CN109609510A - The application of soybean PHR transcription factor encoding gene GmPHRb - Google Patents
The application of soybean PHR transcription factor encoding gene GmPHRb Download PDFInfo
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- CN109609510A CN109609510A CN201811563997.6A CN201811563997A CN109609510A CN 109609510 A CN109609510 A CN 109609510A CN 201811563997 A CN201811563997 A CN 201811563997A CN 109609510 A CN109609510 A CN 109609510A
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Abstract
The invention discloses the applications of soybean PHR transcription factor encoding gene GmPHRb.Soybean PHR transcription factor encoding gene GmPHRb, nucleotide sequence are as follows: SEQ ID NO.1.The plant Overexpression vector pMDC83-GmPHRb of building is subjected to heterogenous expression in the wild type of arabidopsis, it was found that the plant root staple length and lateral root number that are overexpressed increase, 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 GmPHRb of the present invention can increase plant root hair length and lateral root number 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 GmPHRb, belong to genetic engineering field, specifically
It says and is related to increasing plant root hair length and lateral root number from the PHRb transcription factor encoding gene of soybean, and then influence to turn
The application of gene plant Low phosphorus tolerance.
Background technique
Phosphorus is a kind of necessary mineral element of plant growth and development, and phosphorus supply is insufficient to generate not the growth metabolism of plant
Benefit influences.But largely can in anthropogenic soil there is no containing it is sufficient concentrations of meet plant growth and development using phosphorus
Element need to usually apply phosphate fertilizer to supplement phosphorus supply.But crop is to the utilization rate wretched insufficiency of phosphate fertilizer.Therefore, research soybean phosphorus effect
The function of rate related gene, further exploring soybean phosphorus using mechanism has important theory and life for improving stress resistance of plant
Produce meaning.
Biology and abiotic stress are the principal elements for causing crop yield to reduce.Stress responsive gene is mainly being transcribed
Level plays a role, they are plant stress responsing reactions to instantaneous and continuous expression mode the regulation of specific stress gene
An important component.These transcription factors often belong to big gene family, including ERF gene family, WRKY base
Because of family and myb gene family etc..
Plant myb gene family takes part in numerous biological processes, and the nascent and secondary metabolism for such as participating in plant reacts (class
Flavone metabolism approach, cell-wall components synthesis etc.), participate in plant cell form and pattern formation (development of cotton fiber, stomata
Differentiation and form etc.), plant growth and development (nutrition organs development and reproductive development) is participated in, plant Stress response is participated in
(arid, pest and disease damage etc.).As first MYB-CC type relevant to phosphorus efficiency being found in arabidopsis transcription because
Sub- AtPHR1, homologous gene be all accredited in corn, rice and Kidney bean, and further progress itself and phosphorus efficiency
The research work of correlation function.PHR gene family member effect played in P elements signal transduction process in soybean
Urgently excavate.
Summary of the invention
It is an object of the invention to disclose a soybean PHR transcription factor encoding gene GmPHRb 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 GmPHRb, nucleotide sequence are as follows: SEQ ID NO.1.
Soybean PHRb albumen, amino acid sequence are as follows: SEQ ID NO.2.
Recombinant expression carrier containing soybean PHR transcription factor encoding gene GmPHRb of the present invention.
It, can be before its transcription initiation nucleotide plus any enhanced when constructing plant expression vector using GmPHRb
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 GmPHRb is increasing root staple length and lateral root number by genetic engineering, mentions
Application in terms of high genetically modified plants Low phosphorus tolerance；The soybean PHR transcription factor encoding gene GmPHRb, nucleotide
Sequence are as follows: SEQ ID NO.1.
Recombinant expression carrier containing soybean PHR transcription factor encoding gene GmPHRb is increasing root hair by genetic engineering
Length and lateral root number improve the application in terms of genetically modified plants Low phosphorus tolerance；The soybean PHR transcription factor encodes base
Because of GmPHRb, nucleotide sequence are as follows: SEQ ID NO.1.
Carry GmPHRb 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
GmPHRb gene function is to increase root staple length and lateral root number, this is the important machine of plant reply low-phosphorus stress
System.Fluorescent quantitative PCR (Quantitative RT-PCR) analysis shows: GmPHRb in Tolerant to low P material
Reach top (Fig. 3) in low-phosphorus stress 0.5h, shows that the gene can perceive rapidly low-phosphorous signal.It constructs sub- thin
It is transferred in protoplasts of Arabidopsis thaliana broken by ultrasonic, the results showed that GmPHRb turns by born of the same parents positioning carrier pAN580-GmPHRb respectively with empty carrier
The record factor is positioned at nucleus (Fig. 2).Transcriptional activation activity checking carrier pGBKT7-GmPHRb is constructed, it is double miscellaneous using yeast
Method validation GmPHRb does not have transcriptional activation activity (Fig. 4).Plant Overexpression vector pMDC83- is constructed simultaneously
GmPHRb, and it is subjected to heterogenous expression in the wild type of arabidopsis.To the T filtered out3In generation, positive seedling was identified, was found
The plant root staple length and lateral root number of overexpression increase (Fig. 5), show that the gene can be used as target gene and import plant, lead to
It crosses and the change of root system of plant structure is made it easier for absorb P elements, to improve the Low phosphorus tolerance of genetically modified plants.
