CN110205331A - It is a kind of to enhance plant to iron deficiency tolerance and the encoding gene accumulated and application - Google Patents
It is a kind of to enhance plant to iron deficiency tolerance and the encoding gene accumulated and application Download PDFInfo
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- CN110205331A CN110205331A CN201910561614.XA CN201910561614A CN110205331A CN 110205331 A CN110205331 A CN 110205331A CN 201910561614 A CN201910561614 A CN 201910561614A CN 110205331 A CN110205331 A CN 110205331A
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- 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 present invention relates to a kind of enhancing plants to iron deficiency tolerance and the encoding gene accumulated and application.The coding gene sequence that enhancing plant is resistant to iron deficiency and accumulates is as shown in sequence table SEQ ID No:l.The present invention will enhance plant and be transferred in plant to iron deficiency tolerance and the encoding gene accumulated, the encoding gene overexpression for making the above-mentioned enhancing plant in plant be resistant to and accumulate to iron deficiency, to make to show the tolerance to iron deficiency in plant.The present invention, which enhances the encoding gene that plant is resistant to iron deficiency and accumulates, to provide genetic resources and technical support for the resistance to Fe Deficiency breeding of crops.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to coding base of the plant to the GAP-associated protein GAP of resistance to Fe Deficiency
Cause, meanwhile, relate to the use of the genes amplification plant to the tolerance of iron deficiency environment, thus positive may adjust plant iron deficiency
Stress response.
Background technique
Iron is a kind of metallic element, and atomic number 26 is located at the periodic table of elements period 4, is most common in life
One of metal.For the existence of people, animal and plant, iron is essential microelement.In plant, iron member
Element is the microelement that plant has the call, and plays an important role in the vital movement of plant, such as participates in leaves of plants
Synthesis, photosynthesis and the respiration etc. of green element.Although the content of ferro element in the soil is high, chemically due to iron
Matter is very active, and the solubility of iron in the soil is very low, leads to plant object in soil and is difficult to obtain from soil and is sufficiently used for
The iron of growth and development, therefore the state for thering is a large amount of plant to be in iron deficiency.Plant iron deficiency mostly occurs in the alkalinity or lime of pH > 7
In matter soil.Plant is slight, and iron deficiency will affect growth, cause chlorophyll synthesis slowly, content reduce, to influence photosynthetic work
With；In the case where too little iron, will lead to chlorophyll can not be synthesized, and leaf color turns yellow, and photosynthesis is obstructed, and plant is raw
Object amount can decline, and cause plant that can not grow.And plant is the Major Foods of humans and animals, plant edible part ferro element lacks
Normal requirement is not achieved in the weary intake that will lead to humans and animals ferro element, leads to decline and the iron deficiency of humans and animals immunity
Property anaemia take place frequently, asiderosis is one of most common recessive hunger in the world, because asiderosis is difficult to be aware, but same meeting
It is unfavorable for the growth and development and breeding of humans and animals.
Ferro element is essential in the growth and development process of biology, but the characteristics of due to global soil structure,
And the limitation of ferro element itself chemical property, lead to the generally generation of plant iron deficiency phenomenon, the situation of Human Iron Nutrition scarcity
Seriously.The iron absorption of different plants is different with transporting mechanism, and the iron deficiency tolerance of different plants is caused to differ greatly.Grass family is planted
The distinctive iron absorption and transport mechanism of object is more conducive to plant and grows in iron deficiency soil, and the staple food in human diet is predominantly big
Rice, corn and wheat, these crops are all to belong to grass family.The iron deficiency situation for solving plant, by increasing the iron content in soil
This method is unrealistic, and this method takes time and effort and effect is unobvious, and the improvement result for relying on iron supplementary can only remain short
Phase effect, so, by the gene transferred crop to play an important role in plant iron deficiency response, being improved using molecular breeding technology
The iron deficiency tolerance of crop improves the iron content of crop so as to improve the iron deficiency tolerance of crop, could fundamentally solve
The problem of Human Iron Nutrition scarcity.
