGAACCACCGCAAGGAGAAGAAGGGCGAGGCCGTCTGGTACGCCCCCGGCAAGGCCGCCAACTGTGGTGGTGTTGCCGTTTCCGGTCTCGAGATGGCTCAGAACAGCCAGCGCCTCAACTGGACTCAGGCTGAGGTTGACGAGAAGCTCAAGGACATCATGAAGAACGCCTTCTTCAACGGTCTCAACACTGCCAAGATCTACGTCGAGGCTGCTGAGGGCGAGCTTCCTTCCCTCGTTGCTGGCTCCAACATTGCTGGTTTCGTCAAGGTTGCCCAGGCCATGCACGACCAGGGTGACTGGTGGTCCAAGAACTAA
In the method for the present invention; Described glutamte dehydrogenase can be regarded for oneself and be eaten the mould Neurospora sitophila of arteries and veins spore (being called for short Ns), and it has amino acid sequence as follows: MSNLPSEPEFEQAYKELAYTLENSSLFQKHPEYRIALAVASIPERVIQFRVVWEDD NGNVQVNRGYRVQFNSALGPYKGGLRLHPSVNLSILKFLGFEQIFKNALTGLSMGG GKGGADFDPKGKSDAEIRRFCCAFMAELHKHIGADTDVPAGDIGVGGREIGYMFGA YRKAANRFEGVLTGKGLSWGGSLIRPEATGYGLVYYVGHMLEYSGAGSYAGKRVAL SGSGNVAQYAALKLIELGATVVSLSDSKGALVATGESGITVEDINAIMAIKEARQS LTTFQHAGHVKWIEGARPWLHVGKVDIALPCATQNEVSKEEAEGLLAAGCKFVAEG SNMGCTLEAIEVFENNRKEKKGEAVWYAPGKAANCGGVAVSGLEMAQNSQRLNWTQ AEVDEKLKDIMKNAFFNGLNTAKTYAEAAEGELPSLVAGSNIAGFVKVPQAMHDQG DWWSKN
：ATGTCTAACCTTCCCTCTGAGCCCGAGTTCGAGCAGGCCTACAAGGAGTTGGCCTACACTCTCGAGAACTCCTCCCTCTTCCAGAAGCACCCCGAGTACCGCACCGCCCTCGCCGTTGCCTCCATCCCCGAGCGTGTCATTCAGTTCCGTGTTGTCTGGGAGGACGACAACGGCAACGTCCAGGTCAACCGCGGTTACCGTGTCCAGTTCAACTCCGCCCTCGGTCCCTACAAGGGTGGTCTCCGTCTCCATCCCTCCGTCAACCTTTCCATTCTCAAGTTCCTCGGTTTCGAGCAGATCTTCAAGAATGCCCTTACTGGCCTGAGCATGGGTGGTGGCAAGGGTGGTGCCGACTTCGACCCCAAGGGCAAGAGCGATGCTGAGATCCGTCGCTTCTGCTGCGCTTTCATGGCCGAGCTTCACAAGCACATTGGTGCCGATACCGATGTTCCCGCTGGTGATATTGGTGTTGGTGGCCGCGAGATCGGTTACATGTTCGGTGCCTACCGCAAGGCTGCGAACCGTTTCGAGGGTGTCCTTACTGGTAAGGGCCTCTCCTGGGGTGGTTCGCTCATTCGCCCTGAGGCCACTGGTTACGGTCTCGTCTACTACGTCGGCCACATGCTCGAGTACTCTGGCGCCGGCTCCTACGCTGGCAAGCGCGTTGCCCTCTCTGGTTCCGGCAACGTCGCCCAGTACGCCGCCCTCAAGCTCATTGAGCTAGGCGCCACCGTTGTCTCCCTCTCCGACTCCAAGGGTGCCCTTGTCGCCACTGGCGAGTCCGGCATCACTGTTGAGGACATCAACGCCATCATGGCCATCAAGGAGGCCCGTCAGTCTCTTACCACCTTCCAGCACGCTGGCCACGTCAAGTGGATCGAGGGCGCCCGCCCCTGGCTTCACGTTGGCAAGGTTGACATCGCTCTTCCTTGCGCTACCCAGAACGAGGTCTCCAAGGAGGAGGCTGAGGGTCTCCTTGCCGCCGGCTGCAAGTTCGTCGCTGAGGGTTCCAACATGGGCTGCACTCTCGAGGCCATCGAGGTCTTTGAGAACAACCGCAAGGAGAAGAAGGGCGAGGCCGTCTGGTACGCCCCCGGCAAGGCCGCCAACTGTGGTGGTGTTGCCGTTTCCGGTCTCGAGATGGCTCAGAACAGCCAGCGCCTCAACTGGACTCAGGCTGAGGTTGACGAGAAGCTCAAGGACATCATGAAGAACGCCTTCTTCAACGGTCTCAACACTGCCAAGACCTACGCCGAGGCTGCTGAGGGCGAGCTTCCTTCCCTCGTTGCCGGCTCCAACATTGCTGGTTTCGTCAAGGTACCCCAGGCCATGCACGACCAGGGTGACTGGTGGTCCAAGAACTAA
We are Ni-GDH, Ns-GDH, and the mould genus gene clone of three kinds of arteries and veins spores of Nc-GDH advances in the e. coli bl21 (DE3) to carry out abduction delivering, the Ni-GDH of purifying, Ns-GDH, Nc-GDH carries out enzyme assay, and it is mould that the enzymic activity of finding three kinds of GDH is higher than the arteries and veins spore of other genus.It is all stronger to the avidity and the stability of ammonia.To three kinds of GDH gene subclones in plant expression vector pROKII, by Agrobacterium-mediated Transformation, electrization transforms and crops such as pollen tube passage method transformation of tobacco, corn, cotton.Identify through PCR, Southern, Northern and enzymic activity dyeing, filter out P8 ' property transformant.It is moved in the substratum of different N concentration, find at low nitrogen 5mM to lower ammonium ion concentration, the tobacco of expression GDH can both normal growth, and the blade yellow of unconverted control group, impaired development presents the nitrogen stress symptom.Measure plant and hanging down the nitrogenous total amount under the nitrogen condition and do not utilizing the nitrogen residual quantity, find that the nitrogen content of commentaries on classics fungus G DH plant is more unconverted high more than 20%, the nitrogen residual quantity reduces 20-30%.Measure the total nitrogen of plant in poor nitrogen soil, the more unconverted plant of result is high by about 40%.Simultaneously we have made up cash crop such as the corn that changes the GDH gene, paddy rice, cotton, find that its nitrogen content transforms and does that the nitrogen residual quantity reduces more than 20% in the object height 20-30% soil in the end.Detection shows, GDH has obtained high expression level in above-mentioned plant, it has accelerated the reductive amination effect of the oxidative deamination and the α-Tong Wuersuan (2-oxoglutarate) of L-glutamic acid, makes to have started a new nitrogen in the plant and utilize approach, thereby has improved the utilization ratio of ammonia.Though GDH is also arranged in the higher plant, its avidity to ammonia has only 1/10 to 1/100 of fungus G DH, so can not bring into play the effect of assimilate ammonia.Fungus G DH makes the oxidative deamination of L-glutamic acid be accompanied by the release of a large amount of ATP and the formation of α-Tong Wuersuan (2-oxoglutarate), and this provides competent energy and a large amount of carbohydrate that participates in tricarboxylic acid cycle to plant.Nitrogen is the required first nutritive element of plant-growth, and it is that crop absorbs that the nitrogenous fertilizer of being used on the present agricultural only has 30%-40%, and major part all runs off, and causes environmental pollution.And the crop that changes GDH can be improved nitrogen utilization efficiency effectively, thereby can save the utilization of nitrogenous fertilizer, reduces environmental pollution, and its economic benefit is very huge.
Embodiment
Mould genus cultivation of fungi, cereuisiae fermentum and bisporous mushroom of embodiment 1. arteries and veins spores and inducing of glutamate dehydrogenase thereof
