CN110295176A - The polypeptide of poplar PsnERF1 gene cDNA and its coding - Google Patents
The polypeptide of poplar PsnERF1 gene cDNA and its coding Download PDFInfo
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- CN110295176A CN110295176A CN201910667210.9A CN201910667210A CN110295176A CN 110295176 A CN110295176 A CN 110295176A CN 201910667210 A CN201910667210 A CN 201910667210A CN 110295176 A CN110295176 A CN 110295176A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- 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
- C12N15/8273—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 for drought, cold, salt resistance
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Abstract
The cDNA of poplar PsnERF1 gene and its polypeptide of coding, it is related to the cDNA of poplar PsnERF1 gene and its polypeptide of coding.The cDNA of poplar PsnERF1 gene, specific nucleotide sequence is as shown in SEQ ID NO:1.The polypeptide of the cDNA sequence coding of poplar PsnERF1 gene, amino acid sequence include 445 amino acid, and the amino acid sequence of poplar PsnERF1 gene coding is as shown in SEQ ID NO:2.The present invention provides clone's xylophyta poplar PsnERF1 genes, it is transferred to overexpression in poplar, transgenic poplar salt resistance ability can be improved, transgenic salt-tolerant wheat ability improves 10% or more than control, and plant height and fresh weight are obviously higher than compareing under stress conditions.
Description
Technical field
The present invention relates to the cDNA of poplar PsnERF1 gene and its polypeptides of coding.
Background technique
The growth and development for seriously affecting plant such as saline and alkaline in natural environment, arid.The agricultural land in the whole world 45% by
The Irrigation farming land used of the influence of arid in various degree, the whole world 19.5% belongs to wetland with saline-alkaline, it is contemplated that will have by 2028
30% saline Land, the year two thousand fifty there will be over the influence of 50% soil by salinization of soil.Chinese soil arid and soil
Salinization of soil is got worse, and Chinese saline alkali land area reaches whole world saline alkali land area 10%, cultivates drought resisting salt-resistant plant new product
Kind is the important channel for making full use of salt-soda soil and improving the salinization of soil.
Summary of the invention
The invention aims to solve the problems, such as that poplar salt resistance ability is low, and provide the cDNA of poplar PsnERF1 gene
And its polypeptide of coding.
The cDNA of poplar PsnERF1 gene of the present invention includes 5 ' end non-translational region (1-199), protein coding region
(200-1537) and 3 ' ends non-translational region (1538-1584), specific nucleotide sequence is as shown in SEQ ID NO:1.
The polypeptide of the cDNA sequence coding of poplar PsnERF1 gene of the present invention, amino acid sequence include 445 amino
Acid, the amino acid sequence of poplar PsnERF1 gene coding is as shown in SEQ ID NO:2.
The present invention provides clone's xylophyta poplar PsnERF1 genes, are transferred to overexpression in poplar,
Can be improved transgenic poplar salt resistance ability, transgenic salt-tolerant wheat ability improves 10% or more than control, under stress conditions plant height and
Fresh weight is obviously higher than compareing.
Detailed description of the invention
Fig. 1 is the electrophoretogram for carrying out DNA level detection in the present invention to transgenic poplar using PCR, wherein M:
DL2000；P: positive plasmid；W: wild type poplar；T1-T8: transgenic poplar；
Fig. 2 is the electrophoretogram for carrying out rna level detection in the present invention to transgenic poplar using RT-PCR, wherein M:
DL2000；P: positive plasmid；W: wild type poplar；T1-T8: transgenic poplar.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: the cDNA of present embodiment poplar PsnERF1 gene, includes 5 ' end non-translational region (1-
199), protein coding region (200-1537) and 3 ' ends non-translational region (1538-1584), specific nucleotide sequence such as SEQ ID
Shown in NO:1.
Specific embodiment 2: the polypeptide that the cDNA sequence of present embodiment poplar PsnERF1 gene encodes, amino acid
Sequence includes 445 amino acid, and the amino acid sequence of poplar PsnERF1 gene coding is as shown in SEQ ID NO:2.
