CN110305201A - Plant stress-resistance related gene and its coding albumen and application - Google Patents
Plant stress-resistance related gene and its coding albumen and application Download PDFInfo
- Publication number
- CN110305201A CN110305201A CN201810242690.XA CN201810242690A CN110305201A CN 110305201 A CN110305201 A CN 110305201A CN 201810242690 A CN201810242690 A CN 201810242690A CN 110305201 A CN110305201 A CN 110305201A
- Authority
- CN
- China
- Prior art keywords
- gene
- plant
- resistance
- seq
- agl16
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
-
- 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
- C12N15/8273—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 for drought, cold, salt resistance
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention identifies a kind of and anti contravariance related gene and its coding albumen and application.The present invention provides DNA nucleotide sequences shown in a kind of protein and SEQ ID NOs.9-16 that the amino acid sequence shown in SEQ ID NOs.1-8 forms；The experiment proved that Gene A/G L16 provided by the invention plays an important role in resistance, negative regulation resistance, missing causes plant more resistant to salt, drought-enduring and resistance to osmotic stress phenotype.Different plants such as knocked out in rice, corn, wheat, soybean, cotton, rape as described in gene or its homologous gene, can cultivate more resistant to salt, drought-enduring and resistance to osmotic stress new varieties.
Description
Technical field
The invention belongs to field of biotechnology, relates generally to a kind of plant stress-resistance related gene and its coding albumen and answer
With.Specifically, described " degeneration-resistant " to refer to salt tolerant, drought-enduring and resistance to osmotic stress.
Background technique
One mode herbaceous plant of the arabidopsis (Arabidopsis thaliana) as typical scientific research, it belongs to
In Dicotyledoneae, Cruciferae is distributed widely in Eurasia and the African northwestward.The research of arabidopsis gene function can
A theoretical basis is provided with the research for other crops.
The arid and salinization of soil problem of soil increasingly threaten the limited valuable land resource of human survival.Arid is ring
A plant growth factor the most serious is influenced in the stress of border.It counts according to the study, world's arid, semiarid zone account for earth land
The 33% of the ground gross area is equivalent to the summation of other natural calamities to the harm of its world food yield.Drought stress is due to lacking
The supply of few moisture, causes osmotic potential in plant to change, causes the protein structure on cell membrane to change, be metabolized in vivo disorderly
Disorderly, while a large amount of toxicity of biological active oxygen plant can be generated, seriously affects the growth and development and yield of plant.Salt stress has become entirely
The important Environmental Factors of agricultural production are influenced within the scope of ball.Data results are shown according to investigations, the area in global salt-soda soil with
Annual 1.0x106-1.5x106 hectares of speed increases.When facing salt stress, the membrane permeability of cell is changed, cell membrane
Protein structure and activity lose stabilization, while a large amount of inorganic ions of plant interior accumulation, cause the suction to other nutrients
Imbalance is received, intracellular normal metabolism is seriously destroyed, accumulates toxic metabolite, inhibit its root system or tissue growth,
Prevent plant from normally completing life cycle, seriously affects yield and quality.Therefore, understand and parse its degeneration-resistant mechanism, it is right
In the resistance for improving plant, cultivating degeneration-resistant new varieties is a vital tasks of science.
Summary of the invention
It is an object of the invention to identify that participating in the degeneration-resistant related gene of arabidopsis and its coding albumen, missing causes
The more degeneration-resistant phenotype of plant.
In the present invention, " degeneration-resistant phenotype " refers to plant under salt and osmotic stress treatment conditions, in Their Seed Germinating Period
Between compare with control there is higher seed germination rate, seedlings root, which is grown, shows as the root system that has longer or flourishing, with
And seedling growth phase is more resistant to salt and drought-enduring function." adversity gene " refers to that the plant growth and development stage has more resistant to salt, resistance to
The gene of non-irrigated and resistance to osmotic stress function.In the art, " salt tolerant " typically refers to have salt damage in growing process resistance to
The ability received；" drought-enduring " typically refers to the adaptation and resistivity of the lack of moisture caused by arid in growing process；It is " resistance to
Osmotic stress " typically refers to the tolerance changed in growing process to infiltration type caused by osmotic stress.In laboratory
In, it is generally used for adding NaCl in culture medium to examine the salt tolerance of plant, for example, adding final concentration of 100- in the medium
The NaCl of 300mM, also, examine plant resistance to osmotic stress usually by add in the medium a certain amount of mannitol come
It carries out, for example, adding the mannitol of final concentration of 100-400mM in the medium.It adds simultaneously in the medium a certain amount of
NaCl and mannitol can examine simultaneously plant salt tolerant and resistance to osmotic stress ability.It can be tested and be examined by soil drought simultaneously
The drought-resistance ability of measuring plants.For example, plant normal growth surrounding at 22-24 DEG C of temperature and 16h illumination and 8h dark condition is left
Primary rear progress Osmotic treatment (not watering) processing is sufficiently poured after two weeks in the right side, then rehydration (sufficiently pouring), counts different strains
Survival rate.It should be appreciated by those skilled in the art that different plants may have different salt tolerants, drought-enduring and resistance to osmotic stress
Ability, therefore, under the conditions of laboratory simulation, it is generally recognized that compared with the adjoining tree grown under the same conditions, if
Test plant grows well than control, and survival rate is high, then it is assumed that test plant has corresponding tolerance.
In a first aspect, a kind of adversity gene that the present invention identifies is ATAGL16, from arabidopsis, nucleotides sequence
Column selection is from as follows:
(1) nucleotide sequence of amino acid sequence shown in SEQ ID NO.1 is encoded；
(2) DNA sequence dna shown in SEQ ID NO.9；
(3) nucleotide sequence that the DNA sequence dna that can be limited in sequence table 1 under high high stringency conditions hybridizes.
