CN109666677A - The application of soybean PHR transcription factor encoding gene GmPHRa - Google Patents
The application of soybean PHR transcription factor encoding gene GmPHRa Download PDFInfo
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- CN109666677A CN109666677A CN201811566166.4A CN201811566166A CN109666677A CN 109666677 A CN109666677 A CN 109666677A CN 201811566166 A CN201811566166 A CN 201811566166A CN 109666677 A CN109666677 A CN 109666677A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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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- 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
Abstract
The invention discloses the applications of soybean PHR transcription factor encoding gene GmPHRa.Soybean PHR transcription factor encoding gene GmPHRa, nucleotide sequence are as follows: SEQ ID NO.1.The plant Overexpression vector pMDC83-GmPHRa of building is subjected to heterogenous expression in the wild type of arabidopsis, it was found that the plant root staple length being overexpressed increases, show that the gene can be used as target gene and import plant, it makes it easier for absorbing P elements by the change to root system of plant structure, to improve the Low phosphorus tolerance of genetically modified plants.As it can be seen that soybean PHR transcription factor encoding gene GmPHRa of the present invention can increase plant root hair length by genetic engineering, application in terms of genetically modified plants Low phosphorus tolerance is improved.
Description
Technical field
The present invention relates to the applications of soybean PHR transcription factor encoding gene GmPHRa, belong to genetic engineering field, specifically
It says and is related to increasing plant root hair length from the PHRa transcription factor encoding gene of soybean, and then it is resistance to influence genetically modified plants
The application of low-phosphorous ability.
Background technique
Phosphorus is one of three big elements necessary to plant, and phosphorus supply deficiency will have an adverse effect to the growth metabolism of plant.
Available phosphate concentration need to usually apply phosphate fertilizer far from meeting the needs of plant to supplement phosphorus supply in real soil.But crop is to phosphorus
The utilization rate wretched insufficiency of fertilizer.Plant does not parse P elements regulatory mechanism completely yet at present.Study soybean phosphorus efficiency dependency basis
The function of cause, further exploring soybean phosphorus using mechanism has important theory and production meaning for improving stress resistance of plant.
Scholar points out gene transcriptional activation when studying arabidopsis and rice isotype plant or inhibiting effect is plant
Cope with the important step of environment stress, total regulation of the transcription factor as gene expression, in plant to biology and abiotic stress
Responsing reaction in play an important role.Therefore, the research of the relevant transcription factor of Tolerant to low P in soybean is helped to change
Good soybean Low phosphorus tolerance.
Existing functional study work show MYB family transcription factor take part in Secondary Metabolism of Plant, hormone and environment because
Sub- response, and the organ morphologies such as cell differentiation, cell cycle and blade are built up with important adjustment effect；And nearest
It studies, it was also found that MYB family transcription factor is during plant accumulates anthocyanidin, respectively to pericarp, pulp, blade and floral organ etc.
The formation of kind color plays a significant role.Transcription factor AtPHR1 is that find in arabidopsis first is relevant to phosphorus efficiency
The transcription factor of MYB-CC type, and its homologous gene has been found in corn, rice and Kidney bean, and further progress
The research work of itself and phosphorus efficiency correlation function.PHR gene family member institute in P elements signal transduction process in soybean
The effect of performance is also urgently excavated.
Summary of the invention
It is an object of the invention to disclose a soybean PHR transcription factor encoding gene GmPHRa resistance genetic engineering to answer
With the gene can be used as target gene and import plant, the Low phosphorus tolerance of plant be improved, to carry out plant species improvement.
The purpose of the present invention can be achieved through the following technical solutions:
Soybean PHR transcription factor encoding gene GmPHRa, nucleotide sequence are as follows: SEQ ID NO.1.
Soybean PHRa albumen, amino acid sequence are as follows: SEQ ID NO.2.
Recombinant expression carrier containing soybean PHR transcription factor encoding gene GmPHRa of the present invention.
It, can be before its transcription initiation nucleotide plus any enhanced when constructing plant expression vector using GmPHRa
Promoter or inducible promoter.It, can be to plant used for the ease of transgenic plant cells or plant are identified and screened
Object expression vector is processed, such as selected marker (gus gene, luciferase genes) are added in plant.From turn
The safety perspective of gene plant considers, any selected marker can be not added, and screens transformed plant by adverse circumstance.
