CN107384955B - Peanut glutamyl t-RNA reductase and application thereof - Google Patents
Peanut glutamyl t-RNA reductase and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a peanut glutamyl t-RNA reductase and application of the peanut glutamyl t-RNA reductase in regulation of chlorophyll and calmodulin expression. The invention has the beneficial effects that: the invention identifies the function of the glutamyl t-RNA reductase in the peanuts, has the function of regulating the expression of chlorophyll and calmodulin, defines the action relation between the enzyme and calcium signals, and lays a theoretical foundation for later-stage research.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a peanut glutamyl t-RNA reductase and application of the peanut glutamyl t-RNA reductase.
Background
Chlorophyll (Chl), a fat-soluble pigment, is present in chloroplasts in higher plants. When plants are subjected to photosynthesis, chlorophyll plays an extremely important role in absorbing and converting solar energy, and in the process, water molecules are decomposed to generate oxygen molecules and reducing oxygen, so that the chlorophyll plays an important role in a cell electron transfer chain, and thus the energy conversion process is started, and the chlorophyll plays an important role in the growth and development of plants and the formation of the quality and yield of crops. The starting material of chlorophyll biosynthesis is L-glutamyl-tRNA, and L-glutamyl-t RNA is reduced into L-glutamate-1-semialdehyde through the catalytic action of GluTR; then, the alpha-aminolevulinic acid (ALA) is formed under the catalytic action of glutamate-1-semialdehyde transaminase, and the ALA is a key precursor substance of chlorophyll biosynthesis. GluTR is the central controller of chlorophyll biosynthesis, which is encoded by the glutamyl t-RNA reductase (HEMA) gene, and HEMA is therefore a key enzyme gene regulating the chlorophyll synthesis pathway. There may be some variation in the number of HEMA members from plant to plant. In the model plant Arabidopsis thalianaHEMAHas 3 members, each isHEMA1、HEMA2AndHEMA3and only among cucumbersHEMA1AndHEMA2two genes.
But the peanutHEMAThe gene has not been reported at home and abroad, and the regulation function of the gene in the physiological processes of plant adversity and aging is not clear.
Disclosure of Invention
In order to solve the problems of peanuts in the prior artHEMAThe application discloses a peanut glutamyl t-RNA reductase, which solves the problem of blank research on the regulation effect of genes in the physiological processes of plant stress and aging.
The invention also provides application of the peanut glutamyl t-RNA reductase.
The invention is obtained by the following steps:
the application of the peanut glutamyl t-RNA reductase in the regulation of the expression of chlorophyll and calmodulin.
The application is preferably that the amino acid sequence of the peanut glutamyl t-RNA reductase is shown as a sequence 4 in a sequence table.
The application preferably expresses the nucleotide sequence of the peanut glutamyl t-RNA reductase as shown in a sequence 3 in a sequence table.
The application, preferably the application of the peanut glutamyl t-RNA reductase in the regulation of the expression of the calmodulin shows that the expression amount of the calmodulin in a strain which overexpresses the peanut glutamyl t-RNA reductase gene is increased under the condition of salt stress.
The application is preferably the application of the peanut glutamyl t-RNA reductase in the regulation of the expression of chlorophyll and calmodulin, and particularly shows that under normal conditions and after salt stress treatment, the biosynthesis of chlorophyll is also increased after the overexpression of the peanut glutamyl t-RNA reductase gene.
The calcium is one of the essential nutrient elements for plant growth and development, it participates in the whole process from germination to growth, differentiation, flowering and fruiting of plants, and the main function of calcium ion in the plant body is: (1) nutrients, promote the formation of microtubules. (2) Structural organization of cell walls and fluidity of membranes. (3) Alleviating the toxic effects of salt stress. (4) Promoting the growth and elongation of the pollen tube. Peanuts are crops with more calcium, and the quantity of the calcium required by the peanuts is second to that of nitrogen, higher than that of phosphorus and equivalent to that of potassium. When the peanuts are lack of calcium, the growth of the seeds is hindered, the shell tissues are loose, empty fruits, blighted fruits and rotten fruits are increased, and the yield is obviously reduced. Study of Ca2+The regulation mechanism of peanut stress physiology and the relation between the regulation mechanism and the chlorophyll synthesis process are of great significance in determining the utilization rule of calcium for peanut growth and development and ensuring high peanut yield.