The stringent condition are as follows: in 6 × SSC, the solution of 0.5%SDS, hybridize at 65 DEG C, then with 2 × SSC,
It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
DNA sequence dna shown in SEQ ID NO.9 is made of 723 nucleotide.
In second aspect, the present invention relates to the albumen of adversity gene ATAGL16 coding, and the ATAGL16 albumen is as follows
(a) or (b) or (c) albumen:
(a) albumen that the amino acid sequence shown in SEQ ID NO.1 forms；
(b) amino acid sequence shown in SEQ ID NO.1 is passed through to the substitution and missing of one or several amino acid residues
And/or it adds and there is active derivative protein identical as albumen shown in SEQ ID NO.1；
(c) other gene codings with the composition of amino acid sequence shown in SEQ ID NO.1 there is similitude to reach 50%
Above and the active albumen with albumen shown in SEQ ID NO.1；
Amino acid sequence shown in SEQ ID NO.1 is made of 240 amino acid residues.
Using the website NCBI BLAST tool, sent out by being compared with arabidopsis AGL16 amino acid sequence (SEQ ID NO.1)
It is existing rice, corn, tobacco, wheat, cotton, soybean, same with arabidopsis AGL16 amino acid sequence (SEQ ID NO.1) in rape
The albumen (as shown in SEQ ID NOs.2-8) in source, thus it is speculated that they also have and arabidopsis AGL16 amino acid sequence (SEQ ID
NO.1) similar function.Also, the present inventor demonstrates the tool of the homologous sequence (SEQ ID NO.2) in rice in embodiment
There is the similar function with arabidopsis AGL16 amino acid sequence (SEQ ID NO.1).Therefore, with arabidopsis AGL16 amino acid sequence
(SEQ ID NO.1) homologous protein sequence and its coding nucleotide sequence is also within the scope of the invention.It can will be with quasi- south
Mustard AGL16 amino acid sequence (SEQ ID NO.1) is homologous and there is the active albumen of identity function to be referred to as AGL16 albumen, will
The nucleotide sequence for encoding it is referred to as AGL16 gene.
In one embodiment, the AGL16 gene of AGL16 albumen shown in SEQ ID NOs.2-8 is encoded respectively such as
Shown in SEQ ID NOs.9-16.
Adversity gene AGL16 gene and its homologous gene of the table 1. from arabidopsis
In the third aspect, the present invention relates to the primer for any fragment amplification of AGL16, the expression comprising AGL16 gene
Box or recombinant plant expression vector, AGL16 transgenic cell line, the host strain comprising AGL16 gene.Wherein the host strain can
For eukaryon mushroom or protokaryon mushroom, such as, but not limited to, Agrobacterium, saccharomycete, Escherichia coli etc..
In fourth aspect, the present invention relates to adversity gene AGL16 to cultivate the application in the plant with resistance.This hair
Bright people discovery, AGL16 gene is lacked in plant can be improved the salt tolerant of plant, drought-enduring and resistance to osmotic stress ability.
It should be appreciated by those skilled in the art that the plant of missing ATAGL16 gene can pass through gene knockout (knock-
Out) technology obtains.
At the 5th aspect, the present invention relates to a kind of methods cultivated and have the plant variety of resistance, knock out in plant
AGL16 gene or the higher gene of its homology obtain the transgenic line with resistance, wherein " resistance " refers to
Salt tolerant, drought-enduring and resistance to osmotic stress ability.Specifically, the method includes the following steps:
(1) selection coding SEQ ID NO.1 shown in amino acid sequence nucleotide sequence or target plant in SEQ
The highest gene of amino acid alignment similarity shown in ID NO.1 is as homologous gene, by the nucleotide sequence or together
The segment of source gene is building up in gene knockout carrier；
(2) with the cell or tissue of the gene knockout carrier conversion target plant built in step (1), transgenosis is obtained
Plant；
(3) identification that the transgenic plant of acquisition is carried out to expression quantity on resistance screening and transcriptional level, determines purpose base
Because being knocked, so that obtaining transgenosis knocks out strain；
Obtained transgenosis knocks out strain to be had more in the culture medium with 150mM NaCl and 250mM treatment with mannitol
High seed germination rate and more flourishing root system have good salt tolerant and resistance to osmotic stress ability.Meanwhile in soil water experiment
In, with the salt water irrigation containing 250mM NaCl and Osmotic treatment (not watering) after two weeks, survival rate is high, has salt tolerant and drought-enduring
Function.
For example, coding can be used if to cultivate the rice varieties with salt tolerant, drought-enduring and resistance to osmotic stress ability
The nucleotide sequence building gene knockout carrier of amino acid sequence shown in SEQ ID NO.2, the cell or tissue of rice transformation,
Transgenic rice plant is obtained, the transgenic rice plant of acquisition is carried out to the mirror of expression quantity on resistance screening and transcriptional level
It is fixed, determine that target gene has been knocked, so that obtaining transgenosis knocks out strain, obtained transgenosis knock out strain with
There is higher seed germination rate and more flourishing root system on the culture medium of 150mM NaCl and 250mM treatment with mannitol, have
Good salt tolerant and resistance to osmotic stress ability, meanwhile, with salt water irrigation and Osmotic treatment, survival rate is high, has salt resistance and drought resisting
Ability.Thus the rice varieties with salt tolerant, drought-enduring and resistance to osmotic stress are obtained.Cultivation for other species, method
It is similar, in selection target species with the highest gene conduct of amino acid alignment similarity shown in SEQ ID NO.1
The segment of the nucleotide sequence or homologous gene is building up in gene knockout carrier, converts target species by homologous gene
Cell or tissue obtains the plant that transgenosis knocks out, and further verifies resulting transgenosis and knocks out plant with Osmotic Stress Tolerance
Property.