1. three kinds of fungies are changeed in malt extract medium by the solid inclined-plane, 250 rev/mins of shaking tables are cultivated 48 hours centrifugal collection mycelium, and (4% glucose, 0.02MNH4AC induced centrifugal collection mycelium 3 hours in 0.04MKN05) to change the ammonia inducing culture over to.
2. extracting and the reverse transcription of total RNA---polymerase chain reaction (RT-PCR) method amplification gdh gene:
Mycelium after inducing adopts the guanidine isothiocyanate method extracted total RNA to carry out reverse transcription one polymerase chain reaction after the liquid nitrogen grinding fragmentation.
The primer is as follows:
Primer 1:5 ' GCTCAGAATGTCTAACCTTCCCTCTGAG 3 '
Primer 2: 5 ' GCGAGCTCTAGTCTTGGACCACCAGTCACC 3 '
The reverse transcription reaction condition is:
65 ℃ 1 minute,
30 ℃ 5 minutes,
30 ℃-65 ℃ 30 minutes,
98 ℃ 5 minutes,
5 ℃ 5 minutes.
The polymerase chain reaction condition:
94 ℃ 3 minutes,
94 ℃ 1 minute
55 ℃ of 25 circulations in 1 minute
72 ℃ 2 minutes
72 ℃ 10 minutes
3. the mould genus glutamate dehydrogenase of arteries and veins spore (GDH) gene sequencing:
Glutamate dehydrogenase (GDH) gene of RT-PCR method amplification is reclaimed through agarose gel electrophoresis.Get 3 microlitres (ul) and reclaim product, and adding 1ul pGEM-T easy (Strategene, USA) carrier, 5ul 2xT4 ligase enzyme damping fluid, (Strategene, USA), 4 ℃ of enzymes even spend the night the 1ul dna ligase.Next day, enzyme is connected product transformed into escherichia coli DH50a carry out the bacterium colony screening.Screening method adopts bacterium colony PCR and enzyme to cut evaluation, and restriction enzyme site is XbaI, SacI.Enzyme is cut and is produced 3.0kb and the positive clone of 1.4kb two band persons pT-GDH.The picking positive colony carries out sequencing.
4. cereuisiae fermentum and bisporous mushroom glutamate dehydrogenase (GDH) gene sequencing:
Glutamate dehydrogenase (GDH) gene of RT-PCR method amplification is reclaimed through agarose gel electrophoresis.Get 3 microlitres (ul) and reclaim product, and adding 1ul pGEM-T easy (Strategene, USA) carrier, 5ul 2xT4 ligase enzyme damping fluid, the 1ul dna ligase, 4 ℃ of enzymes even spend the night.Next day, enzyme is connected product transformed into escherichia coli DH50a carry out the bacterium colony screening.Screening method adopts bacterium colony PCR and enzyme to cut evaluation, and restriction enzyme site is XbaI, SacI.Enzyme is cut and is produced 3.0kb and the positive clone of 1.4kb two band persons pT-GDH.The picking positive colony carries out sequencing.
5. mould, cereuisiae fermentum of arteries and veins spore and the expression vector pT-GDH of bisporous mushroom gdh gene in intestinal bacteria cut through XbaI, SacI enzyme, agarose electrophoresis reclaims and obtains the GDH fragment, (Strategene USA) even spends the night at 4 ℃ of enzymes with the pBluescript carrier of cutting through same enzyme.Next day, transformed into escherichia coli DH50a cut evaluation positive colony pBlueGDH through bacterium colony PCR and enzyme.PBlueGDH cuts through ECoRV, SacI enzyme, and agarose electrophoresis reclaims and obtains the GDH fragment, connects at 12 ℃ of enzymes with the pET30a carrier of cutting through same EcoRV, SacI enzyme and spends the night.Next day, transformed into escherichia coli DH50a cut evaluation through bacterium colony PCR and enzyme, and positive colony is pETGDH.Next day, the picking positive colony, transformed into escherichia coli BL21 (DE3) (CLONTECH, USA).Be cultured to about OD0.4, induce 4hr, the results thalline through 1mM IPTG.Thalline behind deionized water wash, ultrasonic degradation.Centrifugal, go up cleer and peaceful precipitation and do not carry out the 10%SDS-PAGE electrophoresis.The result proves that expression product thinks that the inclusion body form exists.
6. metal is integrated the affinitive layer purification glutamate dehydrogenase
Prepare in the MCAC-0 solution that contains 8M urea (20mm/L TrisCl, pH7.9,0.5mol/L NaCl, 10% (v/v) glycerine, several PMSF of 1mmol).Cofabrication MCAC-40, MCAC-60, MCAC-80, MCAC-100, MCAC-200, MCAC-500 promptly adds 0.4mol/L, 0.6mol/L, the imidazoles of 0.8mol/L, 1mol/L, 2mol/l, 5mol/L respectively in MCAC-0.
The glutamate dehydrogenase inclusion body is dissolved in the MCAC-0 solution that contains 8M urea, is splined in the NTA chromatography column, wash post, the abandoned stream fluid with 5ml 8M Urea-MCAC damping fluid with the flow velocity of 20-30ml/h.Mode with stepwise elution is washed post with the following damping fluid of 5ml successively, 8MUrea-MCAC40,8M Urea-MCAC60,8M Urea-MCAC80,8MUrea-MCAC100,8M Urea-MCAC200,8M Urea-MCAC500, collect effluent liquid respectively, carry out the 10%SDS-PAGE electrophoresis, silver dyes and carries out the purity evaluation." MCAC200 takes off liquid in later Shen, the renaturation of dialysing to collect 8M Urea.Dialysis buffer liquid divides two kinds, is respectively the 0.1mol/L Tris.HCl of pH8.5 and pH7.4,1mM EDTA.
7. glutamate dehydrogenase determination of activity
The recombinant protein of dialysed overnight is centrifugal, collect supernatant, measure protein concentration, concentration (mg/ml)=A280 * 0.825 through the 280nm ultraviolet detection.Measuring enzyme in system A, system C respectively lives.System A measures the effect of GDH reductive amination.
2.55ml 0.1MTris.HCl，1mMEDTA，pH7.4