Soybean PHR transcription factor encoding gene GmPHRa is increasing root staple length by genetic engineering, improves transgenosis and plants
Application in terms of object Low phosphorus tolerance；The soybean PHR transcription factor encoding gene GmPHRa, nucleotide sequence are as follows: SEQ
ID NO.1。
Recombinant expression carrier containing soybean PHR transcription factor encoding gene GmPHRa is increasing root hair by genetic engineering
Length improves the application in terms of genetically modified plants Low phosphorus tolerance；The soybean PHR transcription factor encoding gene GmPHRa,
Its nucleotide sequence are as follows: SEQ ID NO.1.
Carry GmPHRa of the present invention plant expression vector can by using Ti-plasmids, Ri plasmid, plant viral vector,
The conventional biology methods such as DNA directly converts, microinjection, conductance, mediated by agriculture bacillus conversion plant cell or tissue, and will turn
The plant tissue of change is cultivated into plant.The plant host being converted is either the unifacial leaves such as sorghum, rice, wheat, corn are planted
Object is also possible to the dicotyledons such as peanut, soybean, rape, tomato, poplar, turfgrass, clover.
Beneficial effect
GmPHRa gene function is to increase root staple length, this is a kind of important mechanisms of plant reply low-phosphorus stress.In real time
Fluorescent quantitative poly chain reaction (Quantitative RT-PCR) analysis shows: the low-phosphorous item of GmPHRa in Tolerant to low P material
Expression quantity under part be consistently higher than plus phosphorus under the conditions of (Fig. 3), show that the expression variation of the gene is related to low-phosphorus stress.It constructs
It is transferred in protoplasts of Arabidopsis thaliana broken by ultrasonic by subcellular localization carrier pAN580-GmPHRa respectively with empty carrier, the results showed that
GmPHRa transcription factor is primarily targeted for nucleus, is positioned on cell membrane (Fig. 2) on a small quantity.Construct transcriptional activation activity verifying
Carrier pGBKT7-GmPHRa has transcriptional activation activity (Fig. 4) using yeast double miscellaneous method validations GmPHRa.It constructs simultaneously
Plant Overexpression vector pMDC83-GmPHRa, and it is subjected to heterogenous expression in the wild type of arabidopsis.To filtering out
T3In generation, positive seedling was identified, it is found that the plant root staple length being overexpressed increases (Fig. 5), shown that the gene can be used as purpose
Gene transfered plant makes it easier for absorbing P elements by the change to root system of plant structure, to improve genetically modified plants
Low phosphorus tolerance.
Detailed description of the invention
The PCR amplification of Fig. 1 GmPHRa gene
M:DL2000marker
The subcellular localization of Fig. 2 GmPHRa
Plasmid pAN580-GmPHRa containing GFP is transferred in protoplasts of Arabidopsis thaliana broken by ultrasonic, and is seen under laser confocal microscope
It examines.
Inducing expression of Fig. 3 GmPHRa gene under lasting low-phosphorus stress
CD+P, CD-P: gene normal phosphorus and under the conditions of subtract phosphorus in spring beans CD
YH+P, YH-P: gene normal phosphorus and under the conditions of subtract phosphorus in cloud and honeycomb beans YH
The transcriptional activation activity of Fig. 4 GmPHRa detects
A:SD-trp culture medium；B:SD-trp/X- α-gal culture medium；C:SD-trp/X- α-gal/AbA culture medium
Fig. 5 is overexpressed positive plant and adjoining tree phenotype compares
A and b: wild-type Arabidopsis plants and GmPHRa positive Arabidopsis plant is overexpressed respectively on normal MS culture medium
Phenotype；C and d: wild-type Arabidopsis plants and GmPHRa positive Arabidopsis plant is overexpressed respectively on low-phosphorous MS culture medium
Phenotype；E and f: stereoscope Wildtype Arabidopsis thaliana plant and overexpression GmPHRa positive Arabidopsis plant are cultivated in low-phosphorous MS
Root hair phenotype on base
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and embodiments.
Method therefor is conventional method unless otherwise instructed in following embodiments.
1) clone of soybean PHR transcription factor encoding gene GmPHRa
With soybean varieties spring beans CD (soybean Tolerant to low P germplasm) for material, according to GmPHRa gene Glyma19g35080
The corresponding base sequence of the gene is found in Soybase database, according to the sequence design specific primer, primer sequence
See SEQ ID NO.3 and SEQ ID NO.4.
