CN107384955A - Peanut glutamy t RNA reductases and its application - Google Patents

Peanut glutamy t RNA reductases and its application Download PDF

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CN107384955A
CN107384955A CN201710721435.9A CN201710721435A CN107384955A CN 107384955 A CN107384955 A CN 107384955A CN 201710721435 A CN201710721435 A CN 201710721435A CN 107384955 A CN107384955 A CN 107384955A
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peanut
rna
glutamy
reductases
sequence
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CN107384955B (en
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杨莎
赵璐颖
李新国
万书波
张佳蕾
郭峰
耿耘
孟静静
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Biotechnology Research Center of Shandong Academy of Agricultural Sciences
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Biotechnology Research Center of Shandong Academy of Agricultural Sciences
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    • 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.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/0107—Glutamyl-tRNA reductase (1.2.1.70)

Abstract

The present invention relates to gene engineering technology field, more particularly to one cultivates peanut glutamy t RNA reductases, further relates to application of the peanut glutamy t RNA reductases in regulation chlorophyll and calmodulin expression.Beneficial effects of the present invention：The function of glutamy t RNA reductases in peanut is identified by the present invention, has the function that to adjust in chlorophyll and calmodulin expression, specify that the interactively between the enzyme and Ca2+ oscillations, theoretical foundation has been established for the research in later stage.

Description

Peanut glutamy t-RNA reductases and its application
Technical field
The present invention relates to gene engineering technology field, more particularly to one cultivates peanut glutamy t-RNA reductases, further relates to peanut paddy The application of aminoacyl t-RNA reductases.
Background technology
Chlorophyll（Chlorophyll, Chl）It is a kind of fat-soluble pigment, is present in higher plant in chloroplaset.Plant For thing when carrying out photosynthesis, chlorophyll plays extremely important effect, can absorb and change solar energy, and in this mistake Water-molecule dissociation is produced oxygen molecule and reproducibility oxygen in journey, played a very important role in electronic cell transfer chain, So as to start the conversion process of energy, thus chlorophyll plant grow and the shape of the quality and yield of crops Into all playing highly important effect above.The initial substance of chlcrophyll biosynthesis is L- glutamy-tRNA, is passed through GluTR catalytic action, L- glutamy-t RNA are reduced into as Pidolidone ester -1- semialdehydes；Then glutamate -1- is passed through The catalytic action of semialdehyde transaminase, formation-aminolevulinic acid（ALA）, ALA is the critical precursors of chlcrophyll biosynthesis. GluTR is the center effector of chlcrophyll biosynthesis, and the enzyme is by glutamy t-RNA reductases (HEMA) gene code, therefore HEMA is the key gene of controlling chlorophyll route of synthesis.Different plant HEMA numbers of members may have different.Pattern In plant Arabidopsis thalianaHEMAThere are 3 members, be respectivelyHEMA1、HEMA2WithHEMA3, and in cucumber onlyHEMA1WithHEMA2Two Individual gene.
But peanutHEMAGene there is no report both at home and abroad, and its regulation during plant stress physiology and Senescence Physiology is made With unclear.
The content of the invention
In order to solve above peanut in the prior artHEMARegulation of the gene during plant stress physiology and Senescence Physiology is made The problem of with research blank, cultivated peanut glutamy t-RNA reductases this application discloses one.
Present invention also offers the application of peanut glutamy t-RNA reductases.
What the present invention was obtained through the following steps：
Application of the peanut glutamy t-RNA reductases in regulation chlorophyll and calmodulin expression.
Described application, preferably peanut glutamy t-RNA reduce enzyme amino acid sequence as shown in sequence 4 in sequence table.
Described application, the institute of sequence 3 in the nucleotide sequence such as sequence table of preferred expression peanut glutamy t-RNA reductases Show.
The application of described application, preferably peanut glutamy t-RNA reductases in regulation calmodulin expression is shown as, Under condition of salt stress, overexpress calmodulin expression quantity in the strain of peanut glutamy t-RNA reductase genes and be consequently increased.
The application of described application, preferably peanut glutamy t-RNA reductases in regulation chlorophyll and calmodulin expression, It is embodied in, under normal condition and after salt stress is handled, after peanut glutamy t-RNA reductase gene overexpressions, The biosynthesis of chlorophyll also has increase.