0.1ml 0.1MNH4Cl
0.15ml 0.2M a-ketoglutaric acid
0.2ml 0.15％(W/V)NADPH
The 2ul recombinant protein, 25 ℃ of incubations 10 minutes are measured the variation of system A at the 340nm absorbance value under 25 ℃ of constant temperature.
System C, measure the GDH oxidative deaminization:
2.8ml the 0.16M monosodium glutamate is dissolved in 0.1M Tris.HCl, 1mM EDTA, pH
8.5 damping fluid in, 0.2ml 0.2%NADP, the 2ul recombinant protein, 37 ℃ of incubations were determined at the variation of 340mm absorbance value after 10 minutes.
Activity unit is that per minute reduces each micromole NADP +Be that each micromole NADPH of a unit or per minute oxidation is a unit.
The result is determined as that Ni-GDH activity unit is 109.92U/mg in system A, in system C, for Ni-GDH is 72.93U/mg, Ns-GDH is 95.37U/mg in system A, be 63013U/mg in system C, Nc-GDH is 100.25U/mg in system A, is 65.00U/mg in system C.
8. the mould gdh gene subclone of arteries and veins spore is gone into plant expression vector pROKII and the GDH gene fragment is downcut with XbaI, SacI double digestion from the pT-GDH carrier, after agarose electrophoresis reclaims, (CLONTECH, USA) carrier even spends the night at 4 ℃ of enzymes with the pROKII that cuts through same enzyme.Transformed into escherichia coli DH5a cut through bacterium colony PCR and enzyme and to identify positive colony pROKII-GDH next day, select positive colony be transformed into Agrobacterium LBA4404 (CLONTECH, USA) in.
9. cereuisiae fermentum, bisporous mushroom gdh gene subclone are gone into plant expression vector pROKII and the GDH gene fragment are downcut with XbaI, SacI double digestion from the pT-GDH carrier, after agarose electrophoresis reclaims, (CLONTECH, USA) carrier even spends the night at 4 ℃ of enzymes with the pROKII that cuts through same enzyme.Transformed into escherichia coli DH5a cut through bacterium colony PCR and enzyme and to identify positive colony pROKII-GDH next day, select positive colony be transformed into Agrobacterium LBA4404 (CLONTECH, USA) in.
10. Agrobacterium tumefaciens mediated leaf disc transformation method transformation of tobacco
(1) cultivation of tobacco aseptic seedling
Go up cultivation with cultivating at the MS of no hormone substratum (MS salt+15g/L sucrose+8g/L agar) after the tobacco seed surface sterilization.25-28 ℃, 80uE (m 2.S) illumination is 16 hours, along with the growth (after 1-1.5 month) of seedling, stem apex is downcut, and forwards new MS substratum to and generates plantlet.
(2) tobacco leaf disc is cultivated altogether
The agrobacterium tumefaciens that contains recombinant plasmid is inoculated in 5ml and contains in the substratum of kantlex and Rifampin, 28 ℃ of 200 rev/mins of shaking table overnight incubation, centrifugal collection thalline.Aseptic seedling blade edge and middle arteries and veins are cut with scalpel, and the arteries and veins vertical direction is cut into the wide leaf bar of 5-8mm with blade in the edge, puts into agrobacterium liquid after the blade cuts immediately and soaks 30-40 minute.Take out the blade of cultivating altogether with tweezers, be put into to inhale on the aseptic filter paper and remove too much bacterium liquid, the leaf bar moves into and contains in the plate of common culture medium (MS inorganic salt+0.6mg/l 2,4-D+30g/l sucrose+8g/l agar).With film plate is sealed, to reduce moisture evaporation and pollution.28 ℃ of dark 48hr that cultivate.
11. the screening of transformed plant
To cultivate blade altogether and transfer to callus inducing medium (MS inorganic salt+0.6mg/l 2,4-D+300mg/l kantlex+500mg/l helps joint penicillin+30g/l sucrose+8g/l agar) on, make the conversion blade fully contact the absorption that substratum helps nutrition and hormone, after cultivating for two weeks on the callus inducing medium, blade is transferred to bud substratum (MS inorganic salt, 1mg/L IAA+1mg/L 6-BA+300mg/L kantlex+500mg/L Pyocianil+30g/L sucrose+8g/L agar) go up cultivation.Downcut budlet with scalper and transfer to upward cultivation of root media (MS inorganic salt+0.4mg/11BA+100mg/l kantlex+30g/l sucrose+8g/l agar).
12. particle bombardment maize transformation
Particle gun is positioned in the big super clean bench, is beneficial to aseptic technique.Get the 6ul bag and put in the micropartical carrier center by the metallic particles dehydrated alcohol suspension of DNA (about 0.6ug plasmid and 0.37ug metallic particles), dry in moisture eliminator immediately, or on worktable, dry up.Be layered on one in the target tissue that desire is transformed by the 1-2 metafiltration paper of the wetting mistake of liquid nutrient medium or contain the 9cm culture dish center of solid culture ware, vacuumize, when vacuum tightness reaches desirable value (660-760mmHg, 1mmHg=133, in the time of 322Pa), bombard, shoot about 12 seconds.Change the explant after the bombardment over to callus inducing medium or bud division culture medium, 28 ℃, the dark or the low light level are cultivated down, do not add screening pressures such as microbiotic in this substratum, and general 1-2 week is cultivated in transition.Explant after transition is cultivated is selected to cultivate on the substratum that contains suitable Ka Nalei element, changes over to about 1 month in the subculture expansion breeding culture medium to cultivate.
13. pollen tube passage method mediation fungus G DH gene transformation cotton