Poplar PsnERF1 gene obtains in accordance with the following methods:
1. extracting the total serum IgE of Hybrid poplar (P.simonii × P.nigra) using RNA extracts kit, RNA PCR is utilized
Kit (AMV) Ver.3.0 kit by reverse transcription at cDNA；
2. according to poplar genome sequence design primer,
Primers F 1:5 '-ATGATGCAGAAAGATTTTAGCACTG-3 '；
Primer R1:5 '-TTAACCAGTGGAGGAAGGACGGCG-3 '；
Using poplar cDNA as template, overall length poplar PsnERF1 gene cDNA sequence is obtained with PCR；
3. the cDNA of above-mentioned gained poplar PsnERF1 gene is building up to plant expression vector pBI121 plant expression vector
In, poplar genome is transferred to using agrobacterium-mediated transformation；The result shows that the transgenic poplar strain under condition of salt stress
Plant height and fresh weight it is higher than non-transgenic strain by 10% or more, difference reaches extremely significant level；
Involved in the above process: 4. reverse transcription reaction, 5. pcr amplification reaction and the 6. plant expression vector structure of cDNA synthesis
It is as follows to build process:
4. cDNA reverse transcription reaction
A, inverse transcription reaction liquid forms:
| Ingredient | Dosage (μ l) |
| MgCl2(25mM) | 2 |
| 10xRT Buffer | 1 |
| RNase-free ddH2O | 3.75 |
| dNTP(10mM each) | 1 |
| RNase Inhibitor | 0.25 |
| AMV Reverse Transcriptase | 0.5 |
| Random 9mers | 0.5 |
| Total RNA | 1 |
| Total | 10 |
B, reverse transcription reaction program: 30 DEG C, 10min；42 DEG C, 30min；95 DEG C, 5min；5 DEG C, 5min.
5. pcr amplification reaction
A, PCR reaction solution forms:
B, PCR response procedures: 94 DEG C, 30s；58 DEG C, 30s；72 DEG C, 1min 30s, 35 wheel circulations；
C, it after reaction, takes 5 μ l PCR reaction solutions to carry out agarose gel electrophoresis, confirms band length；
6. plant expression vector construction process is as follows:
Using specific primer F2 and R2 carry out PCR amplification, ERF76 genetic fragment 5 ' and 3 ' hold import XbaI with
SacI restriction enzyme site；
F2:5 '-GCGTCTAGAATGATGCAGAAAGATTTTAGC-3 '；
R2:5 '-GCGGAGCTCTTAACCAGTGGAGGAAGGACG-3 '.
A, PCR reaction solution forms:
B, PCR program is as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 62 DEG C of annealing 30s, 72 DEG C of extension 1min
30s, 30 wheel circulations；72 DEG C of extension 7min, 4 DEG C of preservations；
C, pcr amplification product is passed through into Ago-Gel recovery purifying；
D, according to the form below prepares the double enzyme digestion reaction liquid of pBI121 plasmid and ERF76 genetic fragment, in 37 DEG C of reaction 6h, to enzyme
PBI121 plasmid and target fragment after cutting carry out Ago-Gel recycling；
Endonuclease reaction liquid:
| Ingredient | Dosage (μ l) |
| XbaI | 2 |
| SacI | 2 |
| 10×M Buffer | 4 |
| CDNA or pBI121 carrier | ≤2μg |
| ddH2O to final volume | 40 |
E, using T4DNA ligase by after purification pBI121 digestion products and cDNA digestion products be attached, junctor
It is 10 μ l, comprising 1 μ l of carrier DNA 100ng, cDNA segment 20ng, 10x ligase buffer solution, T4 ligase 1u, 15 DEG C overnight.
Connection product is converted into competent escherichia coli cell JM109.Bacterium solution PCR detection is carried out with specific primer, and will mirror
The positive bacterium solution made sends to sequencing；
Above-mentioned poplar PsnERF1 gene cDNA expresses system using the plant of Caulimovirus (CaMV) 35S promoter driving
System, as a result as follows:
Above-mentioned gained poplar PsnERF1 gene cDNA is building up in plant expression vector pBI121, is situated between using Agrobacterium
Inducing defecation by enema and suppository is transferred to poplar genome.DNA level detection (Fig. 1) is carried out to transgenic poplar using PCR and utilizes RT-PCR
Rna level detection (Fig. 2) is carried out to transgenic poplar, the results showed that external source PsnERF1 gene obtains in transgenic poplar
Correct expression.Wherein M:DL2000；P: positive plasmid；W: wild type poplar；T1-T8: transgenic poplar.