Soybean varieties spring beans CD with low-phosphorous processing 7 days is materials object, takes its root, is ground with mortar, and addition, which fills, to be split
The 1.5mL EP pipe for solving liquid moves in 1.5mL EP pipe sufficiently after oscillation, extracted total RNA (Tiangen, Beijing,
China).Total serum IgE quality, spectrophotometric determination rna content are identified with denaturing formaldehyde gel electrophoresis.Using the total serum IgE of acquisition as mould
The specification of plate, the reverse transcription reagent box provided according to Takara company carries out reverse transcription, after obtaining the first chain of cDNA, carries out
PCR amplification, PCR program are as follows: 95 DEG C initial denaturation 3 minutes, 95 DEG C be denaturalized 15 seconds, 60 DEG C anneal 15 seconds, 72 DEG C extend 1 minute 30
Second, totally 35 recycle, and last 72 DEG C keep the temperature 5 minutes, subsequent 12 DEG C of constant temperature.It then carries out PCR product rubber tapping purifying, connection and turns
Chemical industry is made, the sequencing of picking positive monoclonal.The soybean GmGPHRa for having that the length of complete coding region is 1455bp is obtained after sequencing
The CDS sequence of gene, wherein coding region sequence is shown in SEQ ID NO.1, is named as GmPHRa, forms (Fig. 1) by 1455bp.
2) the subcellular localization research of GmPHRa
With the subcellular localization carrier of double digestion method building GmPHRa, design first is complete containing target gene GmPHRa
The specific primer with specific restriction enzyme site of CDS sequence (being free of terminator), primer sequence is the same as SEQ ID NO.3 and SEQ
ID NO.4, specific PCR process are identical as step 1).Then PCR product is tapped rubber and is purified, PCR product after purification and
PAN580 empty plasmid together with two kinds of selected digestion with restriction enzyme, after digestion by the two with T4 ligase in 22 DEG C of companies
It connects 3 hours, is the subcellular localization carrier pAN580- for obtaining being built into function by conversion, the examining order of positive monoclonal
GmPHRa.Itself and empty carrier are transferred to protoplasts of Arabidopsis thaliana broken by ultrasonic respectively, the results showed that GmPHRa transcription factor is primarily targeted for carefully
Karyon is positioned on cell membrane (Fig. 2) on a small quantity.
3) expression analysis of the GmPHRa under the induction of lasting low-phosphorus stress
By the consistent soybean phosphorus sensitive varieties cloud of growing way and honeycomb beans YH and two kinds of material seedling of Tolerant to low P kind spring beans CD
Containing 0.005mM KH respectively2PO4(low-phosphorous) and 0.5mM KH2PO4It is cultivated in the 1/2Hogland nutrient solution of (normal phosphorus),
In Stress treatment 0h, 0.5h, 1h, 3h, 6h, when 9h, 12h, draw materials the root of CD and YH, save after liquid nitrogen flash freezer in -80 DEG C.Always
The same step 1) of the extraction of RNA.Using the Tubulin of soybean constitutive expression as internal reference, primer sequence is shown in SEQ ID NO.5
With SEQ ID NO.6.Since under the conditions of two kinds of material different disposals of soybean phosphorus sensitive varieties (YH) and Tolerant to low P kind (CD)
The total serum IgE of root is template, is reversed to carry out real-time fluorescence quantitative PCR reaction (Real-time RT-PCR) after cDNA, primer
Sequence is shown in that SEQ ID NO.7 and SEQ ID NO.8, detection GmPHRa gene are changed by the expression quantity of low-phosphorus stress.
It is low-phosphorous sensitivity we have found that being higher than low-phosphorous sensitive material in expression quantity of the GmPHRa gene in Tolerant to low P material
3.64 times of expression quantity in material.In phosphorus sensitive material YH, GmPHRa gene is presented in the Stress treatment time in 0-6h
Subtract out the expression quantity under the conditions of phosphorus be consistently higher than plus phosphorus under the conditions of mode, in Tolerant to low P material C D, GmPHRa gene is subtracting
Expression quantity under the conditions of phosphorus is consistently higher than plus phosphorus condition.The result illustrates that GmPHRa gene and soybean low-phosphorus stress exist centainly
Relationship, expression of the low-phosphorus stress induction of GmPHRa gene.