The pROKII-GDH carrier that contains the GDH gene is dissolved in 1 * SSC solution, select the normotrophic hero of having spent, bagging is isolated, pollination is had one's hair trimmed the cotton filigree the day before yesterday, in sterile petri dish, add skim pollen germination substratum, gather the fresh pollen of sprouting at the end and place substratum,, add 30mm in every 10ml substratum cultivating about 3 minutes under 30 ℃ of conditions 3Pollen, when the pollen when about 1/10 has been sprouted, the careful mixing of dna solution that adds 1/10 volume, the final concentration that mixes back DNA with pollen is 5ug/ml, the mixed solution of DNA and pollen is applied on the column cap, pollen after about 10mm3 handles is awarded a female fringe, after the pollination again bagging be partitioned to seed maturity.
14. agriculture bacillus mediated fungus G DH gene transformation paddy rice
The agrobacterium tumefaciens that contains reorganization GDH gene is inoculated in 5ml and contains in kantlex and the Rifampin liquid, 28 ℃ of 200 rev/mins of overnight incubation, centrifugal collection thalline, soaked 30 minutes putting into agrobacterium liquid immediately after the aseptic seedling cutting, take out blade, remove too much bacterium liquid so inhale to the aseptic filter paper, change in the common substratum and cultivate.28 ℃ of dark 48hr that cultivate.
15. change the screening and the evaluation of fungus G DH gene plant positive transformant
(1) extracting DNA method from plant
Get the 1g plant leaf, add the liquid nitrogen grind into powder.Add 700 with 2 * CTAB extracting solution (2% (W/V) CTAB (cetyltriethylammonium bromide), 100mmol/l TrisCl, pH8.0,20mmol/L EDTA, 1.4mol/l NaCl), shake up gently, 65 ℃ of water-baths 30 minutes are shaken therebetween frequently.Add 700ul phenol/chloroform/primary isoamyl alcohol (25: 24: 1), and jog to solution is emulsified state, 7000 rev/mins centrifugal 5 minutes.Get supernatant liquor and continue to add 2 times of volume dehydrated alcohols with phenol/chloroform/primary isoamyl alcohol (25: 24: 1) extracting 2-3 time, mixing spends the night in-20 ℃ of precipitations.7000 rev/mins centrifugal 10 minutes, collect the nucleic acid precipitation, be dissolved in the deionized water.-20 ℃ of storages are standby.
(2) nucleic acid hybridization is identified positive transformant
The total DNA enzyme of extractive plant cut spend the night, restriction enzyme site is XbaI, and SacI carries out agarose gel electrophoresis next day.Dna fragmentation is transferred to from gel electrophoresis on the nylon membrane, and uv irradiating fixed dna fragmentation in 3 minutes.To hybridize 68 ℃ of hybridization temperatures, 20 hours time with fixed DNA on good probe of digoxin (DIG) mark and the nylon membrane.After hybridization was finished, the alkaline phosphatase (anti DIG-AP) that cleans nylon membrane and anti-digoxin reacted 30 minutes, carried out the NBT/BCIP colour developing after the cleaning.
As a result, a hybrid belt appears in position, the 1400bp left and right sides, and identical with positive control, show that the GDH gene is integrated in the Plant Genome.
16. the transplanting of transformed plant and offspring analysis
Carefully remove the agar of plant root, plant be transferred in the larger container that contains the MS inorganic salt solution,, made gaseous diffusion several hours, add some sterilized waters simultaneously to replenish evaporated liquid with covered opening, afterwards plantlet of transplant in damp soil.The seed of tying measure the offspring with nucleic acid hybridization and the vital staining of GDH enzyme and separate, the transgenic strain that isozygotys until acquisition.
17. gel analysis and the dyeing of paddy hydracid dehydrogenase activity
In liquefied ammonia, grind, extract plant leaf albumen, carry out 5% native gel electrophoresis, 120V72 hour.Gel is immersed in the following staining fluid (50mMTrispH9.3,8mg/ml L-glutamic acid, 0.04mg/ml NADP, 0.04mg/ml MTT, the fen of 0.04mg/ml vitriolic is coughed, 0.08mg/mlCaCl2).After gel soaks, one band is arranged in staining fluid, show that the GDH of conversion goes out to have active glutamate dehydrogenase at the plant expression in vivo at the position that contains GDH.
18. the growth of transfer-gen plant and nitrogen utilization efficiency test
After expressing fungus G DH sun plant results seed, carry out the breeding (with 8 (1)) of aseptic seedling.Along with the growth of seedling, stem apex is downcut, forward on the MS substratum of different nitrogen contents.The concentration of ammonia is respectively 20mM, 10mM, and 5mM, 2.5mM is to test its upgrowth situation.As a result, at 20mM, on the MS substratum of 10mM, positive transformant and control group offspring do not show obvious difference.On 5mM and 2.5mM substratum, the positive transformant upgrowth situation significantly is better than control group, and the sallow nitrogen stress symptom that waits of blade then appears in contrast.After plant-growth 1-1.5 month, the plant cleaning is clean, oven dry, test dry weight.The result shows that positive transformant increases about 20% than control group dry weight.Utilize Kjeldahl determination simultaneously, the nitrogenous rate that records transfer-gen plant increases about 20-30% than control group.Measure the nitrogen utilization efficiency that is grown in plant in the MS substratum simultaneously, the nitrogen utilization efficiency of the more unconverted plant of transformed plant improves about 20-30%.