Using model organism arabidopsis as experimental material, the function of the related gene in the response of plant iron deficiency is studied.It is first
First, arabidopsis only has 5 pairs of chromosomes, number very little；And individual is small, it can be with mass propgation.Secondly, the arabidopsis development growth period
It is exceedingly fast, can breed mostly generation in a short time, meet to variation and the requirement evolved；Third, arabidopsis can produce thousands of every time
Piece seed meets the requirement genetically counted.It is most important to be exactly arabidopsis or stringent close colored self-pollination, it is ensured that
Stablize heredity.Just because of above-mentioned advantage, traditional genetics have carried out many researchs by material of arabidopsis, have accumulated a large amount of moneys
Material, provides convenience for studying from gene level now, and most of genes of arabidopsis can be found in other plants,
Any discovery in relation to arabidopsis can be applied to other plant researches.It therefore can faster more using arabidopsis as research object
Good reaches experiment target, can largely shorten experimental period and simplify experiment condition.
Arabidopsis is to study species the most thorough in the world at present, and a large amount of arabidopsis gene function is set forth, this
Improvement to carry out cultivated crop using mode species information is had laid a good foundation.Pass through the research to mutant, discovery
The gene of some regulation iron transfers relevant to plant, such asNAS、MATE、MYB10Deng.Further appreciate that arabidopsis iron deficiency is rung
Regulated and control network is answered, to provide more theoretical foundations for the use of the agricultural production of molecular breeding technology improvement crop melt quality,
So as to improve Human Iron Nutrition is improved, it is of great immediate significance.
The problem of iron content lacks in soil, finds the functional gene that can adapt to iron deficiency environment and illustrates its function
With important theory and practice significance.It is found and is found according to arabidopsis sequencing data storehouse (www.arabidopsis.org)
New functional gene with independent intellectual property rights is between one of hot spot of International Plant research field and country variant
The focus of science and technology competition.
Summary of the invention
The first purpose of the invention is to provide a kind of enhancing plants to iron deficiency tolerance and promotes the volume of plant iron content
Code gene；A second object of the present invention is to provide the encoding genes in enhancing iron deficiency resistance of plants environment and to increase in plant
Application in terms of iron content.
The encoding gene of the present invention for enhancing plant to iron deficiency tolerance and promote plant iron content, is named asbZIP44
(AT1G75390), from the arabidopsis of Colombia's wild type, title and the gene number of the encoding gene are from quasi-
Southern mustard sequencing data storehouse (www.arabidopsis.org).
DNA sequence dna such as sequence table of the enhancing plant to iron deficiency tolerance and the encoding gene that promotes plant iron content
Shown in SEQ ID No:l.
Polynucleotide sequence such as sequence of the enhancing plant to iron deficiency tolerance and the encoding gene that promotes plant iron content
Shown in list SEQ ID No:2.
The enhancing plant is to iron deficiency tolerance and promotes the encoding gene of plant iron content and includes and SEQ in sequence table
The DNA sequence dna that ID No:l is limited has 90% or more homology and encodes the DNA sequence dna of identical function protein.
The encoding gene that enhancing plant shown in the sequence table SEQ ID No:l is resistant to iron deficiency and accumulates is transferred to plant
In object, the encoding gene overexpression for making the enhancing plant in plant be resistant to and accumulate to iron deficiency, the plant is arabidopsis.
Concrete application of the invention is constructed using technique for gene engineering35S: bZIP44Overexpression vector passes through leaching
Flower method is transferred to WT lines, makes its overexpression in wild type, and plant performance is to iron deficiency environmental resistance.It willbZIP44Base
Because promoter or induction type can be enhanced plus any before its transcription initiation nucleotide when being building up in plant expression vector
Promoter.For the ease of transgenic plant cells or plant are identified and screened, used carrier can be processed,
Make it have the antibiotic marker (kanamycins) of resistance.The plant host that is converted, can also either monocotyledon
To be dicotyledon, such as: rice, wheat, rape, corn, cucumber, tomato, poplar, turfgrass or lucerne place.Carry this
InventionbZIP44The expression vector of gene can by using Ti-plasmids, Ri plasmid, plant viral vector, direct DNA conversion,
The conventional biology methods such as microinjection, conductance, mediated by agriculture bacillus convert plant cell or tissue, and by the plant of conversion through group
Cultivation is knitted into plant.
The encoding gene that enhancing plant of the present invention is resistant to iron deficiency and accumulates can be the breeding of the crops environment of resistance to iron deficiency
Genetic resources and technical support are provided with the iron accumulation increased in crops.