The invention has the beneficial effects that: the invention identifies the function of the glutamyl t-RNA reductase in the peanuts, has the function of regulating the expression of chlorophyll and calmodulin, defines the action relation between the enzyme and calcium signals, and lays a theoretical foundation for later-stage research.
Drawings
FIG. 1 is a drawing ofAhhemAAs a result of the gene cloning,
FIG. 2 isAhhemAThe hydrophobicity analysis of the protein coded by the gene,
FIG. 3 is a drawing showingAhhemAAnalyzing the transmembrane characteristics of the gene coding protein,
FIG. 4 shows AhhemA expression in different organs; f: flower, R is root; s: a stem; l: a blade; fr is the fruit of the plant,
FIG. 5 shows the measurement of AhhEMA gene expression level and chlorophyll content after exogenous calcium application under salt stress condition,
FIG. 6 shows downstream gene expression level after exogenous ALA application under salt stress,
FIG. 7 shows the PCR identification of AhhEMA transgenic plants,
FIG. 8 shows the measurement of chlorophyll content,
FIG. 9 shows CaM gene expression.
Detailed Description
The invention is further illustrated by the following specific examples:
example 1
1.AhhemAIsolation of genes
RNA (purchased from Tiangen Biochemical technology Ltd.) was extracted from peanut leaves using an RNA kit and reverse-transcribed. Designing a primer according to the gene sequence, wherein the primer sequence is as follows: 5'-ATGGCTGTTTCGACGAGC-3' and 5'-TTAGCTGTCACTATGGTT-3' were subjected to Polymerase Chain Reaction (PCR), and the PCR products were subjected to agarose gel electrophoresis to detect that the amplified fragment coincided with the estimated fragment size (1626 bp) (FIG. 1).
The obtained PCR product was ligated with pMD18-T cloning vector to transform E.coli. And (3) rapidly screening by colony PCR to obtain positive clones, extracting plasmids of the positive clones, and cutting the plasmids by utilizing enzyme cutting sites in the vector to obtain strips with the same size as the PCR product. And (3) sending the identified bacterial liquid to Shanghai bioengineering company for sequencing, wherein the sequence is shown as a sequence 3 in a sequence table, and comparing the sequence of the obtained fragment with a known sequence by using Blast, DNAman or DNAclub software to find that the fragment is the target gene to be cloned.
2.AhhemASequence analysis of genes
(a) sequence characterization
1626 base pairs in length
Nucleic acid of type
Double strand
Topology of linear
(b) Molecular type cDNA
(c) Suppose that
(d) Antisense NO
(e) Peanut as the primary source
Sequence 4 in the sequence table:
(a) sequence characterization
Length: 541 amino acid
Type: amino acids
Chain type: single strand
Topology structure: linearity
(b) Molecular type: protein
3.AhhemABiochemical characterization of Gene-encoded proteins
3.1AhhemAHydrophobicity analysis of Gene-encoded proteins
The hydrophobicity of the protein coded by the peanut glutamyl t-RNA reductase gene is analyzed, and the result shows that the proportion of hydrophilic amino acid and hydrophobic amino acid is not greatly different (figure 2). The proportion of hydrophilic amino acid is slightly higher, which indicates that the protein is water-soluble protein.
3.2AhhemAAnalysis of transmembrane characteristics of Gene expression products
By submitting the amino acid sequence of AhhEMA to an online PRED-TMR database on the Internet, no transmembrane signal region was predicted in the structure of the enzyme protein (FIG. 3).
4.AhhemAAnalysis of Gene expression in peanut plants
4.1AhhemAExpression in different organs of peanut
To understandAhhemAEndogenous expression of genes in different organs, we show flowersRaw materialAhhemAA section of cDNA at the 3' end of the gene is used as a primer for carrying out fluorescent quantitative PCR analysis. The results show that the method has the advantages of high yield,AhhemAthe gene is expressed in all organs and is constitutive expression, the expression in leaves is obviously higher than that in other organs, and the expression level in roots is the lowest, which indicates thatAhhemAThe gene was expressed in a high level in tissues with high chlorophyll content (FIG. 4).