In a preferred embodiment, the method for the plant for cultivating salt tolerant, drought-enduring and resistance to osmotic stress includes
Following step: (1) in selection target plant with SEQ ID NO.1 shown in amino acid alignment similarity highest gene make
For homologous gene, the segment of homologous gene is building up in gene knockout carrier, such as CRISPR/Cas9 carrier；
(2) gene knockout carrier built in step (1) is carried by Agrobacterium-mediated genetic transformation, plant virus
The conventional methods such as body is system converting, direct exogenous DNA conversion (conductance, particle gun) carry out turning for different plant cells or tissue
Change, obtains transgenic plant；
(3) transgenic plant of acquisition is carried out to the identification of expression quantity on resistance screening and transcriptional level, it has been determined that the base
Because being knocked, do not have function, so that obtaining transgenosis knocks out strain；
(4) it after the seed of seed and control material that the transgenosis of acquisition knocks out strain being carried out sterile cleaning, is laid on and contains
On the culture medium for having 150mM NaCl and 250mM mannitol, the seed germination rate of comparison and contrast；
(5) it after the seed of seed and control material that the transgenosis of acquisition knocks out strain being carried out sterile cleaning, is laid on just
It in normal culture medium, is transferred to after 4 days to be grown on the culture medium containing 150mM NaCl and 250mM mannitol, or directly
It is laid in the culture medium containing 150mM NaCl and 250mM mannitol, counts under ground portion extent of the root system and overground part leaflet
The size of son；
(6) seedling that the transgenosis of acquisition knocks out strain and control material is carried out in the soil at salt water irrigation or arid
Reason, counts the survival rate of plant, the higher strain of survival rate is as salt tolerant and drought-enduring plant；
(7) by seed germination rate is higher on the culture medium containing 150mM NaCl and 250mM treatment with mannitol, root system more
Prosperity, plant of the more strong strain of aerial part as salt tolerant and resistance to osmotic stress.
Plant suitable for the method includes, but are not limited to arabidopsis, tobacco, rice, wheat, corn, cotton, rape
Or soybean etc., preferably arabidopsis and rice.
Plant expression vector containing AGL16 of the present invention can be by using Agrobacterium-mediated genetic transformation, plant virus
The conventional methods such as gene transfer using various vectors, direct exogenous DNA conversion (conductance, particle gun) carry out turn of different plant cells or tissue
Change.Wherein dicotyledon (arabidopsis, cotton, rape, soybean etc.) and monocotyledon (wheat, rice, corn etc.) all may be used
Using as the host being converted.
In the scheme that the present invention is implemented, the degeneration-resistant Arabidopsis Mutants are Salk_104701, in the present invention
It is named as agl16 deletion mutant；The target plant is wildtype Arabidopsis thaliana (Arabidopsis thaliana).
The function of the plant stress-resistance, which refers to, simulates osmotic stress and salt (NaCl) processing at mannitol (mannitol)
Under conditions of have higher seed germination rate and more flourishing root system.Meanwhile in soil water experiment, with contain 250mM NaCl
Salt water irrigation and Osmotic treatment (not watering) after two weeks, plant have higher survival rate.
We obtain a T-DNA from arabidopsis seed bank ABRC and disclose missing on AT3G57230 gene extron
Mutant Salk_104701.From TAIR query site to the arabidopsis gene is named as ATAGL16, will in later experiments
Salk_104701 experimental material is named as agl16 deletion mutant.Meanwhile constructing 35S promoter driving AGL16 gene table
The overexpression strain OX reached.Using arabidopsis wild type Col-0 as control, implements seed and sprouts experiment and the experiment of root system phenotype,
The ratio and main root that germination rate, the cotyledon of statistics seed turn green are long.As a result, it has been found that AGL16 is the degeneration-resistant transcription of a negative regulation
The factor.In order to further verify the function of ATAGL16, We conducted the soil salt treatment of aerial part and arid experiment, system
Survival rate, stomatal frequency and the stomatal conductance of its different strain are counted, as a result middle agl16 deletion mutant is showed relative to wild type
To be more degeneration-resistant, OX be overexpressed strain occur more intolerant to phenotype, these results all demonstrate ATAGL16 gene and play in degeneration-resistant
Particularly important effect is degeneration-resistant negative regulatory factor, lacks the phenotype for causing plant more degeneration-resistant.
In order to cultivate other anti-adversities, the gene homologous with arabidopsis ATAGL16 has been knocked out we obtain rice
Homozygous lines have carried out functional analysis to it.The result shows that under salt stress and osmotic stress treatment conditions, the AGL16 of rice
Afunction strain has more flourishing root system and more strong aerial part, hence it is evident that has the function of degeneration-resistant.This result is more
It demonstrates to knock out in plant and can cultivate new anti-adversity with arabidopsis ATAGL16 homologous gene.
In conclusion present invention offer is following:
1. a kind of relevant albumen of stress resistance of plant, selected from following:
(a) albumen that the amino acid sequence shown in SEQ ID NOs.1-8 forms；
(b) amino acid sequence shown in SEQ ID NOs.1-8 by the substitution of one or several amino acid residues and is lacked
It loses and/or adds and there is active derivative protein identical as SEQ ID NOs.1-8；Or
(c) other forming with amino acid sequence shown in SEQ ID NOs.1-8 for gene coding reach with similitude
50% or more albumen；
Wherein the resistance refers to the characteristic of salt tolerant, drought-enduring and resistance to osmotic stress.
2. the gene of albumen described in coding the 1st.