4) the transcriptional activation activity verifying of GmPHRa
With recombination method building GmPHRa transcriptional activation activity verify BD carrier, primer sequence see SEQ ID NO.9 and
SEQ ID NO.10, specific PCR process are identical as step 1).Obtained PCR product and pGBKT7 carrier carries out recombining reaction,
By conversion, coated plate and clone identification, transcriptional activation activity checking carrier pGBKT7-GmPHRa is obtained.Recombinant vector is transferred to
In yeast strain Y2HGold competence, it is respectively coated on SD-trp, SD-trp/X- α-gal and SD-trp/X- α-gal/AbA training
It supports in base, the results showed that GmPHRa has transcriptional activation activity (Fig. 4).
The genetic engineering application of 2 gene GmPHRa of embodiment
1) clone of soybean PHR transcription factor encoding gene GmPHRa
Using the root total serum IgE of soybean (Glycine max) Tolerant to low P material C D as template, the first chain of cDNA is synthesized through reverse transcription
Afterwards, PCR amplification is carried out, primer sequence is shown in that SEQ ID NO.3 and SEQ ID NO.4, PCR program is as follows: 95 DEG C of initial denaturations 3 are divided
Clock, 95 DEG C are denaturalized 15 seconds, and 60 DEG C are annealed 15 seconds, and 72 DEG C extend 30 seconds 1 minute, and totally 35 recycle, and last 72 DEG C keep the temperature 5 minutes,
Subsequent 12 DEG C of constant temperature, is cloned into pAN580 carrier for PCR product, and it is 1455bp that obtaining after sequencing, which has the length of complete coding region,
Soybean GmPHRa gene CDS sequence, wherein coding region sequence is shown in SEQ ID NO.1；
2) building of plant expression vector
By GmPHRa gene order and Invitrogen companyTechnology with ClonaseTMII
PDONR221 carrier in kit carries out BP reaction, and carries out bacterium solution PCR sequence verification, and primer sequence is shown in SEQ ID NO.11
With SEQ ID NO.12, specific PCR process is identical as step 1), obtains entry clones；By obtained entry clones with
The purpose expression vector pMDC83 of Invitrogen company exploitation carries out recombination exchange, and it is excessive to obtain pMDC83-GmPHRa plant
Express expression vector, plant conversion carrier pMDC83 contains 2x35S strong promoter, can induced strong target gene GmPHRa by
Expression in body.Then carrier is transferred in Agrobacterium tumefaciens strain EHA105 by freeze-thaw method；
3) acquisition of transgenic plant
The Agrobacterium tumefaciens strain EHA105 for the carrier containing pMDC83-GmPHRa that step 2) is obtained is by using being stained with colored method
Arabidopsis thaliana transformation (Arabidopsis thaliana) Columbia-0 type is environmental, carries out PCR to the transgenic plant of acquisition,
PCR specific amplification is carried out using DNA fragmentation of the target gene specific primer to extraction, primer sequence is shown in SEQ ID NO.11
With SEQ ID NO.12, detect whether gene encoder block is inserted into arabidopsis thaliana genomic dna, specific PCR process and step 1)
Identical, real time fluorescent quantitative qPCR primer sequence is shown in SEQ ID NO.7 and SEQ ID NO.8, and the phenotype of plant is carried out after verifying
Character analysis；
The homozygous T that screening is obtained3For transgenic line plantation in MS culture medium, Arabidopsis thaliana Seedlings are shifted after 1 week
The continued growth into normal phosphorus and low-phosphorous MS culture medium, observes and records the growth and development process and its table of transgenic arabidopsis
Type character.Arabidopsis observes its root hair after cultured on solid medium 10 days, using stereoscope.The result shows that normal
Under the conditions of phosphorus, wild-type Arabidopsis plants and be overexpressed Arabidopsis plant growing way without marked difference, but it is low-phosphorous under the conditions of cross table
Increase (Fig. 5) up to Arabidopsis plant root staple length.It should be the result shows that the gene, which can be used as target gene, imports plant, by right
The change of root system of plant structure makes it easier for absorbing P elements, to improve the Low phosphorus tolerance of genetically modified plants.