4.2 peanut plants grown under Normal conditions for three weeks, treated with 200mM NaCl for 3 days, treated with exogenous calcium, selected from 0, 10, 20, 40, 80mM calcium (denoted as CK, C10, C20, C40, C80, Normal as no salt control), sampled, and subjected to fluorescent quantitative PCR detectionAhhemAExpression, and chlorophyll content was also determined, see fig. 5. The results show that exogenous calcium application to the peanuts under the condition of salt stresshemAThe gene expression is down-regulated, however, the chlorophyll content is not affected after calcium application and is in an ascending trend, indicating that the action site of the calcium signal in the chlorophyll synthesis pathway is probably at the downstream of hemA.
Under the condition of salt stress, after ALA synthesized by AhhEMA catalysis is sprayed exogenously, the expression of catalytic subunits ChlH and ChlD genes of the magnesium chelatase active center is up-regulated, and the expression amount reaches the peak value after 10mg/l ALA treatment, as shown in figure 6.
5.AhhemAConstruction of Gene plant expression vector
5.1 amplificationAhhemAGene
The peanut variety 'Huayu 22' is provided by the academy of agricultural sciences of Shandong province and is amplified by RT-PCRAhhemAThe coding region of the gene.
hemA-F(5'-TCTAGAATGGCTGTTTCGACGAGC-3')(XbaI) (ii) a A sequence 5 in a sequence table, wherein,
hemA-R(5'-GGATCCTTAGCTGTCACTATGGTT-3')(BamHI) (ii) a A sequence 6 in a sequence table, wherein,
the above primer sequences are respectivelyAhhemAThe 1 st-18 th base and the 1612-1626 th base of the gene correspond to each other.
5.2AhhemAConnection of gene, cloning vector pMD18-TSimple and plant expression vector pBI121
When constructing eukaryotic expression vector, PCR resultThe gene is connected with a cloning vector pMD18-TSimple (purchased from TaKaRa) in full length, the ligation product is transformed into Escherichia coli Trans5 α to obtain a colony resistant to kanamycin, the screened positive clone is enriched with bacteria liquid, then the plasmid is extracted, the plasmid is subjected to double enzyme digestion with XbaI and BamHI, and the plasmid is subjected to double enzyme digestion and contains XbaI and BamHIAhhemARecovering the full-length band; the pBI121 vector was then digested with the same enzyme, and the resulting vector fragment was recovered. The full-length sequence and the vector sequence are provided with complementary cohesive ends, and are connected to transform escherichia coli, recombinants are screened out through enzyme digestion identification, and an expression vector pBI121-AhhemA。
5.3 preparation and transformation of Agrobacterium tumefaciens LBA4404 competent cells
The preparation steps of the competent cells are as follows:
(1) picking a little agrobacterium LBA4404, inoculating in 5mLLB liquid culture medium (containing 50 mg/LSTR), culturing overnight at 28 ℃ and 200 rpm;
(2) 2mL of the culture was taken and cultured in LB liquid medium (containing 50 mg/LSTR) until OD800 was about 0.5.
(3) Placing the culture in ice bath for 30min, centrifuging at 4 deg.C and 5000rpm for 5min, and removing supernatant;
(4) 10mL of cold 0.1mol/LNaCl suspension was used;
(5) centrifuging at 4 deg.C and 5000rpm for 5min, and removing supernatant;
(6) with 1mL of cold CaCl2(20 mmol/L) were suspended, split-packed into 50. mu.L/tube, frozen in liquid nitrogen and stored at-80 ℃.
And (3) agrobacterium transformation by a freeze-thaw method:
(1) thawing the competent cells in an ice bath;
(2) adding 2 μ L plasmid DNA into a 1.5mL LEppendorf centrifuge tube, adding 50 μ L melted competent cells, mixing, ice-cooling for 30min, freezing in liquid nitrogen for 1min, and standing in 37 deg.C water bath for 5 min;
(3) adding 950mL of LB liquid culture medium without antibiotics, and oscillating at 28 ℃ and 200rpm for 3 h;
(4) centrifuging at 8000rpm for 1min, removing supernatant, and dissolving thallus with 100 μ LLB;
(5) applying 50 μ L of the bacterial liquid to LB solid medium (containing 50mg/LKan, 100 mg/LRif), and culturing at 28 deg.C for 2-3 d.