3. such as the 2nd gene, wherein the gene be it is following any one:
(1) DNA sequence dna shown in SEQ ID NO.9-16；Or
(2) nucleotide sequence that can hybridize with DNA sequence dna shown in SEQ ID NO.9-16 under high high stringency conditions；
The stringent condition are as follows: in 6 × SSC, the solution of 0.5%SDS, hybridize at 65 DEG C, then with 2 × SSC,
It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
4. plant recombinant vector or expression cassette containing gene described in the 2nd or 3.
5. the host cell comprising plant recombinant vector described in gene described in the 2nd or 3 or the 4th or expression cassette,
Wherein the host cell is microbial cell, preferably agrobatcerium cell or Bacillus coli cells.
6. albumen described in the 1st, gene described in the 2nd or 3, recombinant vector or expression cassette or the 5th described in the 4th
Application of the host cell in the plant for cultivating salt tolerant, drought-enduring and resistance to osmotic stress described in.
7. a kind of method for the plant for cultivating salt tolerant, drought-enduring and resistance to osmotic stress, the method includes the following steps:
(1) selection coding SEQ ID NOs.1-8 shown in amino acid sequence nucleotide sequence or target plant in
The highest gene of amino acid alignment similarity shown in SEQ ID NO.1 is as homologous gene, by the nucleotide sequence
Or the segment of homologous gene is building up in gene knockout carrier；
(2) with the cell or tissue of the gene knockout carrier conversion target plant built in step (1), transgenosis is obtained
Plant；
(3) identification that the transgenic plant of acquisition is carried out to expression quantity on resistance screening and transcriptional level, determines purpose base
Because being knocked, so that obtaining transgenosis knocks out strain；
Obtained transgenosis knocks out strain to be had more in the culture medium with 150mM NaCl and 250mM treatment with mannitol
High seed germination rate and more flourishing root system have good salt tolerant and resistance to osmotic stress ability, in soil water experiment, with containing
After two weeks, n plant survival rate is high for the salt water irrigation and Osmotic treatment (not watering) for having 250mM NaCl, has salt tolerant and drought-enduring
Function.
8. method described in the 7th, wherein gene knockout carrier used in step (1) is CRISPR/Cas9 carrier.
9. method described in the 7th, wherein the plant is monocotyledon or dicotyledon.
10. method described in the 8th, wherein the plant is selected from arabidopsis, tobacco, rice, wheat, corn, cotton, oil
Dish or soybean.
11. method described in the 10th, wherein the plant is arabidopsis or rice.
Detailed description of the invention
The insertion point schematic diagram of Fig. 1 (A) Salk_104701；
(B) it is identified on the genome and rna level of Salk_104701；
(C) determine Salk_104701 for missing Mutants homozygous.
Fig. 2 (A) is overexpressed the amplification of strain AGL16-CDS target fragment；
(B) by Gateway system, the digestion that AGL16-CDS target fragment is building up to final carrier pCB2004 is identified
Figure, arrow indicate digestion positive fragment；
(C) building is correctly named as the map schematic diagram of the final carrier of pCB2004-AGL16；
(D) strain AT3G57230 gene is overexpressed in the expression identification of rna level.
Fig. 3 (A) agl16, OX and Co1-0 seed in normal MS culture medium (preparation method is shown in embodiment 3), contain
120mM NaCl, 150mM NaCl, 250mM mannitol MS culture medium on sprouting situation；
(B) under the conditions of different disposal different strain seed germination rates statistical result；
(C) different strain cotyledons turn the statistical result of green rate under the conditions of different disposal.
Fig. 4 (A) arabidopsis agl16 deletion mutant, AGL16 are overexpressed strain (OX) and wildtype Arabidopsis thaliana Col-0 children
Seedling normal MS culture medium, containing 120mM NaCl, 150mMNaCl, 250mM mannitol MS culture medium on root growth
Situation；
(B) under the conditions of different disposal different strain seedling main root length statistical result.
Fig. 5 (A) arabidopsis agl16 deletion mutant, AGL16 are overexpressed strain (OX) and wildtype Arabidopsis thaliana Col-0 material
Expect in normal soil and with the experiment of 250mM NaCl salt water irrigation；
(B) the survival rate statistics of different strain salt water irrigation experiments；
(C) different strain soil drought processing；
(D) survival rate of seedling counts in different strain soil drought experiments；
(E) statistics of the in vitro percentage of water loss of different strain blades；
(F) statistics of different strain Stoma of Leaves density；
(G) statistical result in abscisic acid (ABA) the processing posterior spiracle aperture of various concentration.
The amplification of Fig. 6 (A) AGL16-pro target fragment；
(B) AGL16pro::GUS vector construction (pCB308R) cleavage map, arrow indicate digestion positive fragment；
(C) building is correctly named as the map schematic diagram of the final carrier of pCB308R-AGL16；
(D)-(O) the different times tissue expression of arabidopsis AGL16 gene.
The amplification of Fig. 7 (A) AGL16-GFP target fragment；
(B) pGWB5-AGL16 vector construction (pGWB5) cleavage map, arrow indicate digestion positive fragment；
(C) building is correctly named as the map schematic diagram of the final carrier of pGWB5-AGL16；
(D) transgenic line of the pGWB5-AGL16 obtained, is observed by laser co-focusing, and arabidopsis AGL16 albumen is fixed
Positioned at nucleus.
Fig. 8 (A) wild type (WT) rice and afunction mutant Osagll6 material in salt and treatment with mannitol item
Phenotypic analysis under part；
(B)-(D) wild type (WT) rice and afunction mutant Osagl16 material in normal condition (MS culture
Base), the root under salt (the MS culture medium of the NaCl containing 100mM) and mannitol (the MS culture medium of the mannitol containing 200mM) treatment conditions
Long statistical chart.
