CN115260299A - Ginseng PgWRKY2 transcription factor and application thereof - Google Patents
Ginseng PgWRKY2 transcription factor and application thereof Download PDFInfo
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- CN115260299A CN115260299A CN202210517926.2A CN202210517926A CN115260299A CN 115260299 A CN115260299 A CN 115260299A CN 202210517926 A CN202210517926 A CN 202210517926A CN 115260299 A CN115260299 A CN 115260299A
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- ginseng
- pgwrky2
- transcription factor
- phosphorus
- sequence
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Abstract
The invention relates to the field of biotechnology, in particular to a ginseng PgWRKY2 transcription factor and application thereof; the invention discloses a regulating effect of ginseng PgWRKY2 transcription factor on phosphorus nutrition, realizes detection of phosphorus nutrition stress in ginseng adversity stress by revealing expression specificity of key enzyme in ginseng phosphorus stress pathway, thereby providing effective regulating measures and having important application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a ginseng PgWRKY2 transcription factor and application thereof.
Background
Phosphorus (P) is a necessary macronutrient element for plants, participates in various metabolic processes in the plants and plays an important role in the growth and development of the plants. Plant adaptation to the phosphorus environment includes morphological physiological basis and gene expression regulation. Such as regulation of Phosphorus Transporters (PTs), secretion of amino acids and derivatives, flavonoids, phosphatases, and alterations of root structure. The metabolic pathway of phosphorus is regulated by a variety of genes, which are expressed differently under different phosphorus conditions. As in rice, osPHR2, a major transcriptional regulator of low phosphorus response, regulates adaptation to the phosphorus environment by activating the binding of the phosphorus starvation-induced gene PHT1 to the P1BS (PHR 1 binding sequence GNATATATNC) motif in the promoter region of the gene.
In addition, the molecular mechanism of plant response to phosphorus stress has also been studied in crops such as corn, soybean, ryegrass, barley and wheat. At present, in the aspect of researching plant phosphorus stress, the research is mainly concentrated in mode plants such as arabidopsis, rice, corn and the like and field crops, the research in medicinal plants is very little, the research in ginseng is not yet reported, meanwhile, the research in WRKY type transcription factors is also less, although the research reports that a plurality of WRKY type transcription factors in ginseng are induced and expressed by hormones such as salicylic acid, abscisic acid, na Cl and the like and salts, the research does not find that the WRKY type transcription factors specifically expressed by ginseng under the induction of phosphorus stress, and a marker and a technical method for detecting the phosphorus stress of ginseng from the gene expression level are not available.
Disclosure of Invention
Aiming at the defects in the prior art, the invention discloses the regulation and control effect of the ginseng PgWRKY2 transcription factor on phosphorus nutrition, and realizes the detection of the phosphorus nutrition stress in the ginseng adversity stress by disclosing the expression specificity of key enzymes in the ginseng phosphorus stress pathway, thereby providing effective regulation and control measures and having important application value.
In order to achieve the purpose, the invention provides the following technical scheme:
the nucleotide sequence of the ginseng PgWRKY2 transcription factor is shown in SEQ ID No. 1;
the coding protein sequence of the ginseng PgWRKY2 transcription factor is shown in SEQ ID No. 2.
Further, the cDNA sequence primer pair for expanding the ginseng PgWRKY2 transcription factor comprises: the sequence of the upstream primer is shown as SEQID No.3, and the sequence of the downstream primer is shown as SEQID No. 4.
Further, the primer pair for fluorescence detection of the ginseng PgWRKY2 transcription factor is as follows: the sequence of the upstream primer is shown as SEQID No.5, and the sequence of the downstream primer is shown as SEQID No. 6.
The invention also provides application of the ginseng PgWRKY2 transcription factor in ginseng organ tissues, wherein the ginseng PgWRKY2 transcription factor is expressed in the main root, the lateral root, the stem and the leaf of the ginseng, the expression level of the ginseng PgWRKY2 transcription factor in the lateral root and the main root is highest, and the expression level of the ginseng PgWRKY2 transcription factor in the leaf and the stem is lowest.