6. Agrobacterium-mediated transformation of tobacco
6.1 Agrobacterium culture:
single colonies were picked and cultured in 5mLLB liquid medium (containing 100mg/LRif and 50 mg/LKAn) for 36h (28 ℃, 200 rpm), 1% of the colonies were inoculated into the same medium, cultured at 28 ℃, 200rpm for 12h, 2mL of the bacterial solution was centrifuged (4 ℃, 4000rpm, 10 min), and the cells were suspended in 20mLMS liquid medium for transformation.
6.2 establishment of tobacco transformation and regeneration system:
(1) accelerating germination of tobacco Nc89 seeds, sowing the seeds in a plastic tray, and performing conventional management till the 2-3 true leaf stage for later use;
(2) sterilizing tobacco leaf with 70% ethanol for 30s, and adding 0.1% HgCl2Sterilizing for 8-10min, washing with sterile water for several times, and cutting into small pieces (0.5 x 0.5 cm)2）；
(3) Placing the cut tobacco leaves in an MS differentiation culture medium, pre-culturing for 2 days at 28 ℃, with the illumination time of 16h/d and the illumination intensity of 2000 LX;
(4) immersing the pre-cultured tobacco leaves into the bacterial liquid for 5-10min, then sucking the redundant bacterial liquid by using sterilized filter paper, and inoculating the residual bacterial liquid into an MS culture medium; dark culture at 28 ℃ for 2 days;
(5) washing the co-cultured explant with sterile water containing 250mg/L of cephalosporin for 3 times, then blotting the explant with sterile filter paper, transferring the explant to a differentiation medium containing 100mg/L of kanamycin and 250mg/L of cephalosporin, and culturing at constant temperature (the conditions are the same as those of pre-culture); changing the culture medium every 15 days;
(6) when the bud grows to about 1cm, the bud is cut off and transferred into an MS rooting medium (50 mg/L kanamycin and 250mg/L cephalosporin) to promote the rooting. After the root system is developed, transferring the root system into a flowerpot filled with sterile soil, moisturizing the root system for 2 days by using a plastic film, and performing conventional greenhouse management.
7. PCR detection of transgenic tobacco plants
7.1 extraction of genomic DNA by CTAB microassay
(1) Taking 0.1-0.2g of fresh leaves, putting the fresh leaves into liquid nitrogen, grinding the fresh leaves into powder, transferring the powder into a 1.5mL centrifuge tube, adding 300 mu L of 2 xCTAB extraction buffer solution, slightly stirring the solution to fully disperse the powder, and preserving the heat in a water bath at 65 ℃ for 20 min;
(2) subsequently, centrifugation was carried out at 10000rpm for 10min at 4 ℃;
(3) transferring the supernatant into a new centrifuge tube, adding 200 μ L chloroform/isoamylol (24: 1), mixing, and standing for 5 min;
(4) centrifuging at 8000rpm for 10min at 4 deg.C;
(5) transferring the supernatant into another centrifuge tube, adding 500 μ L cold isopropanol, mixing, and standing at 4 deg.C for 10 min;
(6) centrifuging at 4 deg.C and 8000rpm for 10min, removing supernatant, and placing the centrifuge tube on absorbent paper;
(7) washing the precipitate with 75% ethanol, and drying in an ultra-clean bench for 20 min;
(8) add 50. mu.LTE to dissolve the DNA and store at-20 ℃ for further use.
2 XCTAB extraction buffer (100 mL) 1M Tris Cl10mL, 0.5M DTA4mL, NaCl8.182g, CTAB2.0g, and PVP3.0g were dissolved in distilled water, and then dissolved in 100mL, sterilized, and 200. mu.L of β -mercaptoethanol (β -mercaptoethanol, β -ME) was added thereto for use.
7.2 PCR detection of transgenic plants
Extracting genome DNA of regenerated plant, using said vector sequence andAhhemAdesigning primers for gene sequence to carry out PCR amplification. The PCR reaction program is: 95 deg.C for 5 min; 95 deg.C, 50s, 55 deg.C, 50s, 72 deg.C, 1min30s, 30 cycles; 72 deg.C, 10 min. The PCR identification of the transgenic plants is shown in figure 7, and the positive rate reaches 76.4%.
