CN106119261A - Salt mustard Tolerant to low P gene ThPHT1 4 and recombinant vector and application - Google Patents

Salt mustard Tolerant to low P gene ThPHT1 4 and recombinant vector and application Download PDF

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CN106119261A
CN106119261A CN201610512106.9A CN201610512106A CN106119261A CN 106119261 A CN106119261 A CN 106119261A CN 201610512106 A CN201610512106 A CN 201610512106A CN 106119261 A CN106119261 A CN 106119261A
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gene
low
tolerant
thpht1
salt mustard
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杨少辉
寇莹莹
王洁华
宋英今
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Tianjin University
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Tianjin University
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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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
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    • 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

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Abstract

The invention discloses salt mustard Tolerant to low P gene ThPHT1 4 and recombinant vector and application, salt mustard Tolerant to low P gene ThPHT1 4 is the nucleotide sequence shown in SEQ ID NO.1 in sequence table.This gene is improving the application of plant tolerant to low-phosphorus performance.The arabidopsis using this gene to transfect shows the tolerance to low-phosphorus stress, illustrates that Tolerant to low P gene ThPHT1 4 that the present invention provides, in terms of the ability of Crop Improvement Tolerant to low P, plays an important role.

Description

Salt mustard Tolerant to low P gene ThPHT1-4 and recombinant vector and application
Technical field
The invention belongs to molecular biology and biological technical field, more particularly, relate to a kind of salt mustard Tolerant to low P base Cause and application.
Background technology
Phosphorus is one of important nutrient of biological growth and development, is protein, nucleic acid, lipid and various important little The important composition composition of molecule, plays highly important role during plant metabolism.But, phosphorus is that one can not The resource of regeneration, major part soil available phosphorus content is relatively low, it is difficult to meet the normal demand of plant growing.It is reported, in the world At least the crop yield of 30%～40% is seriously suppressed (Runge-Metzger A.) by low-phosphorus stress.Agricultural production On, mainly by use phosphate fertilizer solve soil phophorus lack problem, but use phosphate fertilizer be easy to by soil fixing be crop It is difficult to the insoluble phosphorus absorbed, it is difficult to fully meet the crop demand to phosphorus.Therefore, plant is improved to the absorption of phosphorus and utilization Rate, formulates Tolerant to low P crop varieties, for reducing the amount of application of agricultural phosphorus, safeguards the sides such as ecological safety, the yield of raising crop Mask is significant.Along with the molecular mechanism research of plant low-phosphorus stress response is deepened continuously, with arabidopsis it is particularly Object of study, plant research under the conditions of low-phosphorus stress achieves breakthrough progress.At present, to having Tolerant to low P performance The separation of gene is the top priority currently utilizing gene engineering method to carry out Tolerant to low P crop cultivation.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of salt mustard Tolerant to low P gene.
Second object of the present invention is to provide the recombinant vector of a kind of saliferous mustard Tolerant to low P gene.
Third object of the present invention is to provide a kind of host cell containing above-mentioned recombinant vector.
Fourth object of the present invention is to provide a kind of salt mustard Tolerant to low P gene in the application improving plant tolerant to low-phosphorus.
Technical scheme is summarized as follows:
A kind of salt mustard Tolerant to low P gene ThPHT1-4, described gene is the nucleotide shown in SEQ ID NO.1 in sequence table Sequence.
Recombinant vector containing a kind of salt mustard Tolerant to low P gene ThPHT1-4.
Host cell containing above-mentioned recombinant vector.
A kind of salt mustard Tolerant to low P gene ThPHT1-4 is improving the application of plant tolerant to low-phosphorus performance.
The salt mustard Tolerant to low P gene ThPHT1-4 of the present invention, obtains from salt mustard and has SEQ ID NO.1 in sequence table Shown nucleotide sequence, the arabidopsis using this gene to transfect shows the tolerance to low-phosphorus stress, illustrates that the present invention carries The Tolerant to low P gene ThPHT1-4 of confession, in terms of the ability of Crop Improvement Tolerant to low P, plays an important role.
Accompanying drawing explanation