Sequence table
<110>Northeast Forestry University
<120>polypeptide of the cDNA of poplar PsnERF1 gene and its coding
<160> 6
<210> 1
<211> 1584
<212> DNA
<213>Hybrid poplar (P. simonii × P. nigra)
<220>
<221> CDS
<222> (200)...(1537)
<400> 1
atgatgcaga aagattttag cactgtttca atggcggcgg atcaaatttg aattctctcc 60
atatataacc ccctcccttc actataactt cagaaaaggt tctcctacaa tttgacaaaa 120
aacctaaaga aaaaaacccc aaaaaagaaa agaaaaaact tggcttgata tttgtaccct 180
gccttttcat tcaagttac 199
atg tgc gta ttg aag gtg gcg aac cca aga ggc aac tca ggg gag 244
Met Cys Val Leu Lys Val Ala Asn Pro Arg Gly Asn Ser Gly Glu
1 5 10 15
tac agc agg tac cct tac aca gat agc gat gat aac caa gaa gag 289
Tyr Ser Arg Tyr Pro Tyr Thr Asp Ser Asp Asp Asn Gln Glu Glu
20 25 30
aga tcg tac aca gca caa caa ata ttt tct gaa ttg aat caa cca 334
Arg Ser Tyr Thr Ala Gln Gln Ile Phe Ser Glu Leu Asn Gln Pro
35 40 45
ata cag cgg cat gtg gag gta act caa tca cca cat atg ttc atg 379
Ile Gln Arg His Val Glu Val Thr Gln Ser Pro His Met Phe Met
50 55 60
ggg tat agc agt tca gca gag atg tct gca atg gta tca gca ctt 424
Gly Tyr Ser Ser Ser Ala Glu Met Ser Ala Met Val Ser Ala Leu
65 70 75
aca cat gtg gtc tca ggc cac aga gga agt act agc gat tgg gga 469
Thr His Val Val Ser Gly His Arg Gly Ser Thr Ser Asp Trp Gly
80 85 90
tca tat ggg gcc tca ggt cta gga ggt gca aca ata acg tcg act 514
Ser Tyr Gly Ala Ser Gly Leu Gly Gly Ala Thr Ile Thr Ser Thr
95 100 105
ttt gtt cag gca gca cct ggt tct aac act tct ccc gct tct cca 559
Phe Val Gln Ala Ala Pro Gly Ser Asn Thr Ser Pro Ala Ser Pro
110 115 120
tct ttg tct gca tat tct tct act tct ggt tct ggt tcc tgg att 604
Ser Leu Ser Ala Tyr Ser Ser Thr Ser Gly Ser Gly Ser Trp Ile
125 130 135
ggc cag aag aga ggg aga gca aag gaa gcc ggt gca gca gct caa 649
Gly Gln Lys Arg Gly Arg Ala Lys Glu Ala Gly Ala Ala Ala Gln
140 145 150
ttg aag gag tct ttg cca agg gtt cat aga ggg ttt gat gat ttt 694
Leu Lys Glu Ser Leu Pro Arg Val His Arg Gly Phe Asp Asp Phe
155 160 165
aga agt tca ctg ggc gat tcg tcg tca tct ggt gca act gca act 739
Arg Ser Ser Leu Gly Asp Ser Ser Ser Ser Gly Ala Thr Ala Thr
170 175 180
gaa gag gtg tct gct tca act ctg gta ttc tcc acc aca gca aca 784
Thr Ala Thr Glu Glu Val Ser Ala Ser Thr Leu Val Phe Ser Thr
185 190 195
cca tca agt gaa aca gca tct ttg gga gaa aca gga gag cga aaa 829
Pro Ser Ser Glu Thr Ala Ser Leu Gly Glu Thr Gly Glu Arg Lys
200 205 210
aga aga tac aga gga gtt agg caa agg cca tgg ggc aaa tgg gca 874
Arg Arg Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys Trp Ala
215 220 225
gca gag ata cgt gat cca cac aaa gca gca aga gtc tgg cta ggc 919
Ala Glu Ile Arg Asp Pro His Lys Ala Ala Arg Val Trp Leu Gly
230 235 240