Specific embodiment
Technical solution of the present invention is illustrated by the following examples.However, these embodiments are only used for illustrating
Purpose, be not meant to that the scope of the present invention is limited to this.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Experimental material used in following embodiments, reagent etc., are commercially available unless otherwise specified.
Identification of the 1 arabidopsis agl16 deletion mutant of embodiment on DNA and rna level
Firstly, from arabidopsis seed bank ABRC (Arabidopsis Biological Resource Center, The
43210 USA of Ohio State University Rightmire Hall 1060Carmack Road, Columbus, OH)
Obtain deletion mutant Salk_ of the T-DNA insertion AT3G57230 gene on the exon of terminator codon
104701 (Figure 1A).It takes seedling or adult seedling to carry out the extracting of DNA of plants, obtains genomic templates, passed through using primer LP and RP
PCR amplification is crossed, genomic level identification is completed, obtains homozygote strain (Figure 1B).The extracting for passing through RNA again afterwards, is purified
Then RNA afterwards carries out reverse transcription and obtains cDNA template, using primer P1 and P2, using Ubiqutin5 gene as internal reference, pass through
QRT-PCR detection, as a result, it has been found that AT3G57230 gene is hardly expressed in all strains of agl16, i.e. 5 strains of agl16
System is afunction homozygote strain (Fig. 1 C).
The extracting method of genome (DNA): (1) the fresh arabidopsis organization material for weighing about 50-100mg is put into mortar
In, the DNA extraction buffer that 420uL is added thereto grinds material.After the completion of grinding, 5ul is added into mortar
Liquid in mortar is transferred in the EP pipe of 1.5mL rapidly, is mixed by inversion back and forth by the RNA enzyme of 10mg/mL, places 65 DEG C of water
Bath, warm bath 10-15 minutes, taking out to be mixed by inversion every 3 minutes reacted it sufficiently.(2) 65 DEG C of water-baths take out sample, to
Isometric phenol and chloroform are added in EP pipe and removes extra albumen.(3) 12000rpm revolving speed high speed centrifugation 10 minutes, then take
300uL supernatant is transferred to new 1.5mLEP pipe, then Xiang Guanli is separately added into the isopropanol and 0.1 times of volume 3M of 0.7 times of volume
Sodium acetate promotes DNA precipitating.(4) with 12000rpm revolving speed high speed centrifugation 10 minutes, supernatant is abandoned, 1mL70% alcohol is then added
DNA is cleaned to precipitate twice.(5) dry precipitating at room temperature, to pipe in be added 20uL ddH2O dissolving DNA precipitating, as genome
Template.
PCR primer is as follows:
Upstream primer (LR): 5 '-CCATGCATTTTCGGTTTTATG-3 '
Downstream primer (RP): 5 '-CTCTTCTTTTGCCTTTTGCAG-3 '
20uL PCR amplification system is as follows: (wherein 2x Taq Master mix enzyme is only praised biotechnology purchased from Nanjing promise and is had
Limit company)
| 2x Taq Master mix enzyme | 10uL |
| 20uM LP | 0.5uL |
| 20uM RP | 0.5uL |
| DNA profiling | 0.5uL |
| ddH2O | 8.5uL |
PCR program are as follows: first 95 DEG C initial denaturation 3 minutes, 95 DEG C be denaturalized 30 seconds, 56 DEG C of annealing temperature, 72 DEG C of conditions are downward
Stretch the time 30 seconds, recurring number is set as 40, finally keeps 72 DEG C sufficiently to extend 10 minutes, is stored in 25 DEG C after the completion.
The extracting method of RNA: (1) the fresh arabidopsis organization material for weighing about 50-100mg is put into mortar, then
Appropriate liquid nitrogen is added, material is ground to powder, is rapidly added TRIZOL reagent (the limited public affairs of Beijing Quan Shijin biotechnology of 1mL
Department), after melting, continues to grind repeatedly under low temperature, then the tissue fluid ground is transferred in the sterile EP tube of 1.5mL.(2)
The beta -mercaptoethanol that 10uL is added in Xiang Guanzhong helps to remove the albumen in tissue fluid, is stored at room temperature 5 minutes, sufficiently reacts.(3) it fills
After dividing reaction, the chloroform of 200uL is added into pipe, gently overturns and mixes for several times, after being stored at room temperature 3-5 minutes, with 12000rpm
4 DEG C of revolving speed under the conditions of be centrifuged 10-15 minutes.(4) supernatant for drawing about 250uL is transferred to the EP pipe of new 1.5mL, is added
After isometric isopropanol mixes, 10 minutes are placed at room temperature for, is centrifuged 10 minutes under the conditions of 4 DEG C of 12000rpm revolving speed.(5) in removal
Clearly, the ice alcohol washes RNA mono- to twice of 1mL 75% is added into pipe.(6) 12000rpm revolving speed is centrifuged 5 under the conditions of 4 DEG C
Minute, supernatant is abandoned, RNA is dried, the processed deionized water of DEPC of 20uL is added.
The reverse transcription of RNA: it takes suitable RNA template to carry out reverse transcription and forms cDNA, be used for fluorescence quantitative PCR detection.Make
It is TaKaRa (Prime Script RT reagent Kit) reverse transcription reagent box.Reverse transcription system is following (20uL):
By prepared reverse transcription system, put to 37 DEG C water-bath warm bath 2 hours, complete reverse transcription after, after reaction
Template of the cDNA as quantitative fluorescent PCR (qRT-PCR).