8. Rotating shaftAhhemAFunctional identification of gene plants
8.1 chlorophyll content determination. Strains OE2, OE5 and OE8 with high expression level are screened from the transgenic tobacco to measure the chlorophyll content. The result shows that the chlorophyll content in the transgenic plant is obviously higher than that of the wild plant under normal conditionsA type plant; after salt stress treatment, the chlorophyll content is reduced, but the chlorophyll content in the transgenic line is still higher than that of the wild plant. Show thatAhhemAThe biosynthesis of chlorophyll is increased to a certain extent after the gene is over-expressed, which is beneficial to improving the stress resistance of plants. See fig. 8.
8.2 revolutionsAhhemAExpression level of calmodulin gene in gene plant. The expression of the calmodulin gene was detected by fluorescent quantitative PCR after the control wild type plant and the transgenic plant were treated with 200mM NaCl for 3 days. FIG. 9 shows that under salt stress conditions, overexpression is observedAhhemAThe expression level of calmodulin in the gene strain is increased, and further proves that the action site of calcium signal in chlorophyll synthesis pathway is downstream of hemA.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
<110> center for researching biotechnology of academy of agricultural sciences of Shandong province
<120> peanut glutamyl t-RNA reductase and application thereof
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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
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Met Ala Val Ser Thr Ser Phe Ser Gly Ala Lys Leu Glu Ala Leu Phe Leu Lys Cys Cys
5 10 15
Ser Ser Ser Ser Ala Asn Ala Ala Tyr Ser Leu Cys Val Pro Ser Lys Thr Ala Ala Lys
25 30 35 40
Ala Thr Arg Thr Thr Pro Phe Arg Arg Gly Leu Val Arg Cys Asp Ala Ser Ala Ser Pro
45 50 55 60
Asp Val Ile Leu Asp Asn Ala Ala Ala Val Ser Ala Leu Gln Gln Leu Lys Thr Tyr Ala
65 70 75 80
Ala Asp Arg Tyr Thr Lys Glu Lys Ser Ser Ile Val Val Ile Gly Leu Ser Val His Thr
85 90 95 100
Thr Pro Val Glu Met Arg Glu Lys Leu Ala Ile Pro Glu Ala Glu Trp Pro Arg Ala Ile
105 110 115 120
Gly Glu Leu Cys Gly Leu Asn His Ile Glu Glu Ala Ala Val Leu Ser Thr Cys Asn Arg
125 130 135 140
Met Glu Ile Tyr Val Val Ala Leu Ser Gln His Arg Gly Val Lys Glu Val Thr Glu Trp
145 150 155 160
Met Ser Arg Thr Ser Gly Ile Pro Val Ser Glu Leu Cys Gln His Arg Phe Leu Leu Tyr
165 170 175 180
Asn Lys Asp Ala Thr Gln His Leu Phe Glu Val Ser Ala Gly Leu Asp Ser Leu Val Leu
185 190 195 200
Gly Glu Gly Gln Ile Leu Ala Gln Val Lys Gln Val Val Lys Val Gly Gln Gly Val Asn
205 210 215 220
Gly Phe Gly Arg Asn Ile Ser Gly Leu Phe Lys His Ala Ile Thr Val Gly Lys Arg Val
225 230 235 240
Arg Ala Glu Thr Asn Ile Ala Ala Gly Ala Val Ser Val Ser Ser Ala Ala Val Glu Leu
245250 255 260
Ala Leu Met Lys Leu Pro Glu Thr Ser His Gly Asn Ala Lys Met Leu Val Ile Gly Ala
265 270 275 280
Gly Lys Met Gly Lys Leu Val Ile Lys His Leu Val Ala Lys Gly Cys Thr Lys Met Val
285 290 295 300
Val Val Asn Arg Ser Glu Glu Arg Val Ala Glu Ile Arg Glu Glu Leu Lys Asp Val Glu
305 310 315 320
Ile Ile Tyr Lys Pro Leu Ser Glu Met Leu Ala Cys Val Gly Glu Ala Asp Val Val Phe
325 330 335 340
Thr Gly Thr Ala Ser Glu Asn Pro Leu Phe Leu Lys Asp Asp Val Lys Asp Leu Pro Ser
345 350 355 360
Val Ser Gln Asp Ile Gly Gly His Arg Leu Phe Ile Asp Ile Ser Val Pro Arg Asn Val
365 370 375 380
Gly Ser Cys Val Ser Asp Ile Glu Ser Val Arg Val Tyr Asn Val Asp Asp Leu Lys Glu