In addition, the invention also provides application of the ginseng PgWRKY2 transcription factor in improving stress resistance of ginseng under stress of different exogenous phosphorus concentrations, wherein the ginseng PgWRKY2 transcription factor has the highest expression level at the concentration of 2.0mM, has the lower expression level at the concentration of 4.0mM and has the expression level relatively lower at the concentrations of 0mM, 0.5mM and 1.0mM, and the expression level increases from the concentration of 0mM to the concentration of 2.0mM along with the increase of the phosphorus concentration and shows a positive correlation trend.
Advantageous effects
Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:
the invention discloses a regulating effect of a ginseng PgWRKY2 transcription factor on phosphorus nutrition, in particular to a method for detecting phosphorus nutrition stress in ginseng adversity stress by responding to phosphorus stress expression of the ginseng PgWRKY2 transcription factor and disclosing expression specificity of key enzymes in a ginseng phosphorus stress pathway, thereby providing an effective regulating measure and having important application value.
Drawings
FIG. 1 is a schematic diagram of the purification and recovery of a target band according to the present invention;
FIG. 2 is a schematic diagram showing the expression of the PgWRKY2 transcription factor of ginseng in the main root, lateral root, stem and leaf of ginseng;
FIG. 3 is a schematic diagram of the expression of the ginseng PgWRKY2 transcription factor under different phosphorus concentration stresses.
Detailed Description
The invention identifies a WRKY type transcription factor PgWRKY2 responding to phosphorus stress from ginseng through high-throughput sequencing, designs a PgWRKY2 sequence PCR amplification Primer and a real-time fluorescent quantitative PCR (qRT-PCR) detection Primer by using DNAMAN and Primer Premier 6 software based on a transcriptome database, and provides a new strategy for transforming ginseng callus by using genetic engineering in the future by researching the application of the PgWRKY2 transcription factor in responding to the phosphorus stress of the ginseng, thereby improving the stress resistance of the ginseng.
The present invention will be further described with reference to the following examples.
Example 1: RNA extraction of ginseng rhizome tissue
Adopting biennial ginseng seedlings of ginseng planting bases in Fusong county, jilin province, in an intelligent artificial climate chamber (temperature 20 ℃, illumination 2000 lx) in a laboratory, carrying out water planting by using Hoagland nutrient solutions with different phosphorus concentrations, and setting 5 phosphorus concentration levels: p0 (0 mM), P1 (0.5 mM), P2 (1.0 mM), P3 (2.0 mM) and P4 (4.0 mM), and the ginseng seedlings are cultured in water until sampling is carried out once in the eighth week; RNA of ginseng rhizome tissues is extracted by using an RNAscope Total RNA Kit.
Example 2: PCR amplification
After reverse transcription into cDNA by FastKing gDNA dispensing RT SuperMix kit, 2 × Trans Taq was used
Fidelity (HiFi) PCR Supermix II to perform PgWRKY2 sequence amplification (reaction system: 12.5. Mu.L 2 × Trans Taq
Fidelity (HiFi) PCR Supermix II, 1. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), 1. Mu.L of template, 9.5. Mu.L of ddH2O; the reaction procedure is as follows: pre-denaturation at 95 ℃ for 4min; 35 cycles of 95 deg.C, 30s,51 deg.C, 30s,72 deg.C, 1min, 30 s; 72 deg.C, 7min. ).