QRT-PCR primer is as follows:
Upstream primer (P1): 5 '-GACCTCCACAAGAAAGTAAACC-3 '
Downstream primer (P2): 5 '-GCAATGAAGGAAAAATAGTTGAGT-3 '
Ubiqutin5 gene primer sequence are as follows:
Upstream primer LP:5 '-AGAAGATCAAGCACAAGCAT-3 ',
Downstream primer RP:5 '-CAGATCAAGCTTCAACTCCT-3 '.
The SYBR green reagent that fluorescence real-time quantitative PCR uses comes from TaKaRa company, passes through fluorescence real-time quantitative PCR
Instrument (StepOne real-time PCR system) detects ATAGL16 gene in the expression quantity of transcriptional level.(20uL as follows
System):
| SYBR green | 10uL |
| 20uM P1 | 0.5uL |
| 20uM P2 | 0.5uL |
| Template | 0.5uL |
| ddH2O | 9.5uL |
The amplification condition of fluorescence real-time quantitative PCR: 95 DEG C initial denaturation 20 seconds, 95 DEG C be denaturalized 10 seconds, 60 DEG C annealing and extend
30 seconds, 40 recurring numbers.
The acquisition of 2 arabidopsis AGL16 of embodiment overexpression strain OX
The code area AGL16 (CDS) the primer LP and RP of design with full connector (attB) connection first, is obtained by PCR
Target fragment (Fig. 2A), and carried out glue recycling.Using the method (Gateway Technology) of high flux construction carrier
(Wang Zonggui, Zheng Wenling, Marvin's are beautiful；Gateway-cloning system: the new development Chinese biological engineering magazine of DNA recombinant technique
(2003), the 7th phase of volume 23), by the AGL16-CDS target fragment of recycling, utilize GatewayR BP ClonaseTM II
The shuttle vector of AGL16-CDS recombinant clone to pDONR207 (is purchased from by Enzyme Mix kit (Invitrogen)
Invitrogen in), again by GatewayTMClone technology is by intermediate shuttle vector recombinant clone to pCB2004 end carrier
(Xiang et al., 1999；Lei et al., 2007) it between attR1 and attR2, is obtained by digestion identification correct
PCB2004-AGL16 end carrier (Fig. 2 B and C).Again after sequencing determines whole carrier without mutation, it will construct successfully correct
PCB2004-AGL16 carrier electricity is transferred in Agrobacterium C58C1 competence, is converted by dip-flower, and transgenic seed is obtained.It will receive
T0 be taped against herbicide containing 50mg/L (glufosinate ammonium, commercial entitled Liberty, French Aventis for seed
Crop science company) MS culture medium (preparation method sees below embodiment 3) on screening be transferred to the positive strain of AtAGL16 gene
System.The T1 positive seed of antiweed is grown into maturation in the soil, then single plant sowing.Again T1 is taped against to contain for seed and be removed
On the MS culture medium of careless agent, chooses the positive seedling isolated and continue to plant single plant sowing of going down, obtain T2 for seed.It takes containing removing
The homozygous lines sprouted entirely on careless agent MS culture medium carry out the extracting of RNA.The specific behaviour of the extracting of RNA, reverse transcription and qRT-PCR
Make such as embodiment 1, the primer of the upstream primer (P1), downstream primer (P2) of AtAGL16 gene and reference gene is all in qRT-PCR
With it is consistent in embodiment 1.The result shows that identifying the expression quantity of AtAGL16 gene in each strain by qRT-PCR, we are obtained
Multiple overexpression homozygote strain OX (Fig. 2 D).
During experimental plant vector construction, the amplification body for the KOD FX enzyme (purchased from TOYOBO) that target fragment amplification uses
System, following (50uL system):
| 2x KOD buffer | 25uL |
| 2.5mM dNTP | 5uL |
| 20uM P1 | 1uL |
| 20uM P2 | 1uL |
| KOD FX | 1uL |
| Template | 1uL |
| ddH2O | 16uL |
PCR primer is as follows:
Upstream primer (LP) (lowercase indicates attB1 connector):
5’-ggggacaagtttgtacaaaaaagcaggctATGGGAAGGGGCAAGATCGC-3’
Downstream primer (RP) (lowercase indicates attB2 connector):
5’-ggggaccactttgtacaagaaagctgggtTTATGCAATGAAGGAAAAATAG-3’
Use the PCR amplification condition of KOD FX enzyme: first 98 DEG C initial denaturation 3 minutes, 98 DEG C be denaturalized 10 seconds, annealing temperature 60
DEG C, extend 30 seconds under the conditions of 68 DEG C, recurring number is set as 40, finally keeps 68 DEG C sufficiently to extend 10 minutes, is stored in after the completion
25℃。
The experiment of 3 Seed Germination of Arabidopsis Pumila of embodiment
Firstly, agl16 (is purchased from the Salk_104701 mutant of arabidopsis seed bank ABRC, for missing agl16's
Mutants homozygous), OX (embodiment 2 construct overexpression AGL16 homozygote strain), Col-0 (i.e. wildtype Arabidopsis thaliana, make
For experiment contrast material) seed is dispensed into the EP pipe of 2mL, guarantees a small amount of multitube, the 10%Bleach of 1mL is added into pipe
(i.e. 100mL Bleach is made of 84 thimerosal of 10mL blue moon and 90mL pure water) is placed in the revolving speed cleaning 15 of shaking table 230rpm
Minute, then the pure water after superclean bench sterilizing cleans 4 times again, after the completion, is put in vernalization two under 4 DEG C of dark conditions
It.It chooses full seed point after vernalization and is being added with 0mM, 120mM NaCl, 150mM NaCl, 250mM mannitol respectively
In MS culture medium, which completes in superclean bench.After the completion of point seed, ware is placed in 22-24 DEG C of temperature and 16h light
According to and the culture of 8h dark condition between observe culture.It is calculated second day after seed, it is fixed to be stretched out with the radicle of seed
Justice is sprouts, and the seed germination rate and cotyledon of every day turns green number in statistics one week.