Calcium element is one of necessary nutrient of growth and development of plants, and it take part in plant and is bloomed from germination to Growth and Differentiation As a result overall process, the main function of calcium ion is in plant：（1）Nutriment, promote the formation of micro-pipe.（2）Cell The structure organization of wall and the mobility of film.（3）Mitigate the toxic action of salt stress.（4）Promote the growth and elongation of pollen tube.Flower Life is the crop for needing calcium more, and nitrogen is only second to the quantity required of calcium constituent, suitable with potassium higher than phosphorus.During peanut calcium deficiency, seed Impaired development, shell tissue looseness, empty fruit, not plump fruit and decayed fruit increase, yield are decreased obviously.Study Ca2+To peanut stress physiology Regulatory mechanism and its with the calcium element assimilated equations of the clear and definite Development of Peanut of relation pair of Chlorophyll synthesis interprocedual, ensure spend Growing and high yield has very important significance.
Beneficial effects of the present invention：The function of glutamy t-RNA reductases in peanut is identified by the present invention, has and adjusts The effect in chlorophyll and calmodulin expression is saved, specify that the interactively between the enzyme and Ca2+ oscillations, is established for the research in later stage Theoretical foundation is determined.
Brief description of the drawings
Fig. 1 isAhhemAGene cloning result,
Fig. 2 isAhhemAThe hydrophobicity analysis of gene coded protein,
Fig. 3 isAhhemAGene coded protein cross-film specificity analysis,
Fig. 4 is expression of the AhhemA in Different Organs；F：Flower;R:Root；S：Stem；L：Blade；Fr:Fruit,
Fig. 5 is that external source applies AhhemA gene expression amounts and measuring chlorophyll content after calcium under condition of salt stress,
External source applies downstream gene expression amount after ALA under Fig. 6 condition of salt stress,
Fig. 7 is to turn AhhemA gene plants PCR identifications,
Fig. 8 is measuring chlorophyll content,
Fig. 9 is CaM gene expressions.
Embodiment
The present invention is further described with reference to specific embodiment：
Embodiment 1
1. AhhemAThe separation of gene
RNA kits extraction RNA is utilized from peanut leaf（It is purchased from Tiangeng biochemical technology Co., Ltd）And reverse transcription.According to base Because of sequence, primer is designed, primer sequence is：5 '-ATGGCTGTTTCGACGAGC-3 ' and 5 '-TTAGCTGTCACTATGGTT-3 ' Carry out PCR（PCR）, PCR primer detects amplified fragments through agarose gel electrophoresis and the clip size of estimation accords with Close（1626 bp）（Fig. 1）.
The PCR primer of gained is connected with pMD18-T cloning vectors, converts Escherichia coli.Quickly screened by bacterium colony PCR Positive colony is obtained, extracts the plasmid of positive colony, plasmid is cut using the restriction enzyme site of carrier inside, obtains and PCR primer Size identical band.Mail the bacterium solution identified the sequencing of to Shanghai bio-engineering corporation, see sequence 3 in sequence table, use Gained fragment sequence compared with known array, is it is found that the fragment to be cloned by Blast, DNAman or DNAclub software Target gene.
2. AhhemAThe sequence analysis of gene
Sequence 3 in sequence table：
(a) sequence signature
* length:1626 base-pairs
* type:Nucleic acid
* chain:Double-strand
* topological structure:Linearly
(b) molecule type: cDNA
(c) assume:It is no
(d) antisense:It is no
(e) initial source:Peanut
Sequence 4 in sequence table：
（a）Sequence signature
* length：541 amino acid
* type：Amino acid
* chain：It is single-stranded
* topological structure：Linearly
（b）Molecule type：Protein
3. AhhemAThe Analysis of Biochemical Characteristics of gene coded protein
3.1 AhhemAThe hydrophobicity analysis of gene coded protein
The hydrophobicity of albumen coded by peanut glutamy t-RNA reductase genes is analyzed, the results showed that its hydrophilic ammonia Ratio shared by base acid and hydrophobic amino acid is more or less the same（Fig. 2）.Wherein hydrophilic amino acid proportion is slightly higher, shows this Albumen is water-solubility protein.