Wherein, during amplification:
the upstream primer is as follows: pgWRKY2-F:5 'ATGGGAGAATTTGTTAGTATGGAG-3' (SEQ ID No. 3)
The downstream primer is: pgWRKY2-R:5 'TTACCTAAAATGTTCCGTAGAGCA-3' (SEQ ID No. 4);
after the PCR amplification reaction was completed, the TIANgel Midi purification Kit was used for the testPurifying and recovering target strip with kit (shown in FIG. 1); using the destination strip
Connecting a T5 Zero Cloning Kit to a T5 vector, plating transformed escherichia coli, picking monoclonal shake bacteria, extracting plasmids by using a TIANPrep Mini Plasmid Kit, and sending the plasmids to Shanghai bio-chemical company for sequencing; the sequencing results were analyzed using DNAMAN.
Example 3: pgWRKY2 transcription factor
RNA of main root, lateral root, stem and leaf tissue of ginseng is extracted by using an RNAscope Total RNA Kit, and is reversely transcribed into cDNA serving as a template to carry out qRT-PCR detection analysis (the reaction system is FastStart Universal 2X SYBR Green 12.5 mu L, each of an upstream primer and a downstream primer is 0.5 mu L (10 mu mol/L), a cDNA template is 1 mu L, and 10.5 mu L dd H is added2O, simultaneously with 1. Mu.L of dd H2O as a negative control instead of template; the reaction procedure is as follows: 94 ℃ for 2min; 40 cycles of 94 ℃ at 15s,55 ℃ at 20s,72 ℃ at 30 s; 72 deg.C, 7min. ).
The real-time fluorescent quantitative detection primer pair for the PgWRKY2 transcription factor of the ginseng is as follows:
an upstream primer: pgWRKY2q-F:5 'TCATTCGGTTCACTGGATTACA-3' (SEQ ID No. 5)
A downstream primer: pgWRKY2q-R:5 'GCTCATCTAACTCATCCAAAG-doped 3' (SEQ ID No. 6);
each sample was set up for 3 technical and biological replicates, using 2-ΔΔCtRelative expression levels were calculated.
Experimental results show that the full length of the PgWRKY2 transcription factor is 975bp (SEQID No. 1), and 324 amino acids (SEQID No. 2) are coded; the transcription factor is obviously expressed in roots of the ginseng, and the expression quantity of PgWRKY2 and the phosphorus concentration have a positive correlation trend within a certain exogenous phosphorus concentration range, namely the higher the exogenous phosphorus concentration is, the higher the expression quantity of the PgWRKY2 transcription factor is, and accordingly, the condition that the ginseng is stressed by phosphorus can be preliminarily detected.
Example 4: detected expression pattern of PgWRKY2 transcription factor in ginseng organ tissue
As shown in figure 2, the ginseng PgWRKY2 transcription factor is expressed in the main root, lateral root, stem and leaf of ginseng, but the expression is obviously different, the expression level is highest in the lateral root and main root, and the expression level is lowest in the stem and leaf.
Example 5: expression mode of ginseng PgWRKY2 transcription factor under stress of different phosphorus concentrations
As shown in FIG. 3, under exogenous phosphorus treatment, the PgWRKY2 transcription factor was expressed in the highest amount at the P3 (2.0 mM) concentration, the next time at the P4 (4.0 mM) concentration, the expression levels were relatively low at the P0 (0 mM), P1 (0.5 mM) and P2 (1.0 mM) concentrations, and the expression increased from the P0 (0 mM) concentration to the P3 (2.0 mM) concentration with increasing phosphorus concentration, with a positive correlation trend.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> university of Chengdu