As a result, it has been found that the number of germination rate and cotyledon greening of 4 strains on MS culture dish is substantially without difference.However
In the salt containing various concentration and mannitol culture medium, the seed germination rate of agl16 deletion mutant and cotyledon greening
Ratio is significantly higher than wild type, while two OX overexpression strains are bright relative to the germination rate of wild type and the number of cotyledon greening
Show lower (Fig. 3 A, B, C).The result shows AyAGL16 gene plays very important function in osmotic stress, one
The denier gene delection, plant to extraneous osmotic stress show as with more resistant to function；If the gene overexpression, if plant
Osmotic stress is shown as more sensitive, is unfavorable for the sprouting and growth of plant.
MS culture medium prescription is following (1L):
10x a great number of elements (MSmax) it is formulated following (1L):
| NH4NO3 | 16.5g |
| KNO3 | 19g |
| CaCl2·2H2O | 4.40g |
| MgSO4·7H2O | 3.7g |
| KH2PO4 | 1.7g |
| Water | Complement to 1L |
100x microelement (MSmin) it is formulated following (1L):
| KI | 0.083g |
| H3BO3 | 0.62g |
| MnSO4·4H2O | 2.23g |
| ZnSO4·7H2O | 0.86g |
| Na2MoO4·2H2O | 0.025g |
| CuSO4·5H2O | 0.0025g |
| CoCl2·6H2O | 0.0025g |
| Water | Complement to 1L |
100x molysite is formulated following (1L):
| FeSO4·7H2O | 2.78g |
| Na2-EDTA·2H2O | 3.73g |
| Water | Complement to 1L |
4 arabidopsis root system phenotypic analysis of embodiment
Firstly, being put in 4 DEG C of dark conditions after cleaning agl16, OX, Col-0 seed 15 minutes as the step of embodiment 3
Lower vernalization two days.After completing vernalization, seed is first layered on MS culture medium ware by we, by growth in 4 days, then selects cotyledon
With the consistent different strain seedling of root long be transferred into respectively containing various concentration 0mM, 120mM NaCl, 150mM NaCl and
On the MS culture medium ware of 250mM mannitol, guarantee that the seedling numbers of 4 strains on the ware of each processing are identical.Work as from what is transplanted seedlings
It starts, and observes daily and the main root for counting different materials is long, persistently counts one week.
As a result, it has been found that main root length of 4 strains on normal MS culture dish is consistent with aerial part cotyledon growing way, do not have
Notable difference.However in the processing culture dish of salt (NaCl) and mannitol containing various concentration, agl16 deletion mutant
Main root is considerably longer than wild type, and the main root that two OX are overexpressed strain is relatively shorter wild type, and aerial part also has identical
Trend (Fig. 4 A, B).The result shows AtAGL16 genes to play the growth of root system in salt stress and osmotic stress
Important role.Once the gene delection, the main root of seedling has the function more resistant to salt and resistance to osmotic stress；If the gene
It is overexpressed, the main root of seedling then shows as salt stress and osmotic stress more sensitive, inhibits the elongation of main root.This result is more
Demonstrate in embodiment 3 as a result, illustrating that AtAGL16 gene plays a part of negative regulation in salt stress and osmotic stress.
The phenotypic analysis of 5 soil salt treatment of embodiment and Osmotic treatment arabidopsis aerial part
The result of embodiment 2 and 3 sufficiently demonstrates arabidopsis AtAGL16 gene in seed sprouting and growth of seedling stage pair
Osmotic stress has the function of negative regulation, and on this basis, we continue to explore in seedling period, and whether AtAGL16 gene
Similarly play important function.Firstly, we are taped against MS culture medium for after agl16, OX and Col-0 wash seeds vernalization
In, it is to be generated grow to 7 days after, select seedling state consistency and preferably to move in soil, guarantee each small basin soil slackness as far as possible
Unanimously, identical per small basin seedling numbers, it keeps growing under 22-24 DEG C of temperature and 16h illumination and 8h dark condition.When growing into 4
Or so week and the also non-bolting of Miao Shang, photograph to record the growth conditions of seedling under normal growing conditions, then use 250mM NaCl salt
Water pours, and guarantees that each small basin fully absorbs salt water naturally, then observes the variation (Fig. 5 A) of different strain aerial part blades,
The adult seedling that leaf largely whitens is defined as death.When saline treatment after two weeks, count the survival rate of 4 strain materials simultaneously
(Fig. 5 B) is photographed to record to it, the results showed that agl16 deletion mutant yellow leaf bleaches until dead number is far low
In WT strain, and two overexpression strain mortalities are much higher than control simultaneously.This result verification agl16 missing is prominent
Variant has the function of salt tolerant.
In soil drought experiment, the material preparation of early period is identical with salt water irrigation experiment, to growth of seedling to a 4th week left side
The right side is poured once with water, is made sufficiently to absorb water per small basin, is photographed to record the growth conditions of seedling under normal growing conditions.Exclude other
Disturbing factor carries out Osmotic treatment afterwards, observes the variation of different strain aerial part blades.It is different after Osmotic treatment 15 days
Occur apparent phenotype between strain aerial part and photographs to record.Abundant watering pours water again makes its adult seedling rehydration, counts 4
The survival rate of strain material simultaneously photographs to record (Fig. 5 C, D).Find that the survival rate of agll6 deletion mutant strain is far high after rehydration
In wild type, and the survival rate of two overexpression strains is lower than control simultaneously.The in vitro dehydration experiment of blade is carried out simultaneously, as far as possible
Guarantee the blade for taking the size of different strain same positions close, is placed in the environment of temperature and humidity stability, early period was every 15 minutes
The weight of blade is weighed respectively, and the later period can slow down frequency.Its statistical result shows that after dehydration 3 hours, agl16 is lacked
The leaves water loss rate of mutant is substantially less than wild type, is overexpressed OX 17-5 strain percentage of water loss and is significantly higher than wild type, and crosses table
It is slightly below OX 17-5 strain up to OX 24-15 strain, but is higher than control, equal significant difference (Fig. 5 E).This result is further tested
The function that agl16 deletion mutant has drought resisting is demonstrate,proved.