Fig. 1 is salt mustard Tolerant to low P ThPHT1-4 gene clone's electrophoresis schematic diagram of the present invention.
Fig. 2 is the expression vector schematic diagram of the gene ThPHT1-4 of the salt mustard Tolerant to low P comprising the present invention.
Fig. 3 is that pCAMBIA3301_ThPHT1-4 converts Agrobacterium C58PCR the selection result.
Fig. 4 is ThPHT1-4 transgenic arabidopsis T3 homozygote semiquantitive PCR result.
Fig. 5 is ThPHT1-4 transgenic arabidopsis T3 homozygote Tolerant to low P root system experiment effect.
Detailed description of the invention
The experiment material, the agents useful for same source that relate in the present invention are as follows:
Salt mustard: led by CAS Botanical Institute professor Li Yinxin, picks up from limit, saliferous farmland, Daxing District.
PJET1.2: purchased from Sai Mofei company http://www.thermofisher.com/cn/zh/home.html
PDONR201: purchased from Sai Mofei company http://www.thermofisher.com/cn/zh/home.html
PCAMBIA3301: purchased from Sai Mofei company http://www.thermofisher.com/cn/zh/home.html
DH5 α competent cell, Reverse Transcription box: purchased from Beijing, Beijing Quan Shijin Bioisystech Co., Ltd,
http://www.transgen.com.cn/shop.html。
Glue reclaims test kit: purchased from Bao Bio-Engineering Company, http://www.takara.com.cn/.
Agrobacterium strains C58: purchased from China plasmid vector strain cell pnca gene preservation center,
http://biovector.blog.163.com。
The reaction system related in the present invention:
BP reacts:
attB-PCR 1ul
PDonr 201 0.5ul
1×TE Buffer(PH8.0)to 4ul
LR reacts:
Entry clone(100ng/ul) 1ul
Destination vector(150ng/ul) 0.5ul
1×TE Buffer(PH8.0) 2.5ul
The experimental technique of unreceipted actual conditions in embodiment, generally according to the bar described in normal condition and handbook Part, or according to the condition proposed by manufacturer.
Technical scheme is further illustrated below in conjunction with specific embodiment.
Embodiment 1
A kind of acquisition of salt mustard Tolerant to low P gene
First, with pick up from limit, farmland salt mustard as raw material, utilize gene clone technology, obtain the salt mustard Tolerant to low P of the present invention
ThPHT1-4 gene.
Take the fresh salt mustard plant that grown five weeks, use plant RNeasy Plant Mini Kit (Trans gene Code#EP101-0150rxns) extract total RNA, and utilize EasyScript First-Strand cDNA Synthesis SuperMix (Trans gene Code#AE301-03 100rxns) removes genomic DNA interference, reverse transcription Go out cDNA.
In Phytozome and TAIR data base, find out arabidopsis whole PHT1 gene (9), then by these AtPHTs puts in the salt mustard data base of phytozome and retrieves.The salt mustard PHT1 gene that recycling obtains, Phytozome retrieves again, finds 14 salt mustard PHT1 genes altogether.
ThPHT1s gene is carried out the contrast of albumen complete sequence, has depicted salt mustard and arabidopsis PHT1 family gene and be System is set, and by the salt mustard Thhalv10006497m named ThPHT1-4 nearest with AtPHT1-4 sibship, and carries out base Because of clone.This experiment according to salt mustard ThPHT1-4 gene order design forward primer: PHT-F (shown in SEQ ID NO.2,5'- CTCATTCCACTTCCTTTCTCTCTC-3') and downstream primer: PHT-R (shown in SEQ ID NO.3,5'- AAAAGGCGTATTGTCACCTAAACTA-3'), with salt mustard leaf cDNA as template, carry out gene clone, obtain complete ThPHT1-4 full length gene be 1791bp (shown in SEQ ID NO.1) be heretofore described salt mustard Tolerant to low P gene, see Fig. 1.
Embodiment 2
The structure of the recombinant vector pCAMBIA3301_ThPHT1-4 of saliferous mustard Tolerant to low P gene
Build intermediate carrier pJET1.2_ThPHT1-4, pDONR201_ of the salt mustard Tolerant to low P gene containing the present invention ThPHT1-4 and plant expression vector pCAMBIA3301_ThPHT1-4, is shown in Fig. 2.
Glue is reclaimed purpose fragment after purification, salt mustard Tolerant to low P gene (ThPHT1-4 gene) after purification, utilizes CloneJETPCR Cloning Kit (Clone JET PCR Cloning Kit#K1231 20rxns, Thermo) is carried out The structure of pJET1.2_ThPHT1-4 cloning vehicle, and convert DH5 α competent cell.
Utilize the upstream and downstream primer PHT-F (SEQ ID NO.2) and PHT-R (SEQ ID NO.3) of purpose fragment to bacterium colony Carrying out PCR screening, positive bacteria send order-checking.The plasmid extracting the correct bacterium solution that checks order carries out BP reaction, carrier required in BP reaction For pDONR201, filter out positive colony (pDONR201_ThPHT1-4), check order.
Meanwhile, extracting the correct BP bacteria plasmid that checks order and carry out LR reaction, in LR reaction, required carrier is pCAMBIA3301 table Reach carrier, screening positive clone after reaction, the positive colony amplification culture that will filter out, extract plasmid, convert C58 competence thin Born of the same parents.The i.e. structure of the plant expression vector pCAMBIA3301_ThPHT1-4 of salt mustard Tolerant to low P gene.
Embodiment 3