Sequence table
<110>Agricultural University Of Nanjing
<120>application of soybean PHR transcription factor encoding gene GmPHRa
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<170> SIPOSequenceListing 1.0
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cctggatatt caactgatct tcatcactca tccttttcat ctcatgaaaa acaacctaga 300
aatactcatt ttatttcaca gtcattaagt aacatggctt cattgccatt atcttattct 360
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aatagtcaag tagaaagcag tgactgcaat atcatggcaa ctgaagagta ttctaagcga 540
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tctcaacttc caataggaca ccaatcccaa agccatcaac aacttcctgc ttcatctgga 720
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atgcgctgga cacctgagct tcatgaggca tttgtggagg ctgtcaacca acttggtggg 840
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taccatgtta aaagccacct acagaagtac aggacagcta gatatagacc tgagtcatct 960
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gccatcgaaa gtcaatctgg agtgtcttct gatgccatca aagattctcc agccaaaact 1260
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Pro Val Phe Met Glu Lys Glu Leu Lys Thr Lys Pro Tyr Thr His Ser
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Ser His Leu Thr Ser Ser Gly Ala Val Gly His Met Phe Ser Ser Ser
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Lys Gln Pro Arg Asn Thr His Phe Ile Ser Gln Ser Leu Ser Asn Met
100 105 110
Ala Ser Leu Pro Leu Ser Tyr Ser Ser Asn Ser Glu Pro Ile Pro Ser
115 120 125
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Asn Ser Gln Val Glu Ser Ser Asp Cys Asn Ile Met Ala Thr Glu Glu
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Tyr Ser Lys Arg Asn Asp Trp Gln Glu Trp Ala Asp Gln Leu Ile Ser
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Asp Val Asp Pro Leu Thr Ser Asn Trp Asn Asp Leu Leu Ala Asp Asn
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Ile Gly His Gln Ser Gln Ser His Gln Gln Leu Pro Ala Ser Ser Gly
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Glu Asn Arg Val Gly Val Ala Pro Thr Ser Ser Thr Asn Ser Ala Pro
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Ser His Leu Gln Lys Tyr Arg Thr Ala Arg Tyr Arg Pro Glu Ser Ser
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Glu Gly Ala Ala Glu Lys Lys Leu Ser Pro Ile Glu Glu Met Ser Ser
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Met Glu Val Gln Lys Arg Leu His Glu Gln Leu Glu Ile Gln Arg Asn
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Leu Gln Leu Arg Ile Glu Glu Gln Gly Arg Tyr Leu Gln Met Met Phe
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Glu Lys Gln Cys Lys Pro Gly Ile Glu Thr Phe Lys Ala Ser Ser Ser
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Claims (2)
1. soybean PHR transcription factor encoding gene GmPHRa is increasing root staple length by genetic engineering, genetically modified plants are improved
Application in terms of Low phosphorus tolerance；The soybean PHR transcription factor encoding gene GmPHRa, nucleotide sequence are as follows: SEQ
ID NO.1。
2. the recombinant expression carrier containing soybean PHR transcription factor encoding gene GmPHRa is increasing root staple length by genetic engineering
Degree improves the application in terms of genetically modified plants Low phosphorus tolerance；The soybean PHR transcription factor encoding gene GmPHRa,
Nucleotide sequence are as follows: SEQ ID NO.1.
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| CN201811566166.4A CN109666677A (en) | 2018-12-20 | 2018-12-20 | The application of soybean PHR transcription factor encoding gene GmPHRa |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112010954A (en) * | 2020-07-10 | 2020-12-01 | 浙江省农业科学院 | PHR1 transcription factor of tea tree and coding gene and application thereof |
| CN112552383A (en) * | 2020-12-07 | 2021-03-26 | 中国科学院遗传与发育生物学研究所 | Application of transcription factor HINGE1 in regulation and control of plant nitrogen-phosphorus homeostasis |
| CN115261404A (en) * | 2021-04-29 | 2022-11-01 | 中国科学院分子植物科学卓越创新中心 | Application of phosphorus starvation response factor PHR2 in symbiosis of plants and arbuscular mycorrhiza and improvement of phosphorus nutrition |
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| CN112552383A (en) * | 2020-12-07 | 2021-03-26 | 中国科学院遗传与发育生物学研究所 | Application of transcription factor HINGE1 in regulation and control of plant nitrogen-phosphorus homeostasis |
| CN115261404A (en) * | 2021-04-29 | 2022-11-01 | 中国科学院分子植物科学卓越创新中心 | Application of phosphorus starvation response factor PHR2 in symbiosis of plants and arbuscular mycorrhiza and improvement of phosphorus nutrition |
| CN115261404B (en) * | 2021-04-29 | 2024-01-26 | 中国科学院分子植物科学卓越创新中心 | Application of phosphorus starvation response factor PHR2 in symbiosis of plants and arbuscular mycorrhiza and improvement of phosphorus nutrition |
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