According to the genome sequence of arabidopsis warehouse publication,bZIP44 (AT1G75390) be arabidopsis bZIP transcription because
Member in sub-family,bZIPIt is transcription regulatory factor generally existing in plant in recent years, belongs to the transcription of bZIP family
Regulatory factor.It is found by the applicant that iron deficiency is handledbZIP44Gene delection plant shows as sensitive to iron deficiency, this showsbZIP44Base
Because being related to the regulation to iron deficiency tolerance.For this purpose, we have studied the gene function, result of study showsbZIP44Gene
Iron deficiency processing can inhibit when missingNAS2Gene and gene transcript expression, andbzip44Iron content in mutant in stem is significantly low
In wild type, this illustrates genebZIP44Gene passes through niacinamide synzyme in regulation arabidopsisNASThe expression of encoding gene from
And regulate and control iron in the intracorporal transhipment of arabidopsis, and then adjust plant and iron deficiency is resistant to and is accumulated.
Detailed description of the invention
Fig. 1 is T-DNA insertion point schematic diagram；
Fig. 2 is bzip44Plant qRT-PCR analysis chart；
Fig. 3 is that qRT-PCR is analyzed under different time processingbZIP44The expression variation diagram of gene；
Fig. 4 isbzip44Mutant plants phenotypic analysis figure in the case where adding iron and iron deficiency；
Fig. 5 isbZIP44It is overexpressed plant phenotypic analysis figure in the case where adding iron and iron deficiency；
Fig. 6 is WT,bzip44WithbZIP44It is overexpressed plant chlorophyll content detection figure；
Fig. 7 be wild type withbzip44Iron content compare figure；
Fig. 8 be under different time Fe Deficiency wild type andbzip44In plant bodyAtNAS2Gene expression figure.
Specific embodiment
With reference to the accompanying drawing, the present invention is further described by embodiment.
A kind of encoding gene that enhancing plant is resistant to iron deficiency and accumulates shown in sequence table SEQ ID No:1 is from the U.S.
The mutant for the Arabidopsis Mutants seed screening response plant Fe Deficiency that arabidopsis germplasm resource bank obtains.
Embodiment 1,bZIP44Transgenic plant transcriptional level is identified and its is responded to iron deficiency
Compare and arabidopsis seed mutant library screened using without containing the MS culture medium of iron, obtain one plant it is quick to iron deficiency
The mutant of sense, screening and identification find that the mutant isbZIP44Gene lacks functionality mutant, is named asbzip44.Because should
Seed is T-DNA insertion mutation, carries out PCR amplification by special primer and is sequenced, sequencing result is in ncbi database Blast ratio
Right, comprehensive analysis obtains T-DNA insertion point information, referring to Fig. 1.It is right using qRT-PCRbzip44Mutant carries out transcription water
Flat identification, finds in the two mutantbZIP44The expression of gene is substantially less than wild type, sees Fig. 2.It is referring to Fig. 3
It further determines thatbZIP44Whether gene responds plant Fe Deficiency, we are extracted the wild type of different time iron deficiency processing
Simultaneously reverse transcription becomes cDNA to arabidopsis RNA, is carried out using qRT-PCRbZIP44The transcription analysis of gene, discoverybZIP44Gene
It is significantly induced by Fe Deficiency.
Referring to fig. 4, by wild type (WT) withbzip44It is seeded in the culture dish that diameter is 90 mm simultaneously, culture medium is
Add iron and the solid medium that iron is not added, be placed in 22 DEG C of constant temperature illumination boxs (photoperiod be illumination in 16 hours, 8 hours are black
Secretly) vertical culture.After two weeks, it can be observed thatbzip44Mutant with WT lines (WT) are vertical on culture dish cultivates,
Direct dibbling is shown in Fig. 4 in the comparison photo figure for vertically cultivating 2 weeks on the culture medium of MS and-Fe under normal lighting conditions respectively
Middle A；Wherein MS culture medium be to impinge upon the WT that is grown on MS culture medium andbzip44In root long (see B in Fig. 4), fresh weight (see Fig. 4
Middle C) etc. without significant difference.It is cultivated on the culture medium without containing iron in the direct dibbling of plant,bzip44It shows bright
The character of aobvious iron deficiency sensitivity.Under Fe Deficiency,bzip44Root long and fresh weight obviously than WT high.The above results showbzip44Obvious sensitivity is shown to Fe Deficiency compared with WT.