Salt mustard Tolerant to low P gene ThPHT1-4 is improving the application of plant tolerant to low-phosphorus aspect of performance
Utilize the pCAMBIA3301_ThPHT1-4 positive expression carrier (weight of salt mustard Tolerant to low P gene that embodiment 2 is screened Group carrier) utilize electric shocking method to convert Agrobacterium C58 competent cell, C58 competent cell has rifampicin resistance (Rif), auxiliary Plasmid is helped to have gentamicin resistance (Gen).
After pCAMBIA3301_ThPHT1-4 is converted C58, picking individual colonies, carry out bacterium colony PCR with cloning primer, such as figure Shown in 3, with the gel electrophoresis of 1%, point sample is 5 μ L 2 × loading buffer+5 μ L PCR primer.Stripe size is 1791bp. Leftmost side band is Marker DL2000.PCR is accredited as the bacterium colony of the positive in order to infect arabidopsis.
The activation of Agrobacterium and amplification culture: single bacterium colony of the positive Agrobacterium choosing preservation is placed in 3mL containing corresponding antibiotic YEB fluid medium in Gen (25 μ g/ml)；Rif(10μg/ml)；Grass fourth phosphine (50 μ g/ml) incubated overnight about 15 hours (to OD600About=0.8), 170rpm, 28 DEG C.The amplification culture of Agrobacterium: add in the YEB fluid medium of fresh 10ml (Gen antibiotic, concentration is 25 μ g/ml to enter appropriate antibiotic；Rif antibiotic, concentration is 10 μ g/ml；Grass fourth phosphine antibiotic, Concentration is 50 μ g/ml), then inoculation 3ml above-mentioned gained Agrobacterium bacterium solution, trains in 50ml fresh YEB fluid medium Support, 170rpm, cultivate to OD at 28 DEG C600=0.6.It is obtained by culture the host cell containing some recombinant vectors.
Positive Agrobacterium (containing the host cell of recombinant vector) through above-mentioned steps screening preserves, and chooses preservation Single bacterium colony incubated overnight of positive Agrobacterium is (to OD600About=0.8) supernatant is abandoned, with the liquid sucrose of 5% after, bacterium solution is centrifugal Soak after resuspended thalline the whole inflorescence about 30-45 second of arabidopsis to be transformed, take out arabidopsis so that it is couch in pallet, Covering moisturizing, and dark treatment 12h with plastic sheeting, then temperature 25 DEG C, photoperiod 16h illumination/8h is dark, relative humidity: Its normal growth is made under under the condition of culture of 70%, until seed maturity.37 DEG C of oven for drying it are put into two weeks, after seed collection Standby follow-up test uses.
By the T1 that collects for seed after sterilization, be placed on 4 DEG C of vernalization of refrigerator three days, then by super-clean bench Uniformly sow in the 1/2MS solid screening culture medium containing 50 μ g/ml grass fourth phosphines through the arabidopsis seed that vernalization is good, at 22- 25 DEG C, 140 μm ol/ (s), when transformed plant grows to 3-4 sheet true leaf, are transplanted in soil, about receive after one and a half months Collection seed is T2 for transformed the seed.Use same procedure to screen, obtain T3 after a continued growth generation for seed, by Containing grass fourth phosphine antibiotic 1/2MS culture medium on screen, 100% survival rate be T3 for homozygote seed from seed.
After salt mustard Tolerant to low P gene transformation arabidopsis, T3 homozygote PCR measures expression result, does half with cloning primer Quantitatively, the expression of detection gene, partial results is as shown in Figure 4.Choose 2 high independent transformation strains of expression and open country The seed of raw type arabidopsis (WT) aseptic process simultaneously, tiles on MS solid plate, 4 DEG C of vernalization two days, 22 DEG C of illumination cultivation Case is sprouted 5 days, then moves on to vertical in low-phosphorous (50 μMs of Pi) culture medium cultivation 5 days, observes its phenotypic difference.As it is shown in figure 5, Under normal phosphorus treatment conditions, turn salt mustard Tolerant to low P gene arabidopsis (ThPHT1-4) and wild type control is basically identical.
The salt mustard Tolerant to low P gene of the above-mentioned experimental verification present invention has Tolerant to low P performance.The purpose of experiment is that checking is above-mentioned Gene has Tolerant to low P performance, said gene is forwarded in plant, if can express in plant, then this kind of plant has resistance to low The characteristic of phosphorus.
It is demonstrated experimentally that the gene of the present invention may apply to crop Semen sojae atricolor, Semen Maydis, in Cotton Gossypii, and there is Tolerant to low P Characteristic.
Above the present invention is done exemplary description, it should explanation, in the situation of the core without departing from the present invention Under, any simple deformation, amendment or other those skilled in the art can not spend the equivalent of creative work equal Fall into protection scope of the present invention.