3.2 AhhemAGene expression product cross-film specificity analysis
By the amino acid sequence that AhhemA is submitted to the online PRED-TMR databases on internet, it is predicted that the zymoprotein knot There is no transmembrane signal area in structure（Fig. 3）.
4.AhhemAExpression analysis of the gene in peanut plant
4.1AhhemAExpression in peanut Different Organs
To understandAhhemAEndogenous expression of the gene in Different Organs, we are with peanutAhhemAOne section of the end of gene 3 ' CDNA is that primer has carried out quantitative fluorescent PCR analysis.As a result show,AhhemAGene has expression in all organs, belongs to Constitutive expression, the expression in blade is apparently higher than other organs, and the expression quantity in root is minimum, explanationAhhemAGene exists The high tissue expression amount of chlorophyll content is higher（Fig. 4）.
After 4.2 peanut plants grow three weeks under normal operation, 200mM NaCl salt treatment 3 days, external source applies Calcium treatment, Select 0 respectively, 10,20,40,80mM various concentrations calcium（CK, C10, C20, C40, C80 are designated as, Normal is to be not added with salt control group） Sampled after processing, fluorescence quantitative PCR detectionAhhemAExpression, while chlorophyll content is determined, see Fig. 5.As a result show, salt Under stress conditions, external source applies calcium to peanuthemAPlay negative regulation effect, the down regulation of gene expression, however, chlorophyll content exists Apply it is after calcium and unaffected, it is in rising trend, show that action site of the Ca2+ oscillations in Chlorophyll synthesis approach is likely to HemA downstreams.
Under condition of salt stress, after external source sprays the ALA that AhhemA is catalyzed and synthesized, the catalytic subunit in magnesium chelatase activated centre ChlH and ChlD gene expressions are raised, and expression quantity reaches peak value after 10mg/l ALA processing, sees Fig. 6.
5. AhhemAThe structure of gene plant expression vector
5.1 amplificationAhhemAGene
Peanut varieties " educate 22 " and provided by Shandong Academy of Agricultural Sciences, is expanded by RT-PCR by flowerAhhemAThe coding of gene Area.
hemA-F(5'-TCTAGAATGGCTGTTTCGACGAGC-3')(XbaI)；Sequence 5 in sequence table,
hemA-R(5'-GGATCCTTAGCTGTCACTATGGTT-3')(BamHI)；Sequence 6 in sequence table,
Above-mentioned primer sequence respectively withAhhemAGene 1-18 bases, 1612-1626 base-pairs should.
5.2 AhhemAThe connection of gene and cloning vector pMD18-TSimple and plant expression vector pBI121
When building carrier for expression of eukaryon, by full length gene obtained by PCR connect into cloning vector pMD18-TSimple (buy in TaKaRa), connection product conversion Escherichia coli Trans5 α obtain anti-Ka Na bacterium colony.The positive colony screened is carried out Plasmid is extracted after bacterium solution enrichment, and plasmid is subjected to double digestion with XbaI and BamHI, will be included after digestionAhhemAThe bar of total length Band is reclaimed；Then pBI121 carriers are subjected to digestion with identical enzyme, resulting carrier segments is reclaimed.So The cohesive end of complementation has been taken on resulting full length sequence and carrier sequence, then after the two is connected, has converted Escherichia coli, Recon is gone out by digestion evaluation and screening, builds expression vector pBI121-AhhemA。
The preparation and conversion of 5.3 agrobacterium tumefaciens lba4404 competent cells
The preparation process of competent cell is as follows：
（1）The a little Agrobacterium LBA4404 of picking, is inoculated in 5mLLB fluid nutrient mediums（Containing 50mg/LSTR）In, 28 DEG C, 200rpm Overnight incubation；
（2）2mL cultures are taken in LB fluid nutrient mediums（Containing 50mg/LSTR）In continue to cultivate, until OD800 be 0.5 or so.
（3）Culture puts 30min in ice bath, 4 DEG C, 5000rpm, centrifuges 5min, abandoning supernatant；
（4）With the 0.1mol/LNaCl suspension bacteria liquids that 10mL is cold；
（5）4 DEG C, 5000rpm, centrifuge 5min, abandoning supernatant；
（6）With the CaCl that 1mL is cold2（20mmol/L）Suspend, be distributed into 50 μ L/ pipes, to -80 DEG C of preservations after being freezed in liquid nitrogen.