Detailed description of the invention
The PCR amplification of Fig. 1 GmPHRb gene
M:DL2000marker
The subcellular localization of Fig. 2 GmPHRb
Plasmid pAN580-GmPHRb 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 GmPHRb 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 GmPHRb 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 GmPHRb positive Arabidopsis plant is overexpressed respectively on normal MS culture medium
Phenotype；C and d: wild-type Arabidopsis plants and GmPHRb positive Arabidopsis plant is overexpressed respectively on low-phosphorous MS culture medium
Phenotype；E and f: stereoscope Wildtype Arabidopsis thaliana plant and overexpression GmPHRb positive Arabidopsis plant are cultivated in low-phosphorous MS
Root hair phenotype on base；G: wild-type Arabidopsis plants and overexpression GmPHRb positive Arabidopsis plant lateral root number histogram
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 GmPHRb
With soybean varieties spring beans CD (soybean Tolerant to low P germplasm) for material, according to GmPHRb gene number
Glyma.09g017400 finds the corresponding base sequence of the gene in Phytozome database, according to sequence design spy
Specific primer, primer sequence are shown in 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,
Totally 35 circulations, last 72 DEG C keep the temperature 5 minutes, subsequent 12 DEG C of constant temperature.Then carry out PCR product rubber tapping purifying, connection and conversion
Work, the sequencing of picking positive monoclonal.The soybean GmGPHRb base for having that the length of complete coding region is 990bp is obtained after sequencing
The CDS sequence of cause, wherein coding region sequence is shown in SEQ ID NO.1, is named as GmPHRb, forms (Fig. 1) by 990bp.
2) the subcellular localization research of GmPHRb
With the subcellular localization carrier of double digestion method building GmPHRb, design first is complete containing target gene GmPHRb
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
GmPHRb.Itself and empty carrier are transferred to protoplasts of Arabidopsis thaliana broken by ultrasonic respectively, the results showed that GmPHRb transcription factor is positioned at nucleus
(Fig. 2).
3) expression analysis of the GmPHRb 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 GmPHRb gene are changed by the expression quantity of low-phosphorus stress.
We have found that with the extension of low-phosphorus stress processing time, other than the time point of 9h, in Tolerant to low P material
Expression quantity of GmPHRb gene under the conditions of subtracting phosphorus is consistently higher than plus phosphorus condition, and when low-phosphorus stress handles 0.5h,
GmPHRb gene expression reaches top.The result illustrates that GmPHRb gene can rapidly perceive low-phosphorous signal, to make phase
The responsing reaction answered.
4) the transcriptional activation activity verifying of GmPHRb
With recombination method building GmPHRb 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-GmPHRb 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 GmPHRb does not have transcriptional activation activity (Fig. 4).
The genetic engineering application of 2 gene GmPHRb of embodiment
1) clone of soybean PHR transcription factor encoding gene GmPHRb
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 1 minute, and totally 35 recycle, and last 72 DEG C keep the temperature 5 minutes, then
PCR product is cloned into pAN580 carrier by 12 DEG C of constant temperature, and it is the big of 990bp that obtaining after sequencing, which has the length of complete coding region,
The CDS sequence of beans GmPHRb gene, wherein coding region sequence is shown in SEQ ID NO.1；
2) building of plant expression vector
By GmPHRb gene order and Invitrogen companyTechnology with ClonaseTM
PDONR221 carrier in II kit carries out BP reaction, and carries out bacterium solution PCR sequence verification, and primer sequence is shown in SEQ ID
NO.11 and 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-GmPHRb plant
Express expression vector, plant conversion carrier pMDC83 contains 2x35S strong promoter, can induced strong target gene GmPHRb 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-GmPHRb 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
To normal phosphorus and low-phosphorous (0.005mM KH2PO4) MS culture medium in continued growth, observe and record the life of transgenic arabidopsis
Long growth course and its phenotypic character.Arabidopsis is after cultured on solid medium 10 days, using stereoscope to its root Mao Jinhang
Observation.The result shows that under the conditions of normal phosphorus, wild-type Arabidopsis plants and Arabidopsis plant growing way is overexpressed without marked difference, but
Be it is low-phosphorous under the conditions of be overexpressed Arabidopsis plant lateral root number and root staple length and increase (Fig. 5).It should be the result shows that the gene can be with
Gene transfered plant as a purpose is made it easier for absorbing P elements by the change to root system of plant structure, be turned to improve
The Low phosphorus tolerance of gene plant.