Claims (4)

1. a salt mustard Tolerant to low P gene ThPHT1-4, is characterized in that described gene is in sequence table shown in SEQ ID NO.1 Nucleotide sequence.
2. the recombinant vector containing a claim 1 salt mustard Tolerant to low P gene ThPHT1-4.
3. containing the host cell of recombinant vector described in claim 2.
4. claim 1 salt mustard Tolerant to low P gene ThPHT1-4 is improving the application of plant tolerant to low-phosphorus performance.
CN201610512106.9A 2016-07-01 2016-07-01 Salt mustard Tolerant to low P gene ThPHT1 4 and recombinant vector and application Pending CN106119261A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108866073A (en) * 2017-05-10 2018-11-23 天津大学 Salt mustard Tolerant to low P gene ThPHT1-8 and recombinant vector and application
CN110607308A (en) * 2019-09-01 2019-12-24 天津大学 Sedum lineare drought-resistant gene SlERF and application thereof
CN114736904A (en) * 2022-06-14 2022-07-12 中国农业科学院作物科学研究所 Maize PILNCR2 gene and application thereof in regulation and control of maize low-phosphorus stress resistance

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105441458A (en) * 2015-12-18 2016-03-30 天津大学 Thellungiella salsuginea low-phosphorus-tolerant gene ThPHT1-1, and recombinant vector and applications thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105441458A (en) * 2015-12-18 2016-03-30 天津大学 Thellungiella salsuginea low-phosphorus-tolerant gene ThPHT1-1, and recombinant vector and applications thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENBANK: "XM_006411055.1", 《NCBI GENBANK》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108866073A (en) * 2017-05-10 2018-11-23 天津大学 Salt mustard Tolerant to low P gene ThPHT1-8 and recombinant vector and application
CN110607308A (en) * 2019-09-01 2019-12-24 天津大学 Sedum lineare drought-resistant gene SlERF and application thereof
CN114736904A (en) * 2022-06-14 2022-07-12 中国农业科学院作物科学研究所 Maize PILNCR2 gene and application thereof in regulation and control of maize low-phosphorus stress resistance

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