Freeze-thaw method Agrobacterium-mediated Transformation step：
（1）Melt competent cell in ice bath；
（2）2 μ L DNAs are added in 1.5mLEppendorf centrifuge tubes, add the competent cell that 50 μ L have melted, are mixed Ice bath 30min after even, 1min is freezed in liquid nitrogen, then places 5min in 37 DEG C of water-baths；
（3）Add the LB fluid nutrient mediums of 950mL antibiotic-frees, 28 DEG C, 200rpm vibrations 3h；
（4）8000rpm centrifuges 1min, supernatant is outwelled, with 100 μ LLB back dissolving thalline；
（5）50 μ L bacterium solutions are taken to be coated onto on LB solid mediums（Contain 50mg/LKan, 100mg/LRif）, 28 DEG C are inverted culture 2- 3d。
6. utilize agrobacterium mediation converted tobacco
6.1 Agrobacteriums are cultivated：
Picking single bacterium falls within 5mLLB fluid nutrient mediums（Contain 100mg/LRif and 50mg/LKan）Middle culture 36h（28℃、 200rpm）, then take 1% to be seeded to above-mentioned same culture medium, 28 DEG C, 200rpm culture 12h, take 2mL bacterium solutions to centrifuge（4℃、 4000rpm、10min）, then with 20mLMS fluid nutrient medium suspension thallines, for conversion test.
The foundation of 6.2 Transformation of tobacco, regenerating system：
（1）Tobacco Nc89 seeds, after vernalization, are seeded in vinyl disc, Routine Management, standby to 2-3 piece leaf periods；
（2）Tobacco leaf is taken, with 70% ethanol disinfection 30s, then 0.1%HgCl2Sterilize 8-10min, with aseptic water washing for several times after, It is cut into small pieces（0.5*0.5cm2）；
（3）The tobacco leaf sheared is placed in MS differential mediums, 28 DEG C, light application time 16h/d, intensity of illumination 2000LX, Preculture 2 days；
（4）Tobacco leaf after preculture is immersed into bacterium solution 5-10min, then blots unnecessary bacterium solution with the filter paper of sterilizing, is accessed MS culture mediums；28 DEG C of light cultures 2 days；
（5）Explant after the co-cultivation sterilizing water washing 3 times of the cephalo containing 250mg/L, is then blotted with sterilizing filter paper, is transferred to It is incubated on cephalo 250mg/L differential medium containing kanamycins 100mg/L（The same preculture of condition）；Every 15 days more Change a subculture；
（6）When bud length to 1cm or so, cut, move into MS root medias（Additional kanamycins 50mg/L, cephalo 250mg/ L）In, promote it and take root.After root system development is good, move into the flowerpot for filling sterile soil, after plastic sheeting moisturizing 2 days, greenhouse Routine Management.
7. the PCR detections of transgenic tobacco plant
7.1 CTAB micromethods extract genomic DNA
（1）The fresh blades of 0.1-0.2g are taken, puts in liquid nitrogen and pulverizes, are transferred in 1.5mL centrifuge tubes, 300 μ L2 of addition × CTAB Extraction buffers, are gently agitated for, and powder is fully scattered, and 20min is incubated in 65 DEG C of water-baths；
（2）Then, 4 DEG C, 10000rpm centrifugations 10min；
（3）Supernatant is transferred in a new centrifuge tube, adds 200 μ L chloroforms/isoamyl alcohol（24:1）, gently mix, place 5min；
（4）4 DEG C, 8000rpm centrifugations 10min；
（5）Supernatant is transferred in another centrifuge tube, the cold isopropanols of 500 μ L is added, mixes, 4 DEG C of placement 10min；
（6）4 DEG C, 8000rpm centrifugation 10min, remove supernatant, centrifuge tube is inverted on blotting paper；
（7）75% ethanol washes precipitation, puts 20min in superclean bench and dries up；
（8）50 μ LTE dissolving DNAs are added, -20 DEG C save backup.
2 × CTAB Extraction buffers（100mL）：Take 1MTrisCl10mL；0.5MEDTA4mL；NaCl8.182g； CTAB2.0g；PVP3.0g.It is calmly molten to arrive 100mL after mentioned component is dissolved with distilled water.Beta -mercaptoethanol is added after sterilizing （β-mercaptoethanol,β-ME）200 μ L, it is standby.