<120> ginseng PgWRKY2 transcription factor and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 975
<212> DNA
<213> Ginseng radix (Panax ginseng C.A. Meyer)
<400> 1
atgggagaat ttgttagtat ggaggaagat tggggtctgc aagctattgt cagaggatcc 60
accgattatt ttgatgatcc gactatttct tcattcggtt cactggatta cattcagaat 120
aataataacg atgatgattt tcatttcact tttcctaatc tttttgaagc caattataac 180
agtaatgata aagttcttgt ggatgagtta gatgagcttt acaagccctt ttatcccatg 240
tttaattctt tttccccaca agctcccatt attacctcct cctcctccga ttctttccct 300
gaagaagtcc tgaagttaaa gccagaaaat caagagatcg aagttatcca gaagcacaca 360
attcctgcta aagctgctag tcctaataaa agtaatactg cccatgcagc taaatataaa 420
aggaagaacc aacacaaaag ggtggtggtt caagtaactg ctgatggtct ttcttctgat 480
ttgtgggctt ggcgtaaata tggtcagaaa cccattaaag gatcacctta tccaaggagc 540
tattataggt gtagtagctt aaaaggatgt ttggcaagga agcaagttga acaaagctgt 600
aatgatcccg gaatgtttat cataacctat tccggggaac atagccacag ccatccaact 660
cgacggagtt ctcttgccgg tacaaaccgg cacaagttca tcaccccgaa aagcccctca 720
tcggtcaata ccagtactat tgtggcaaca cccaaagact ccagttgctc tccaacatca 780
gatatcagtg aagaagtagt aattccccag cagcccatta aacatgaaga agaggctgaa 840
ggaatcggca aggatgctga atttgttata tcagatatga tcttgaacga cgatttcttt 900
gtggaattag aggagttgga ttccctaatt tcaacttcaa ccttttacaa ttgctctacg 960
gaacatttta ggtaa 975
<210> 2
<211> 324
<212> PRT
<213> Ginseng radix (Panax ginseng C.A. Meyer)
<400> 2
Met Gly Glu Phe Val Ser Met Glu Glu Asp Trp Gly Leu Gln Ala Ile
1 5 10 15
Val Arg Gly Ser Thr Asp Tyr Phe Asp Asp Pro Thr Ile Ser Ser Phe
20 25 30
Gly Ser Leu Asp Tyr Ile Gln Asn Asn Asn Asn Asp Asp Asp Phe His
35 40 45
Phe Thr Phe Pro Asn Leu Phe Glu Ala Asn Tyr Asn Ser Asn Asp Lys
50 55 60
Val Leu Val Asp Glu Leu Asp Glu Leu Tyr Lys Pro Phe Tyr Pro Met
65 70 75 80
Phe Asn Ser Phe Ser Pro Gln Ala Pro Ile Ile Thr Ser Ser Ser Ser
85 90 95
Asp Ser Phe Pro Glu Glu Val Leu Lys Leu Lys Pro Glu Asn Gln Glu
100 105 110
Ile Glu Val Ile Gln Lys His Thr Ile Pro Ala Lys Ala Ala Ser Pro
115 120 125
Asn Lys Ser Asn Thr Ala His Ala Ala Lys Tyr Lys Arg Lys Asn Gln
130 135 140
His Lys Arg Val Val Val Gln Val Thr Ala Asp Gly Leu Ser Ser Asp
145 150 155 160
Leu Trp Ala Trp Arg Lys Tyr Gly Gln Lys Pro Ile Lys Gly Ser Pro
165 170 175
Tyr Pro Arg Ser Tyr Tyr Arg Cys Ser Ser Leu Lys Gly Cys Leu Ala
180 185 190
Arg Lys Gln Val Glu Gln Ser Cys Asn Asp Pro Gly Met Phe Ile Ile
195 200 205
Thr Tyr Ser Gly Glu His Ser His Ser His Pro Thr Arg Arg Ser Ser
210 215 220
Leu Ala Gly Thr Asn Arg His Lys Phe Ile Thr Pro Lys Ser Pro Ser
225 230 235 240
Ser Val Asn Thr Ser Thr Ile Val Ala Thr Pro Lys Asp Ser Ser Cys
245 250 255
Ser Pro Thr Ser Asp Ile Ser Glu Glu Val Val Ile Pro Gln Gln Pro
260 265 270
Ile Lys His Glu Glu Glu Ala Glu Gly Ile Gly Lys Asp Ala Glu Phe
275 280 285
Val Ile Ser Asp Met Ile Leu Asn Asp Asp Phe Phe Val Glu Leu Glu
290 295 300
Glu Leu Asp Ser Leu Ile Ser Thr Ser Thr Phe Tyr Asn Cys Ser Thr
305 310 315 320
Glu His Phe Arg
Claims (5)
1. The ginseng PgWRKY2 transcription factor is characterized in that the nucleotide sequence of the ginseng PgWRKY2 transcription factor is shown as SEQ ID No. 1;
the coding protein sequence of the ginseng PgWRKY2 transcription factor is shown in SEQ ID No. 2.