aca ttt gaa act gcg gaa gct gca gct aga gcc tat gat gag gct 964
Thr Phe Glu Thr Ala Glu Ala Ala Ala Arg Ala Tyr Asp Glu Ala
245 250 255
gct tta aga ttc aga gga agt aga gct aaa ctt aac ttc cct gaa 1009
Arg Phe Arg Gly Ser Arg Ala Lys Leu Asn Phe Pro Glu Asn Ala
260 265 270
aat gct agg cta ctg cca gct caa atg caa aat gtt act gct tct 1054
Ala Leu Arg Leu Leu Pro Ala Gln Met Gln Asn Val Thr Ala Ser
275 280 285
caa gtt ccc gtt tct cgt tca caa tta cct tct cat cac cag tta 1099
Gln Val Val Ser Arg Ser Gln Leu Pro Ser His His Gln Leu Pro
290 295 300
cag cca atc tca tct cca agg caa caa gca ctg cgg ccg cag gtg 1144
Gln Pro Ile Ser Ser Pro Arg Gln Gln Ala Leu Arg Pro Gln Val
305 310 315
cca gca cct gca ttg ttt caa tct caa cct gat att ata aga gac 1189
Pro Ala Pro Ala Leu Phe Gln Ser Gln Pro Asp Ile Ile Arg Asp
320 325 330
tac tgg gag tac tcc cag ttg ttg cag agt tct ggg gac ttt cat 1234
Tyr Trp Glu Tyr Ser Gln Leu Leu Gln Ser Ser Gly Asp Phe His
335 340 345
gga caa caa cca ccg cct tca aat ttg tta gag cag atg ttt tat 1279
Gly Gln Gln Pro Pro Pro Ser Asn Leu Leu Glu Gln Met Phe Tyr
350 355 360
aat ccc cag ctg gcc tct cta caa tca tca gct ttg tct tca ttc 1324
Asn Pro Gln Leu Ala Ser Leu Gln Ser Ser Ala Leu Ser Ser Phe
365 370 375
tct tca tta cct tca ggt tct tca ttt gca gca ata cct tct ggt 1369
Ser Ser Leu Pro Ser Gly Ser Ser Phe Ala Ala Ile Pro Ser Gly
380 385 390
tcg ata tca tca aca cta tct cca tcc gct tct tca ttt cct ctg 1414
Ser Ile Ser Ser Thr Leu Ser Pro Ser Ala Ser Ser Phe Pro Leu
395 400 405
ctt ttt gct ggt caa cag ttg ggt tat ttt cgg cca cca gaa aat 1459
Leu Phe Ala Gly Gln Gln Leu Gly Tyr Phe Arg Pro Pro Glu Asn
410 415 420
cag aat cca gct gct agt tcc gat ttt ccg gtg tcc cca tgg aca 1504
Gln Asn Pro Ala Ala Ser Ser Asp Phe Pro Val Ser Pro Trp Thr
425 430 435
gat tct agt cgc cgt cct tcc tcc act ggt taa aat ctc tag agg 1549
Asp Ser Ser Arg Arg Pro Ser Ser Thr Gly
440 445
atc ccc ggg tac cga gct cga atc act gcc cac gc 1584
<210> 2
<211> 445
<212> PRT
<213>Hybrid poplar (P. simonii × P. nigra)
<400> 2
Met Cys Val Leu Lys Val Ala Asn Pro Arg Gly Asn Ser Gly Glu
5 10 15
Tyr Ser Arg Tyr Pro Tyr Thr Asp Ser Asp Asp Asn Gln Glu Glu
20 25 30
Arg Ser Tyr Thr Ala Gln Gln Ile Phe Ser Glu Leu Asn Gln Pro
35 40 45
Ile Gln Arg His Val Glu Val Thr Gln Ser Pro His Met Phe Met
50 55 60
Gly Tyr Ser Ser Ser Ala Glu Met Ser Ala Met Val Ser Ala Leu
65 70 75
Thr His Val Val Ser Gly His Arg Gly Ser Thr Ser Asp Trp Gly
80 85 90
Ser Tyr Gly Ala Ser Gly Leu Gly Gly Ala Thr Ile Thr Ser Thr
95 100 105
Phe Val Gln Ala Ala Pro Gly Ser Asn Thr Ser Pro Ala Ser Pro
110 115 120
Ser Leu Ser Ala Tyr Ser Ser Thr Ser Gly Ser Gly Ser Trp Ile
125 130 135
Gly Gln Lys Arg Gly Arg Ala Lys Glu Ala Gly Ala Ala Ala Gln