The PCR detections of 7.2 transfer-gen plants
Extract the genomic DNA of regeneration plant, using above-mentioned carrier sequence andAhhemAGene order design primer enters performing PCR expansion Increase.PCR response procedures are：95℃、5min；95 DEG C, 50s, 55 DEG C, 50s, 72 DEG C, 1min30s, 30 circulations；72℃、 10min.Fig. 7 is shown in transfer-gen plant PCR identifications, and positive rate reaches 76.4%.
8. turnAhhemAGene plant Function Identification
8.1 measuring chlorophyll content.Expression quantity higher strain OE2, OE5 and OE8 are filtered out from transgene tobacco and carries out leaf The measure of chlorophyll contents.As a result show, under normal condition, transfer-gen plant Determination of Chlorophyll content is significantly higher than WT lines； After salt stress is handled, chlorophyll content has decline, but transgenic line Determination of Chlorophyll content is still below WT lines. ShowAhhemAThe biosynthesis of chlorophyll has increase to a certain extent after gene overexpression, is advantageous to improve the anti-of plant Inverse property.See Fig. 8.
8.2 turnAhhemACalmodulin Gene expression quantity in gene plant.Compare WT lines and transfer-gen plant passes through 200mM NaCl salt treatment utilizes the expression of fluorescence quantitative PCR detection Calmodulin Gene after 3 days.Fig. 8 results show, salt stress Under the conditions of, overexpressionAhhemACalmodulin expression quantity is consequently increased in the strain of gene, further demonstrates Ca2+ oscillations in leaf Action site in green plain route of synthesis is in hemA downstreams.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not limited by embodiment System, it is other it is any without departing from the present invention Spirit Essences with made under principle change, modification, combine, replacement, simplification should be Equivalence replacement mode, is included within protection scope of the present invention.
<110>Biotechnology Research Center, Shandong Academy of Agricultural Sciences
<120>Peanut glutamy t-RNA reductases and its application
<160>6
<210>1
<211>18
<212>DNA
<213>It is artificial synthesized
<400>1
ATGGCTGTTT CGACGAGC 18
<210>2
<211>18
<212>DNA
<213>It is artificial synthesized
<400>2
TTAGCTGTCA CTATGGTT 18
<210>3
<211>1626
<212>DNA
<213>Peanut（Arachis hypogaea）
<400>3
atggctgttt cgacgagctt ttcgggggct aagttggagg ctttgttcct caaatgttgt 60
tcctcttcct ctgccaatgc tgcttattct ttgtgtgtgc cttccaaaac cgccgccaag 120
gccaccagaa cgacgccgtt tcggagaggc ctggttcgtt gtgacgcttc ggcttctcct 180
gatgttattc ttgacaatgc tgctgccgtc tctgctcttc agcaacttaa gacttatgcc 240
gccgataggt atacgaagga aaagagcagc atcgtggtga ttggactcag cgtgcatact 300
acacctgtgg aaatgcgtga aaagcttgcc attccagaag cagaatggcc cagagccatt 360
ggagagcttt gcggcctcaa tcatattgaa gaagcagctg ttctcagcac ctgtaaccga 420
atggagatat atgttgttgc tctctctcag catcgcggtg taaaagaagt caccgagtgg 480
atgtcaagaa ctagtggaat ccctgtttca gaactttgcc agcatcgatt tttgttgtat 540
aacaaagatg ccacacagca tcttttcgaa gtctcagctg gtcttgactc tcttgtgctg 600
ggagaaggcc aaatccttgc ccaggttaag caagttgtca aagttggaca aggagtcaat 660
ggctttggga ggaacatcag cggcctattc aagcatgcga ttactgtcgg gaaaagggtt 720