Claims (6)

1. method that improves plant nitrogen assimilation efficiency, comprise: (a) will can in plant, guide the promotor of exogenous gene expression to link to each other with a kind of from the gene of the glutamate dehydrogenase of the mould Neurospora intermedia of middle arteries and veins spore or the food arteries and veins spore mould Neurospora sitophila that regards for oneself, chimeric genetic construct: (b) mosaic gene that makes up is imported in the vegetable cell, screen and turn out by plants transformed.
2. in accordance with the method for claim 1, wherein, described plant is tobacco, corn, cotton or paddy rice.
3. in accordance with the method for claim 1; Wherein, described glutamte dehydrogenase from the mould Neurospora intermedia of middle arteries and veins spore has amino acid sequence as follows: MSNLPSEPEFEQAYKELAYTLENSSLFQKHPEYRIALTVASIPERVIQFRVVWEDD NGNVQVNRGYRVQFNSALGPYKGGLRLHPSVNLSILKFLGFEQIFKNALTGLSMGG GKGGADFDPKGKSDAEIRRFCCAFMAELHKHIGADTDVPAGDIGVGGREIGYMFGA YRKAANRFEGVLTGKGLSWGGSLIRPEATGYGLVYYVGHMLEYSGAGSYAGKRVAL SGSGNVAQYAALKLIELGATVVSLSDSKGALVATGESGITVEDINAVMAIKEARQS LTSFQHAGHLKWIEGARPWLHVGKVDIALPCATQNEVSKEEAEGLLAAGCKFVAEG SNMGCTLEAIEVFENHRKEKKGEAVWYAPGKAANCGGVAVSGLEMAQNSQRLNWTQ AEVDEKLKDIMKNAFFNGLNTAKIYVEAAEGELPSLVAGSNIAGFVKVAQAMHDQG DWWSKN.
4. in accordance with the method for claim 1; ,Neurospora intermedia：ATGTCTAACCTTCCCTCTGAGCCCGAGTTTGAGCAGGCCTACAAGGAGTTGGCCTACACTCTCGAGAACTCCTCCCTCTTCCAGAAGCACCCCGAGTACCGCACTGCCCTCACCGTTGCCTCCATCCCCGAGCGTGTCATTCAGTTCCGTGTTGTCTGGGAGGACGACAACGGCAACGTCCAGGTCAATCGCGGTTACCGTGTCCAGTTCAACTCCGCCCTCGGTCCCTACAAGGGTGGTCTCCGTCTCCACCCCTCCGTCAACCTTTCCATTCTCAAGTTCCTCGGTTTCGAGCAGATCTTCAAGAATGCCCTCACTGGCTTGAGCATGGGTGGTGGCAAGGGTGGTGCCGACTTCGACCCCAAGGGCAAGAGCGACGCTGAGATCCGTCGCTTCTGCTGCGCTTTCATGGCTGAGCTTCACAAGCACATTGGTGCCGATACCGATGTTCCCGCTGGTGACATCGGTGTTGGTGGCCGTGAGATCGGCTACATGTTCGGTGCCTACCGCAAGGCCGCGAACCGTTTCGAGGGTGTCCTTACTGGCAAGGGCCTCTCCTGGGGTGGTTCGCTCATTCGCCCTGAGGCCACTGGTTACGGCCTCGTCTACTACGTCGGCCACATGCTCGAGTACTCTGGCGCCGGATCCTACGCTGGCAAGCGCGTTGCCCTCTCCGGTTCCGGCAACGTCGCCCAGTACGCCGCCCTCAAGCTCATTGAGCTAGGCGCCACCGTTGTCTCCCTCTCCGACTCCAAGGGTGCTCTTGTCGCCACTGGCGAGTCCGGCATCACCGTTGAGGACATCAACGCCGTTATGGCCATCAAGGAGGCCCGTCAGTCCCTTACCAGCTTCCAGCACGCTGGTCACCTGAAGTGGATCGAGGGCGCCCGCCCCTGGCTTCACGTTGGCAAGGTTGACATCGCTCTTCCTTGCGCTACCCAGAACGAGGTCTCCAAGGAGGAGGCTGAGGGTCTCCTTGCCGCCGGCTGCAAGTTCGTCGCTGAGGGTTCCAACATGGGCTGCACTCTCGAGGCCATTGAGGTCTTTGAGAACCACCGCAAGGAGAAGAAGGGCGAGGCCGTCTGGTACGCCCCCGGCAAGGCCGCCAACTGTGGTGGTGTTGCCGTTTCCGGTCTCGAGATGGCTCAGAACAGCCAGCGCCTCAACTGGACTCAGGCTGAGGTTGACGAGAAGCTCAAGGACATCATGAAGAACGCCTTCTTCAACGGTCTCAACACTGCCAAGATCTACGTCGAGGCTGCTGAGGGCGAGCTTCCTTCCCTCGTTGCTGGCTCCAACATTGCTGGTTTCGTCAAGGTTGCCCAGGCCATGCACGACCAGGGTGACTGGTGGTCCAAGAACTAA。