Embodiment 2 cultivates the arabidopsis of resistance to iron deficiency
1、bZIP44Gene overexpression transgenic linebZIP44-OE1、OE2Acquisition
Further to verify function of the gene in the regulation of plant Fe Deficiency, we are constructedbZIP44Gene overexpression
Carrier (35S: bZIP44).Target fragment amplification is carried out first.Wildtype Arabidopsis thaliana is normally cultivated two on MS culture medium
Week plant total serum IgE is extracted, reverse transcription synthesizes cDNA, using the cDNA of synthesis as template, carries out PCR, amplifies the purpose of sufficient amount
Product.Again using PCR product as template, carries out second and expand, it is therefore an objective to introduce restriction enzyme site.By PCR product and carrier
PCAMBIA1301 carries out digestion recycling.The good target DNA fragment of recovery purifying was connected with carrier with T4 DNA ligase again
Night.Above-mentioned connection liquid is transferred in DH5a, detection filters out positive colony and is sequenced.Sequencing result determine it is errorless after, utilize
Electroporated method is transferred to Agrobacterium GV3101.Agrobacterium GV3101 after electroporated, be coated on after activated containing it is dual anti-(Kan,
Gen LB culture medium flat plate).Random picking single colonie, spreads cultivation in the LB culture solution containing dual anti-(kan, Gen) and extracts matter
Grain.It is errorless with NcoI and II double digestion of BstE identification recombinant vector, using flower-dipping method arabidopsis thaliana transformation WT lines, to obtain
?bZIP44Gene overexpression transgenic line.
2、bZIP44It is overexpressed the identification of transgenic plant transcriptional level and compared with the tolerant to iron deficiency of WT lines
By rightbZIP44It is overexpressed transgenic plant and has carried out the identification of transcriptional level, final choiceOE1WithOE2It carries out next
Step experiment.Referring to Fig. 5, by wild type (WT) withbZIP44-OE1WithOE2It is seeded in the culture dish that diameter is 90 mm simultaneously,
Culture medium is the solid medium for adding iron and iron being not added, be placed in 22 DEG C of constant temperature illumination boxs (photoperiod is illumination in 16 hours,
8 hours dark) vertically culture.After two weeks, it can be observed that transgenic plantbZIP44-OE1WithOE2It hangs down with WT lines
Straight culture, direct dibbling is in the comparison of vertical culture 2 weeks is shone under normal lighting conditions on the culture medium for contain or not contain iron
Piece figure (see A in Fig. 5)；Wherein MS culture medium is control.The WT that is grown on MS culture medium andbZIP44-OE1、OE2In root long
(see B in Fig. 5), fresh weight (see C in Fig. 5) are lower than wild type；It is trained on the culture medium without containing iron in the direct dibbling of plant
It supports,bZIP44-OE1、OE2All show the character of apparent cadmium tolerance.Under Fe Deficiency,bZIP44-OE1、OE2Root long with
Wild type is without significant change, but fresh weight will be significantly higher than WT.The above results showbZIP44-OE1、OE2Iron deficiency is coerced compared with WT
Compel to show obviously to be resistant to.
3. bZIP44The chlorophyll content of associated materials detects
Referring to Fig. 6,bZIP44Mutant, overexpression plant and WT lines (WT) are vertically cultivated on culture dish, straight respectively
Contact kind is cultivated 2 weeks in vertical under normal lighting conditions on the culture medium for adding iron and iron deficiency.It detects respectively each under different condition
Chlorophyll content in material, as a result, it has been found that grown on adding iron culture mediumbzip44Mutant is overexpressed plant and wild
Type plant (WT) is on chlorophyll content without significant difference；And under iron deficiency condition of culture,bzip44In chlorophyll content
(see A in Fig. 6) is significantly lower than wild type；AndbZIP44-OE1、OE2In chlorophyll content (see B in Fig. 6) to be significantly higher than
Wild type.
4. wild type andbzip44Iron content analysis in mutant
Referring to Fig. 7,bzip44Mutant and WT lines (WT) are vertically cultivated on culture dish, respectively direct dibbling Yu Jiatie
It is cultivated 2 weeks with vertical under normal lighting conditions on the culture medium of iron deficiency.It is detected under different condition respectively in each material root and stem
Iron content.As a result, it has been found that under normal operation,bzip44Iron content (see A in Fig. 7) and wild type in root is without obvious poor
It is different, andbzip44Iron content (see B in Fig. 7) in stem is significantly lower than wild type；Under the conditions of iron deficiency,bzip44In stem and root
Iron content be below wild type.