agagccgaga ctaatattgc tgcaggagct gtttctgtta gctcagctgc cgttgaattg 780
gccttgatga agctacctga aacttcacat ggtaatgcta agatgttggt tattggagct 840
ggaaagatgg ggaagcttgt gatcaaacat ttggttgcaa agggttgcac aaagatggtg 900
gttgtcaata gaagtgagga gagagttgct gaaatccgtg aagagctaaa ggatgttgag 960
ataatctaca aacccctctc agaaatgctt gcttgtgtag gtgaagcaga tgtagttttc 1020
accggtacag cctcagaaaa cccattgttc ttgaaagatg atgttaaaga ccttccttct 1080
gtgagtcaag acattggagg ccatcgcctc tttattgata tctcagttcc tcggaacgtg 1140
ggttcatgtg tctcagatat cgagtctgtg cgagtttaca atgttgatga ccttaaagag 1200
gttgtagctg caaataaaga ggatcggctg agaaaagcaa tggaagctca ggcaatcatt 1260
ggtgaagaat cacaacaatt cgaagcttgg agggactcgc ttgaaaccgt tcctaccata 1320
aaaaaattga gggcttatgc tgaaagaatc agggctgctg agcttgagaa atgcttaggt 1380
aagatgggtg atgatatctc gaagaagaca cggcgtgccg tggatgatct tagccgtggc 1440
atagtcaata agttgcttca tggtccaatg cagcacctga ggtgcgacgg cagtgatagc 1500
cggaccctga ccgagaccct cgagaacatg catgctttga atagaatgtt tagccttgag 1560
actgaaatat cagtgttgga gcagaagatt cgagccaagg ttgaacaaaa ccatagtgac 1620
agctaa 1626
<210>4
<211>541
<212>PCR
<213>Peanut（Arachis hypogaea）
<400>4
MetAlaValSerThr SerPheSerGlyAla LysLeuGluAlaLeu PheLeuLysCysCys
5 10 15 20
SerSerSerSerAla AsnAlaAlaTyrSer LeuCysValProSer LysThrAlaAlaLys
25 30 35 40
AlaThrArgThrThr ProPheArgArgGly LeuValArgCysAsp AlaSerAlaSerPro
45 50 55 60
AspValIleLeuAsp AsnAlaAlaAlaVal SerAlaLeuGlnGln LeuLysThrTyrAla
65 70 75 80
AlaAspArgTyrThr LysGluLysSerSer IleValValIleGly LeuSerValHisThr
85 90 95 100
ThrProValGluMet ArgGluLysLeuAla IleProGluAlaGlu TrpProArgAlaIle
105 110 115 120
GlyGluLeuCysGly LeuAsnHisIleGlu GluAlaAlaValLeu SerThrCysAsnArg
125 130 135 140
MetGluIleTyrVal ValAlaLeuSerGln HisArgGlyValLys GluValThrGluTrp
145 150 155 160
MetSerArgThrSer GlyIleProValSer GluLeuCysGlnHis ArgPheLeuLeuTyr
165 170 175 180
AsnLysAspAlaThr GlnHisLeuPheGlu ValSerAlaGlyLeu AspSerLeuValLeu
185 190 195 200
GlyGluGlyGlnIle LeuAlaGlnValLys GlnValValLysVal GlyGlnGlyValAsn
205 210 215 220
GlyPheGlyArgAsn IleSerGlyLeuPhe LysHisAlaIleThr ValGlyLysArgVal
225 230 235 240
ArgAlaGluThrAsn IleAlaAlaGlyAla ValSerValSerSer AlaAlaValGluLeu
245 250 255 260
AlaLeuMetLysLeu ProGluThrSerHis GlyAsnAlaLysMet LeuValIleGlyAla
265 270 275 280
GlyLysMetGlyLys LeuValIleLysHis LeuValAlaLysGly CysThrLysMetVal
285 290 295 300
ValValAsnArgSer GluGluArgValAla GluIleArgGluGlu LeuLysAspValGlu
305 310 315 320
IleIleTyrLysPro LeuSerGluMetLeu AlaCysValGlyGlu AlaAspValValPhe
325 330 335 340
ThrGlyThrAlaSer GluAsnProLeuPhe LeuLysAspAspVal LysAspLeuProSer
345 350 355 360
ValSerGlnAspIle GlyGlyHisArgLeu PheIleAspIleSer ValProArgAsnVal
365 370 375 380
GlySerCysValSer AspIleGluSerVal ArgValTyrAsnVal AspAspLeuLysGlu
385 390 395 400
ValValAlaAlaAsn LysGluAspArgLeu ArgLysAlaMetGlu AlaGlnAlaIleIle
405 410 415 420
GlyGluGluSerGln GlnPheGluAlaTrp ArgAspSerLeuGlu ThrValProThrIle
425 430 435 440
LysLysLeuArgAla TyrAlaGluArgIle ArgAlaAlaGluLeu GluLysCysLeuGly
445 450 455 460
LysMetGlyAspAsp IleSerLysLysThr ArgArgAlaValAsp AspLeuSerArgGly
465 470 475 480
IleValAsnLysLeu LeuHisGlyProMet GlnHisLeuArgCys AspGlySerAspSer
485 490 495 500
ArgThrLeuThrGlu ThrLeuGluAsnMet HisAlaLeuAsnArg MetPheSerLeuGlu
505 510 515 520