5. in accordance with the method for claim 1; Wherein, described glutamte dehydrogenase of regarding for oneself the mould Neurospora sitophila of food arteries and veins spore has amino acid sequence as follows: MSNLPSEPEFEQAYKELAYTLENSSLFQKHPEYRTALAVASIPERVIQFRVVWEDD NGNVQVNRGYRVQFNSALGPYKGGLRLHPSVNLSILKFLGFEQIFKNALTGLSMGG GKGGADFDPKGKSDAEIRRFCCAFMAELHKHIGADTDVPAGDIGVGGREIGYMFGA YRKAANRFEGVLTGKGLSWGGSLIRPEATGYGLVYYVGHMLEYSGAGSYAGKRVAL SGSGNVAQYAALKLIELGATVVSLSDSKGALVATGESGIVEDINAIMAIKEARQSL TTFQHAGHVKWIEGARPWLHVGKVDIALPCATQNEVSKEEAEGLLAAGCKFVAEGS NMGCTLEAIEVFENNRKEKKGEAVWYAPGKAANCGGVAVSGLEMAQNSQRLNWTQA EVDEKLKDIMKNAFFNGLNTAKTYAEAAEGELPSLVAGSNIAGFVKVPQAMHDQGD WWSKN.
6. in accordance with the method for claim 1; ,Neurospora sitophila：ATGTCTAACCTTCCCTCTGAGCCCGAGTTCGAGCAGGCCTACAAGGAGTTGGCCTACACTCTCGAGAACTCCTCCCTCTTCCAGAAGCACCCCGAGTACCGCACCGCCCTCGCCGTTGCCTCCATCCCCGAGCGTGTCATTCAGTTCCGTGTTGTCTGGGAGGACGACAACGGCAACGTCCAGGTCAACCGCGGTTACCGTGTCCAGTTCAACTCCGCCCTCGGTCCCTACAAGGGTGGTCTCCGTCTCCATCCCTCCGTCAACCTTTCCATTCTCAAGTTCCTCGGTTTCGAGCAGATCTTCAAGAATGCCCTTACTGGCCTGAGCATGGGTGGTGGCAAGGGTGGTGCCGACTTCGACCCCAAGGGCAAGAGCGATGCTGAGATCCGTCGCTTCTGCTGCGCTTTCATGGCCGAGCTTCACAAGCACATTGGTGCCGATACCGATGTTCCCGCTGGTGATATTGGTGTTGGTGGCCGCGAGATCGGTTACATGTTCGGTGCCTACCGCAAGGCTGCGAACCGTTTCGAGGGTGTCCTTACTGGTAAGGGCCTCTCCTGGGGTGGTTCGCTCATTCGCCCTGAGGCCACTGGTTACGGTCTCGTCTACTACGTCGGCCACATGCTCGAGTACTCTGGCGCCGGCTCCTACGCTGGCAAGCGCGTTGCCCTCTCTGGTTCCGGCAACGTCGCCCAGTACGCCGCCCTCAAGCTCATTGAGCTAGGCGCCACCGTTGTCTCCCTCTCCGACTCCAAGGGTGCCCTTGTCGCCACTGGCGAGTCCGGCATCACTGTTGAGGACATCAACGCCATCATGGCCATCAAGGAGGCCCGTCAGTCTCTTACCACCTTCCAGCACGCTGGCCACGTCAAGTGGATCGAGGGCGCCCGCCCCTGGCTTCACGTTGGCAAGGTTGACATCGCTCTTCCTTGCGCTACCCAGAACGAGGTCTCCAAGGAGGAGGCTGAGGGTCTCCTTGCCGCCGGCTGCAAGTTCGTCGCTGAGGGTTCCAACATGGGCTGCACTCTCGAGGCCATCGAGGTCTTTGAGAACAACCGCAAGGAGAAGAAGGGCGAGGCCGTCTGGTACGCCCCCGGCAAGGCCGCCAACTGTGGTGGTGTTGCCGTTTCCGGTCTCGAGATGGCTCAGAACAGCCAGCGCCTCAACTGGACTCAGGCTGAGGTTGACGAGAAGCTCAAGGACATCATGAAGAACGCCTTCTTCAACGGTCTCAACACTGCCAAGACCTACGCCGAGGCTGCTGAGGGCGAGCTTCCTTCCCTCGTTGCCGGCTCCAACATTGCTGGTTTCGTCAAGGTACCCCAGGCCATGCACGACCAGGGTGACTGGTGGTCCAAGAACTAA。
CNB011044322A 2000-07-06 2001-02-26 Method for raising plant nitrogen assimilation efficiency Expired - Fee Related CN1137265C (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CNB011044322A CN1137265C (en) 2000-07-06 2001-02-26 Method for raising plant nitrogen assimilation efficiency
AU2001246330A AU2001246330A1 (en) 2000-07-06 2001-02-27 A method of improving nitrogen assimilation efficiency in plants
PCT/CN2001/000294 WO2002002776A1 (en) 2000-07-06 2001-02-27 A method of improving nitrogen assimilation efficiency in plants