140 145 150
Leu Lys Glu Ser Leu Pro Arg Val His Arg Gly Phe Asp Asp Phe
155 160 165
Arg Ser Ser Leu Gly Asp Ser Ser Ser Ser Gly Ala Thr Ala Thr
170 175 180
Glu Glu Val Ser Ala Ser Thr Leu Val Phe Ser Thr Thr Ala Thr
185 190 195
Pro Ser Ser Glu Thr Ala Ser Leu Gly Glu Thr Gly Glu Arg Lys
200 205 210
Arg Arg Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys Trp Ala
215 220 225
Ala Glu Ile Arg Asp Pro His Lys Ala Ala Arg Val Trp Leu Gly
230 235 240
Thr Phe Glu Thr Ala Glu Ala Ala Ala Arg Ala Tyr Asp Glu Ala
245 250 255
Ala Leu Arg Phe Arg Gly Ser Arg Ala Lys Leu Asn Phe Pro Glu
260 265 270
Asn Ala Arg Leu Leu Pro Ala Gln Met Gln Asn Val Thr Ala Ser
275 280 285
Gln Val Pro Val Ser Arg Ser Gln Leu Pro Ser His His Gln Leu
290 295 300
Gln Pro Ile Ser Ser Pro Arg Gln Gln Ala Leu Arg Pro Gln Val
305 310 315
Phe Gln Ser Gln Pro Asp Ile Ile Arg Asp Tyr Trp Glu Tyr Ser
320 325 330
Gln Pro Ala Pro Ala Leu Leu Leu Gln Ser Ser Gly Asp Phe His
335 340 345
Gly Gln Gln Pro Pro Pro Ser Asn Leu Leu Glu Gln Met Phe Tyr
350 355 360
Asn Pro Gln Leu Ala Ser Leu Gln Ser Ser Ala Leu Ser Ser Phe
365 370 375
Ser Ser Leu Pro Ser Gly Ser Ser Phe Ala Ala Ile Pro Ser Gly
380 385 390
Ser Ile Ser Ser Thr Leu Ser Pro Ser Ala Ser Ser Phe Pro Leu
395 400 405
Leu Phe Ala Gly Gln Gln Leu Gly Tyr Phe Arg Pro Pro Glu Asn
410 415 420
Gln Asn Pro Ala Ala Ser Ser Asp Phe Pro Val Ser Pro Trp Thr
425 430 435
Asp Ser Ser Arg Arg Pro Ser Ser Thr Gly
440 445
<210> 3
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223>primers F 1
<400> 3
atgatgcaga aagattttag cactg 25
<210> 4
<211> 24
<212> DNA
<213>artificial sequence
<220>
<223>primer R1
<400> 4
ttaaccagtg gaggaaggac ggcg 24
<210> 5
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>primers F 2
<400> 5
gcgtctagaa tgatgcagaa agattttagc 30
<210> 6
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>primer R2
<400> 6
gcggagctct taaccagtgg aggaaggacg 30
Claims (2)
1. the cDNA of poplar PsnERF1 gene, it is characterised in that its nucleotide sequence is as shown in SEQ ID NO:1.
2. the polypeptide that the cDNA sequence of poplar PsnERF1 gene encodes, it is characterised in that the amino acid sequence of polypeptide includes 445
Amino acid, the amino acid sequence of poplar PsnERF1 gene coding is as shown in SEQ ID NO:2.
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|---|---|---|---|
| CN201910667210.9A CN110295176A (en) | 2019-07-23 | 2019-07-23 | The polypeptide of poplar PsnERF1 gene cDNA and its coding |
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| Publication Number | Publication Date |
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Application publication date: 20191001 |