ThrGluIleSerVal LeuGluGlnLysIle ArgAlaLysValGlu GlnAsnHisSerAsp
525 530 535 540
Ser
541
<210>5
<211>24
<212>DNA
<213>It is artificial synthesized
<400>5
TCTAGAATGG CTGTTTCGAC GAGC 24
<210>6
<211>24
<212>DNA
<213>It is artificial synthesized
<400>6
GGATCCTTAG CTGTCACTAT GGTT 24

Claims (5)

1. application of the peanut glutamy t-RNA reductases in regulation chlorophyll and calmodulin expression.
2. application according to claim 1, it is characterised in that peanut glutamy t-RNA reduces enzyme amino acid sequence such as sequence In table shown in sequence 4.
3. application according to claim 2, it is characterised in that the nucleotide sequence of expression peanut glutamy t-RNA reductases As shown in sequence 3 in sequence table.
4. application according to claim 1, it is characterised in that peanut glutamy t-RNA reductases are in regulation calmodulin expression In application show as, under condition of salt stress, overexpress peanut glutamy t-RNA reductase genes strain in calmodulin table It is consequently increased up to amount.
5. application according to claim 1, it is characterised in that peanut glutamy t-RNA reductases are in regulation chlorophyll and calcium The application in element expression is adjusted, is embodied in, under normal condition and after salt stress is handled, peanut glutamy t-RNA reduction After enzyme gene overexpression, the biosynthesis of chlorophyll also has increase.
CN201710721435.9A 2017-08-22 2017-08-22 Peanut glutamyl t-RNA reductase and application thereof Expired - Fee Related CN107384955B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0731480A (en) * 1993-07-27 1995-02-03 Cosmo Sogo Kenkyusho:Kk Dna fragment coding l-glutamyl-trna reductase
WO1998024920A2 (en) * 1996-12-04 1998-06-11 Institut für Pflanzengenetik und Kulturpflanzenforschung How to affect the chlorophyll biosynthesis in plants
CN104004701A (en) * 2014-06-18 2014-08-27 江南大学 Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains
CN104830748A (en) * 2015-06-02 2015-08-12 江南大学 Method for weakening hemB gene expression to increase yield of 5-aminolevulinic acid synthesized by escherichia coli

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0731480A (en) * 1993-07-27 1995-02-03 Cosmo Sogo Kenkyusho:Kk Dna fragment coding l-glutamyl-trna reductase
WO1998024920A2 (en) * 1996-12-04 1998-06-11 Institut für Pflanzengenetik und Kulturpflanzenforschung How to affect the chlorophyll biosynthesis in plants
CN104004701A (en) * 2014-06-18 2014-08-27 江南大学 Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains
CN104830748A (en) * 2015-06-02 2015-08-12 江南大学 Method for weakening hemB gene expression to increase yield of 5-aminolevulinic acid synthesized by escherichia coli

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BERNHARD GRIMM: "Novel insights in the control of tetrapyrrole metabolism of higher plants", 《CURRENT OPINION IN PLANT BIOLOGY》 *
NONE: "glutamyl-tRNA reductase 1, chloroplastic [Arachis ipaensis]", 《NCBI REFERENCE SEQUENCE: XP_016166511.1》 *
李爽等: "谷氨酰t-RNA还原酶基因( hemA)的高效表达", 《中国生物工程杂志》 *
束胜等: "植物生长调节物质提高蔬菜作物抗逆性的研究进展", 《长江蔬菜》 *
王平荣等: "高等植物叶绿素生物合成的研究进展", 《西北植物学报》 *

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