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN00109779.2 2000-07-06
CN00109779 2000-07-06
CNB011044322A CN1137265C (en) 2000-07-06 2001-02-26 Method for raising plant nitrogen assimilation efficiency

Publications (2)

Publication Number Publication Date
CN1333349A CN1333349A (en) 2002-01-30
CN1137265C true CN1137265C (en) 2004-02-04

Family

ID=25739331

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB011044322A Expired - Fee Related CN1137265C (en) 2000-07-06 2001-02-26 Method for raising plant nitrogen assimilation efficiency

Country Status (3)

Country Link
CN (1) CN1137265C (en)
AU (1) AU2001246330A1 (en)
WO (1) WO2002002776A1 (en)

Families Citing this family (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0707413A2 (en) * 2006-02-09 2011-05-03 Pioneer Hi Bred Int genes for improving nitrogen utilization efficiency in crops
US8889950B2 (en) 2006-07-28 2014-11-18 The George Washington University Repression of AtGLR3.2 increases plant biomass
CA2722889A1 (en) 2008-04-30 2009-11-05 Danisco Us Inc. New chimeric alpha-amylase variants
US7964774B2 (en) 2008-05-14 2011-06-21 Monsanto Technology Llc Plants and seeds of spring canola variety SCV384196
MX2011013224A (en) 2009-06-09 2012-06-01 Pioneer Hi Bred Int Early endosperm promoter and methods of use.
US8440891B2 (en) 2009-09-22 2013-05-14 Board of Trustees of the University of Akransas, N.A. Rice cultivar CL 142-AR
US8440892B2 (en) 2009-10-15 2013-05-14 Board Of Trustees Of The University Of Arkansas, N.A. Rice cultivar CL 181-AR
EP2494056A1 (en) 2009-10-26 2012-09-05 Pioneer Hi-Bred International Inc. Somatic ovule specific promoter and methods of use
NZ601341A (en) 2010-01-22 2014-02-28 Bayer Ip Gmbh Acaricide and/or insecticide active substance combinations
JP2013542215A (en) 2010-11-02 2013-11-21 バイエル・インテレクチユアル・プロパテイー・ゲー・エム・ベー・ハー N-hetarylmethylpyrazolyl carboxamides
BR112013012080A2 (en) 2010-11-15 2016-07-19 Bayer Ip Gmbh n-aryl pyrazole (thio) carboxamides
US20130289077A1 (en) 2010-12-29 2013-10-31 Juergen Benting Fungicide hydroximoyl-tetrazole derivatives
WO2013020985A1 (en) 2011-08-10 2013-02-14 Bayer Intellectual Property Gmbh Active compound combinations comprising specific tetramic acid derivatives
WO2013096810A1 (en) 2011-12-21 2013-06-27 The Curators Of The University Of Missouri Soybean variety s05-11482
US20130167262A1 (en) 2011-12-21 2013-06-27 The Curators Of The University Of Missouri Soybean variety s05-11268
US9006515B2 (en) 2012-01-06 2015-04-14 Pioneer Hi Bred International Inc Pollen preferred promoters and methods of use
CA2860783A1 (en) 2012-01-06 2013-07-11 Pioneer Hi-Bred International, Inc. Ovule specific promoter and methods of use
US8878009B2 (en) 2012-04-26 2014-11-04 Monsanto Technology, LLP Plants and seeds of spring canola variety SCV318181
US8859857B2 (en) 2012-04-26 2014-10-14 Monsanto Technology Llc Plants and seeds of spring canola variety SCV259778
US8835720B2 (en) 2012-04-26 2014-09-16 Monsanto Technology Llc Plants and seeds of spring canola variety SCV967592
CA2887571A1 (en) 2012-10-11 2014-04-17 Pioneer Hi-Bred International, Inc. Guard cell promoters and uses thereof
WO2014159306A1 (en) 2013-03-13 2014-10-02 Pioneer Hi-Bred International, Inc. Glyphosate application for weed control in brassica
UA121847C2 (en) 2013-03-14 2020-08-10 Піонір Хай-Бред Інтернешнл Інк. Compositions and methods to control insect pests
EP2971000A4 (en) 2013-03-15 2016-11-23 Pioneer Hi Bred Int Phi-4 polypeptides and methods for their use
EA030896B1 (en) 2013-08-16 2018-10-31 Пайонир Хай-Бред Интернэшнл, Инк. Insecticidal proteins and methods for their use
EP4159028A1 (en) 2013-09-13 2023-04-05 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
CN103820408B (en) * 2014-01-26 2016-08-24 湖南大学 Improve fungus PcGDH albumen and application thereof that nitrogen efficiently utilizes
EP3705489A1 (en) 2014-02-07 2020-09-09 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
CA2955828A1 (en) 2014-08-08 2016-02-11 Pioneer Hi-Bred International, Inc. Ubiquitin promoters and introns and methods of use
CA2961733A1 (en) 2014-09-17 2016-03-24 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
RU2733898C2 (en) 2014-10-16 2020-10-09 Пайонир Хай-Бред Интернэшнл, Инк. Insecticidal proteins and methods of using them
WO2016099916A1 (en) 2014-12-19 2016-06-23 E. I. Du Pont De Nemours And Company Polylactic acid compositions with accelerated degradation rate and increased heat stability
BR112017024948A2 (en) 2015-05-19 2018-07-31 Pioneer Hi Bred Int insecticide proteins and methods for their use
US10647995B2 (en) 2015-06-16 2020-05-12 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
CA2994676A1 (en) 2015-08-06 2017-02-09 Pioneer Hi-Bred International, Inc. Plant derived insecticidal proteins and methods for their use
WO2017040343A1 (en) 2015-08-28 2017-03-09 Pioneer Hi-Bred International, Inc. Ochrobactrum-mediated transformation of plants
CN108575091A (en) 2015-12-18 2018-09-25 先锋国际良种公司 Insecticidal protein and its application method
US11104911B2 (en) 2015-12-22 2021-08-31 Pioneer Hi-Bred International, Inc. Embryo-preferred Zea mays promoters and methods of use
EP3451837B1 (en) 2016-05-04 2021-08-25 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
CA3022858A1 (en) 2016-06-16 2017-12-21 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
RU2019101793A (en) 2016-06-24 2020-07-24 Пайонир Хай-Бред Интернэшнл, Инк. PLANT REGULATORY ELEMENTS AND METHODS OF THEIR APPLICATION
EP3954202A1 (en) 2016-07-01 2022-02-16 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
US20210292778A1 (en) 2016-07-12 2021-09-23 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
CA3038806A1 (en) 2016-11-01 2018-05-11 Pioneer Hi-Bred International, Inc. Insecticidal proteins and methods for their use
CN106906191B (en) * 2017-03-02 2020-06-12 湖南大学 Fungus TrGDH protein for improving efficient utilization of nitrogen and application thereof
AU2018337756A1 (en) 2017-09-25 2020-04-02 Pioneer Hi-Bred, International, Inc. Tissue-preferred promoters and methods of use
US11525144B2 (en) 2018-03-14 2022-12-13 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
WO2019178038A1 (en) 2018-03-14 2019-09-19 Pioneer Hi-Bred International, Inc. Insecticidal proteins from plants and methods for their use
BR112020023800A2 (en) 2018-05-22 2021-02-23 Pioneer Hi-Bred International, Inc. plant regulatory elements and methods of using them
US20210277409A1 (en) 2018-06-28 2021-09-09 Pioneer Hi-Bred International, Inc. Methods for selecting transformed plants
CA3111090A1 (en) 2018-10-31 2020-05-07 Pioneer Hi-Bred International, Inc. Compositions and methods for ochrobactrum-mediated plant transformation
CN111321153B (en) * 2020-04-26 2021-08-17 广西大学 Dark response GD2 gene from corn and application thereof
BR112022027035A2 (en) 2020-07-14 2023-04-11 Pioneer Hi Bred Int INSECTICIDAL PROTEINS AND METHODS FOR THE USE OF THEM