5, under Fe Deficiencybzip44In WT linesNAS2Gene expression is compared
Referring to Fig. 8, take under the processing of different time iron deficiencybzip44With WT lines, RNA is extracted, carries out RT- after reversion
PCR is measured related gene expression level, discoverybzip44In plant bodyAtNAS2Gene is significantly lower than WT, showsbzip44Plant pair iron deficiency sensitivity may be withAtNAS2The inhibition of gene is related.
SEQUENCE LISTING
<110>HeFei University of Technology
<120>a kind of enhancing plant is to iron deficiency tolerance and the encoding gene accumulated and application
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<170> PatentIn version 3.3
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atcaatctca tgaccaatct ccccgaaacc ctaatctcgt ctttgtcctc atcaaagcaa 300
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gtcttagatg atggatatag atggagaaag tatggccaga aagttgtcaa aaacacccaa 720
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agattagcag atgacccaag aatggtaatc actacttacg aaggaagaca ccttcactct 840
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Claims (7)
1. the encoding gene that a kind of enhancing plant is resistant to iron deficiency and accumulates, it is characterised in that: the enhancing plant is resistance to iron deficiency
By and accumulation encoding gene DNA sequence dna as shown in sequence table SEQ ID No:l.
2. the encoding gene that a kind of enhancing plant according to claim 1 is resistant to iron deficiency and accumulates, it is characterised in that: institute
Stating the encoding gene that enhancing plant is resistant to iron deficiency and accumulates includes having with DNA sequence dna shown in sequence table SEQ ID No:l
There is 90% or more homology and encodes the DNA sequence dna of identical function protein.
3. the application for the encoding gene that a kind of enhancing plant according to claim 1 is resistant to iron deficiency and accumulates, feature
It is: the encoding gene that enhancing plant shown in the sequence table SEQ ID No:l is resistant to iron deficiency and accumulates is transferred to plant
In, the encoding gene overexpression for making the enhancing plant in plant be resistant to and accumulate to iron deficiency, the plant is arabidopsis.
4. application according to claim 3, it is characterised in that: planted enhancing shown in the sequence table SEQ ID No:l
The encoding gene that object is resistant to iron deficiency and accumulates is transferred to WT lines by flower-dipping method, makes its overexpression in wild type,
Plant performance is enhancing plant to Fe Deficiency tolerance.
5. according to the method described in claim 3, it is characterized by: the plant is monocotyledon or dicotyledon.
6. according to the method described in claim 5, it is characterized by: the monocotyledon is rice, wheat, corn or herds
Grass.
7. according to the method described in claim 5, it is characterized by: the dicotyledon is cucumber, tomato, rape, poplar
Or lucerne place.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112779270A (en) * | 2021-03-24 | 2021-05-11 | 合肥工业大学 | Functional gene for enhancing iron deficiency tolerance and iron accumulation of plants and application |
| CN112779272A (en) * | 2021-03-31 | 2021-05-11 | 合肥工业大学 | Coding gene for enhancing iron deficiency tolerance of plants and increasing iron content of plants and application |
| CN115011600A (en) * | 2022-06-17 | 2022-09-06 | 哈尔滨师范大学 | Inducible promoter GmbHLH47 promoter responding to iron deficiency stress and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112779270A (en) * | 2021-03-24 | 2021-05-11 | 合肥工业大学 | Functional gene for enhancing iron deficiency tolerance and iron accumulation of plants and application |
| CN112779270B (en) * | 2021-03-24 | 2022-03-18 | 合肥工业大学 | Functional gene for enhancing iron deficiency tolerance and iron accumulation of plants and application |
| CN112779272A (en) * | 2021-03-31 | 2021-05-11 | 合肥工业大学 | Coding gene for enhancing iron deficiency tolerance of plants and increasing iron content of plants and application |
| CN112779272B (en) * | 2021-03-31 | 2022-03-18 | 合肥工业大学 | Coding gene for enhancing iron deficiency tolerance of plants and increasing iron content of plants and application |
| CN115011600A (en) * | 2022-06-17 | 2022-09-06 | 哈尔滨师范大学 | Inducible promoter GmbHLH47 promoter responding to iron deficiency stress and application thereof |
| CN115011600B (en) * | 2022-06-17 | 2023-08-22 | 哈尔滨师范大学 | Inducible promoter GmbHLH47 promoter responding to iron deficiency stress and application thereof |
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