Sequence table
<110>Agricultural University Of Nanjing
<120>application of soybean PHR transcription factor encoding gene GmPHRb
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ggcaacaact ctaatctcgc ttcaaaacaa cgtttacggt ggacacatga attacatgag 180
cgctttgttg atgctgtggc tcaacttggt gggccagatc gtgccacacc caaaggtgtc 240
ctcagagtta tgggtgtaca aggcttgacc atttaccatg tcaaaagcca tttacagaaa 300
taccgacttg caaaatattt acctgactcc tcatctgatg aagggaaaaa ggctgacaag 360
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cagctacaat tacggataga ggcccagggt aaatacctga aaaagataat cgaagaacag 540
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ggggatgcat gccaagaacc tgataataag actgacccgt caacccctga ccctgaaaag 660
gccgccaaag accgtgctcc ggcaaaaagt ctttcaatag aatctttttc atctcacctt 720
gaaccgatga caccagattc tggctgccat gttggttccc ctgctgaaag ccctaaaggg 780
gagagatcag ccaagaagca acgagtaatc atggatggcg tgtattctaa accagaaatg 840
gtgcttccac atcagatact ggagtcaagc atgtcattat accagcaacc taacactgtt 900
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Lys Gln Arg Leu Arg Trp Thr His Glu Leu His Glu Arg Phe Val Asp
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Ala Val Ala Gln Leu Gly Gly Pro Asp Arg Ala Thr Pro Lys Gly Val
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Leu Arg Val Met Gly Val Gln Gly Leu Thr Ile Tyr His Val Lys Ser
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His Leu Gln Lys Tyr Arg Leu Ala Lys Tyr Leu Pro Asp Ser Ser Ser
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Asp Glu Gly Lys Lys Ala Asp Lys Lys Glu Thr Gly Asp Met Leu Ser
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Asn Leu Asp Gly Ser Ser Gly Met Gln Ile Thr Glu Ala Leu Lys Leu
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Gln Met Glu Val Gln Lys Arg Leu His Glu Gln Leu Glu Val Gln Arg
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Gln Leu Gln Leu Arg Ile Glu Ala Gln Gly Lys Tyr Leu Lys Lys Ile
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Ile Glu Glu Gln Gln Arg Leu Ser Gly Val Leu Ser Glu Thr Pro Gly
180 185 190
Ser Gly Val Ala Ala Val Ala Pro Gly Asp Ala Cys Gln Glu Pro Asp
195 200 205
Asn Lys Thr Asp Pro Ser Thr Pro Asp Pro Glu Lys Ala Ala Lys Asp
210 215 220
Arg Ala Pro Ala Lys Ser Leu Ser Ile Glu Ser Phe Ser Ser His Leu
225 230 235 240
Glu Pro Met Thr Pro Asp Ser Gly Cys His Val Gly Ser Pro Ala Glu
245 250 255
Ser Pro Lys Gly Glu Arg Ser Ala Lys Lys Gln Arg Val Ile Met Asp
260 265 270
Gly Val Tyr Ser Lys Pro Glu Met Val Leu Pro His Gln Ile Leu Glu
275 280 285
Ser Ser Met Ser Leu Tyr Gln Gln Pro Asn Thr Val Phe Leu Gly Gln
290 295 300
Asp Gln Phe Asp Pro Ser Leu Gly Ile Ser Thr Arg Ser Gly Glu Glu
305 310 315 320
Leu Asp Lys Val Gly Gly Gly Asn Leu
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cgctgcaggt cgacggatcc tcacagatta ccaccaccaa c 41
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Claims (2)
1. soybean PHR transcription factor encoding gene GmPHRb is increasing root staple length and lateral root number by genetic engineering, improve
Application in terms of genetically modified plants Low phosphorus tolerance；The soybean PHR transcription factor encoding gene GmPHRb, nucleotides sequence
It is classified as: SEQ ID NO.1.
2. the recombinant expression carrier containing soybean PHR transcription factor encoding gene GmPHRb is increasing root staple length by genetic engineering
Degree and lateral root number improve the application in terms of genetically modified plants Low phosphorus tolerance；The soybean PHR transcription factor encoding gene
GmPHRb, nucleotide sequence are as follows: SEQ ID NO.1.
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|---|---|---|---|---|
| CN109666677A (en) * | 2018-12-20 | 2019-04-23 | 南京农业大学 | The application of soybean PHR transcription factor encoding gene GmPHRa |
| 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 |
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