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5879941A (en) * 1995-10-06 1999-03-09 University Of Florida Polypeptides and polynucleotides relating to the α-and β-subunits of a glutamate dehydrogenase and methods of use
US5998700A (en) * 1996-07-02 1999-12-07 The Board Of Trustees Of Southern Illinois University Plants containing a bacterial Gdha gene and methods of use thereof
ES2143408B1 (en) * 1998-04-02 2000-12-01 Urquima Sa PROMOTER AND CONSTRUCTIONS FOR THE EXPRESSION OF RECOMBINANT PROTEINS IN FILAMENTAL FUNGI.
US6653099B1 (en) * 1998-11-05 2003-11-25 E. I. Du Pont De Nemours And Company Plant UDP-glucose dehydrogenase

Also Published As

Publication number Publication date
CN1333349A (en) 2002-01-30
WO2002002776A1 (en) 2002-01-10
AU2001246330A1 (en) 2002-01-14

Similar Documents

Publication Publication Date Title
CN1137265C (en) Method for raising plant nitrogen assimilation efficiency
CN110117560B (en) Rhizosphere growth-promoting bacterium for enhancing salt tolerance of crops, microbial fertilizer and application thereof
CN113388526B (en) Endophytic fungus FO-R20 and application thereof
CN116478870B (en) Maltophilous oligotrophic single spore fungus OLR3-17 strain and application thereof
CN1055198C (en) Process for selectively breeding and producing high-efficiency crop disease prevention and output increasing strain
CN112725191B (en) Inonotus tumefaciens strain for promoting germination of orchidaceae seeds and application thereof
CN1763207A (en) A kind of corn borer resistant transgenetic ultra-sweet corn regeneration system and establishment method thereof
CN112021073A (en) Morchella esculenta external aid nutrition bag ingredient, nutrition bag and preparation method thereof
CN110122482A (en) A kind of preparation process of toVerticilliumdahliaActivitie Activitie S of Relative PeaT1 plant immune protein soluble powder
CN101333550A (en) Method for preparing CYCLIC DIPEPTIDES compounds and use thereof
CN111011139A (en) Planting method of selenium-rich pollution-free potatoes
CN110184293A (en) A method of increasing phytomass or yield by improving photosynthetic efficiency
CN112442449B (en) Ramaria original strain culture medium and application thereof as well as Ramaria original strain and culture method thereof
CN112063538A (en) Trichoderma hamatum, microbial agent containing trichoderma hamatum and application of trichoderma hamatum
WO2023109774A1 (en) Method for genetic transformation of panicum plant
CN101569269A (en) Production process for culturing king oyster mushroom by utilizing biological magnetized water
CN103636505A (en) Complex breeding method of high-chlorophyll multi-tiller barley
CN107354191B (en) Biological agent for promoting plant growth and preventing and treating plant diseases and insect pests
CN103571788A (en) Collection and storage method of cordyceps militaris ascospores
CN112673900B (en) A strain of Rumex crispus and its cultivation, picking and preservation method
CN114350559B (en) Salt-tolerant growth-promoting Liaoning slow rhizobium RY6 strain and application thereof
CN1429904A (en) Method for gene conversion of corn
CN107372107A (en) The fast culture process of ground loquat
CN113862281A (en) Application of wheat TaLCT1 gene silencing in regulation and control of wheat cadmium stress tolerance
CN111771722A (en) Pure breeding method of quinoa

Legal Events

Date Code Title Description
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C06 Publication
PB01 Publication
C14 Grant of patent or utility model
GR01 Patent grant
C17 Cessation of patent right
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20040204