2. The ginseng PgWRKY2 transcription factor as claimed in claim 1, wherein the cDNA sequence primer pair for expanding the ginseng PgWRKY2 transcription factor is: the sequence of the upstream primer is shown as SEQ ID No.3, and the sequence of the downstream primer is shown as SEQ ID No. 4.
3. The ginseng PgWRKY2 transcription factor as claimed in claim 1, wherein the primer pair for fluorescence detection of the ginseng PgWRKY2 transcription factor is as follows: the sequence of the upstream primer is shown as SEQ ID No.5, and the sequence of the downstream primer is shown as SEQ ID No. 6.
4. The application of the ginseng PgWRKY2 transcription factor in the organ tissues of the ginseng is characterized in that the ginseng PgWRKY2 transcription factor is expressed in the main root, the lateral root, the stem and the leaf of the ginseng, the expression level of the ginseng PgWRKY2 transcription factor in the lateral root and the main root is the highest, and the expression level of the ginseng PgWRKY2 transcription factor in the leaf and the stem is the lowest.
5. The application of the ginseng PgWRKY2 transcription factor to improvement of the stress resistance of ginseng under the stress of different exogenous phosphorus concentrations is characterized in that the ginseng PgWRKY2 transcription factor has the highest expression level at the concentration of 2.0mM, has the lower expression level at the concentration of 4.0mM and has the expression level relatively lower at the concentrations of 0mM, 0.5mM and 1.0mM, and the expression level increases from the concentration of 0mM to the concentration of 2.0mM along with the increase of the phosphorus concentration and shows a positive correlation trend.
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| US20180319852A1 (en) * | 2017-05-02 | 2018-11-08 | Intelligent Synthetic Biology Center | Increased production of ginsenosides through yeast cell organelle improvement |
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| CN111909252A (en) * | 2020-09-25 | 2020-11-10 | 中国农业科学院特产研究所 | Ginseng PgbHLH149 transcription factor and application thereof |
| CN112625103A (en) * | 2021-01-20 | 2021-04-09 | 上海交通大学 | Alfalfa WRKY transcription factor and application thereof in aluminum toxicity and salt stress resistance |
| CN113234734A (en) * | 2021-03-22 | 2021-08-10 | 成都大学 | Sweet orange gene CsMYB30 capable of improving plant resistance and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180319852A1 (en) * | 2017-05-02 | 2018-11-08 | Intelligent Synthetic Biology Center | Increased production of ginsenosides through yeast cell organelle improvement |
| CN110607316A (en) * | 2019-08-23 | 2019-12-24 | 兰州理工大学 | Adversity stress response related gene in Lycium ruthenicum Murr, and encoding protein and cloning method thereof |
| CN111217896A (en) * | 2020-02-28 | 2020-06-02 | 天津大学 | Application of ginseng PgWRKY4X transcription factor in regulating and controlling ginsenoside compound content in ginseng |
| CN111909252A (en) * | 2020-09-25 | 2020-11-10 | 中国农业科学院特产研究所 | Ginseng PgbHLH149 transcription factor and application thereof |
| CN112625103A (en) * | 2021-01-20 | 2021-04-09 | 上海交通大学 | Alfalfa WRKY transcription factor and application thereof in aluminum toxicity and salt stress resistance |
| CN113234734A (en) * | 2021-03-22 | 2021-08-10 | 成都大学 | Sweet orange gene CsMYB30 capable of improving plant resistance and application thereof |
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