385 390 395 400
Val Val Ala Ala Asn Lys Glu Asp Arg Leu Arg Lys Ala Met Glu Ala Gln Ala Ile Ile
405 410 415 420
Gly Glu Glu Ser Gln Gln Phe Glu Ala Trp Arg Asp Ser Leu Glu Thr Val Pro Thr Ile
425 430 435 440
Lys Lys Leu Arg Ala Tyr Ala Glu Arg Ile Arg Ala Ala Glu Leu Glu Lys Cys Leu Gly
445 450 455 460
Lys Met Gly Asp Asp Ile Ser Lys Lys Thr Arg Arg Ala Val Asp Asp Leu Ser Arg Gly
465 470 475 480
Ile Val Asn Lys Leu Leu His Gly Pro Met Gln His Leu Arg Cys Asp Gly Ser Asp Ser
485 490 495 500
Arg Thr Leu Thr Glu Thr Leu Glu Asn Met His Ala Leu Asn Arg Met Phe Ser Leu Glu
505 510 515 520
Thr Glu Ile Ser Val Leu Glu Gln Lys Ile Arg Ala Lys Val Glu Gln Asn His Ser Asp
525 530 535 540
Ser
541
<210>5
<211>24
<212>DNA
<213> Artificial Synthesis
<400>5
TCTAGAATGG CTGTTTCGAC GAGC 24
<210>6
<211>24
<212>DNA
<213> Artificial Synthesis
<400>6
GGATCCTTAG CTGTCACTAT GGTT 24
Claims (3)
1. The application of the peanut glutamyl t-RNA reductase in regulating the expression of calmodulin is characterized in that the amino acid sequence of the peanut glutamyl t-RNA reductase is shown as a sequence 4 in a sequence table.
2. The use according to claim 1, characterized in that the nucleotide sequence for expressing the peanut glutamyl t-RNA reductase is shown as sequence 3 in the sequence table.
3. The use according to claim 1, wherein the peanut glutamyl t-RNA reductase is used for regulating the expression of calmodulin, wherein the expression level of calmodulin in the strain overexpressing the peanut glutamyl t-RNA reductase gene is increased under the condition of salt stress.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710721435.9A CN107384955B (en) | 2017-08-22 | 2017-08-22 | Peanut glutamyl t-RNA reductase and application thereof |
| AU2018100546A AU2018100546A4 (en) | 2017-08-22 | 2018-04-26 | Peanut glutamoyl t-RNA reductase and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710721435.9A CN107384955B (en) | 2017-08-22 | 2017-08-22 | Peanut glutamyl t-RNA reductase and application thereof |
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| CN107384955A CN107384955A (en) | 2017-11-24 |
| CN107384955B true CN107384955B (en) | 2020-06-09 |
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| AU (1) | AU2018100546A4 (en) |
Citations (4)
| 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 |
-
2017
- 2017-08-22 CN CN201710721435.9A patent/CN107384955B/en not_active Expired - Fee Related
-
2018
- 2018-04-26 AU AU2018100546A patent/AU2018100546A4/en not_active Ceased
Patent Citations (4)
| 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 |
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| Title |
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| glutamyl-tRNA reductase 1, chloroplastic [Arachis ipaensis];NONE;《NCBI Reference Sequence: XP_016166511.1》;20170513;氨基酸序列，CDS * |
| Novel insights in the control of tetrapyrrole metabolism of higher plants;Bernhard Grimm;《Current Opinion in Plant Biology》;19981231;245-250 * |
| 植物生长调节物质提高蔬菜作物抗逆性的研究进展;束胜等;《长江蔬菜》;20131231;第16卷;1-12 * |
| 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》.2009,第29卷(第3期),摘要，第631页右栏第2.1节第二段. * |
| 谷氨酰t-RNA还原酶基因( hemA)的高效表达;李爽等;《中国生物工程杂志》;20071231;第27卷(第6期);82-86 * |
| 高等植物叶绿素生物合成的研究进展;王平荣等;《西北植物学报》;20091231;第29卷(第3期);摘要，第631页右栏第2.1节第二段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107384955A (en) | 2017-11-24 |
| AU2018100546A4 (en) | 2018-05-24 |
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