The mechanism of plant drought is directly related with the degree of leading of the density of stomata and stomata.Take the surrounding planted in soil it is big,
Agl16, OX and Col-0 the adult seedling leaf of non-bolting are several, and lower epidermis of tearing, film-making is placed under microscope, choose identical
Position observation statistics stoma number.Fig. 5 F's the result shows that agl16 deletion mutant stomatal frequency be substantially less than compare, cross table
Stomatal frequency up to OX 17-5 strain is significantly higher than control.Stomata is carried out simultaneously to the anti-of abscisic acid (ABA is purchased from Sigma)
Answer sensitivity level to test, first with stomatal opening buffer handle blade 4-8 hour, open stomata sufficiently, then addition 0,
5,20uMABA continues with blade, and the aperture of stomatal opening is counted after reaction 2 hours.When 0uM ABA processing, four strains
Air vent aperture indifference, however when with 5,20uM ABA handle when, the air vent aperture size of agl16 deletion mutant is significantly small
In wild type, and the air vent aperture for being overexpressed OX 24-15 strain is noticeably greater than wild type (Fig. 5 G).Agl16 deletion mutant
Stomatal frequency is less, and air vent aperture is smaller more to save more moisture, prevents more moisture from evaporating, so more drought resisting.
The stronger AtAGL16 gene that demonstrates of all results in Fig. 5 plays a part of negative regulation in degeneration-resistant.
Stomatal opening buffer formulation is as follows: (1L) PH is adjusted to 6.15
| KCl | 3.726g |
| K+-MES | 1.952g |
| CaCl2 | 0.0012g |
| Water | Complement to 1L |
That is, the stomatal opening buffer includes 50mM KCl, 10mM K+- MES and 10uM CaCl2, pH 6.15.
ABA handles buffer formulation: (1L) PH is adjusted to 5.6
| KCl | 0.746g |
| K+-MES | 0.976g |
| CaCl2 | 0.006g |
| Water | Complement to 1L |
That is, the ABA processing buffer includes 10mM KCl, 5mM K+- MES and 50uM CaCl2, pH 5.6.
The different times tissue expression of 6 arabidopsis AGL16 gene of embodiment
Design primer at AGL16 promoter region 1500-2000bp is chosen first, and the front end upstream primer LP band attB1 connects
Head, the front end downstream primer RP band attB2 connector, using the KOD enzyme PCR amplification system in embodiment 2, acquisition size is 1700bp
Target fragment (AGL16-pro) (Fig. 6 A), and carried out glue recycling.Utilize GatewayR BP ClonaseTM II
AGL16-pro is passed through 42 DEG C of heat-shock transformed recombinant clone wearing to pDONR207 by Enzyme Mix kit (Invitrogen)
In shuttle carrier (being purchased from Invitrogen), again by GatewayTMClone technology arrives intermediate shuttle vector recombinant clone
PCB308R end carrier (Xiang et al., 1999；Lei et al., 2007) it between attR1 and attR2, is reflected by digestion
Surely correct pCB308R-AGL16 end carrier (Fig. 6 B, C) is obtained.Determine whole carrier without mutation through sequencing again.It will construct successful
PCB308R-AGL16 carrier electricity is transferred in Agrobacterium C58C1 competence, is converted by dip-flower, and transgenic seed is obtained.It will receive
T0 be taped against for seed and filter out the report strain containing resistant fusion gus gene on the MS culture medium of the herbicide containing 50mg/L
System.The T1 positive seed of antiweed is grown into maturation in the soil, then single plant sowing.Again T1 is taped against to contain for seed and be removed
On the MS culture medium of careless agent, chooses the positive seedling isolated and continue to plant single plant sowing acquisition T2 for seed.It takes containing removing
The homozygote strain sprouted entirely on careless agent MS culture medium carries out subsequent GUS dyeing, and observation arabidopsis is sprouted in seed, seedling and
The spatial and temporal expression situation of seedling different times.By GUS dyeing the results show that seed sprout the stage radicle stretch out can
Color (Fig. 6 D, E)；In seedling two panels, four, six, eight leaf periods, mainly root main root point and center pillar and and blade all
There is expression (Fig. 6 F-I, M, N)；In seedling period, in lotus throne leaf, stem leaf, flower, fruit folder and stomata have certain expression (figure
6J-L, O).
PCR primer is as follows:
Upstream primer (LR) (lowercase indicates attB1 connector):
5’-ggggacaagtttgtacaaaaaagcaggctAACTATGAACTTGGTAGCTCTTG-3’
Downstream primer (RP) (lowercase indicates attB2 connector):
5’-ggggaccactttgtacaagaaagctgggt TTCTGCTTCTATCACTTTTACAC-3’
The preparation ingredient (100mL) of GUS dye liquor:
| X-Gluc | 0.05g |
| N-N- dimethylformamide | 1mL |
| 0.1M phosphate buffer (PH=7.0) | 78mL |
| The 5mM potassium ferricyanide | 10mL |
| 5mM potassium ferrocyanide | 10mL |
| 1%Triton-X 100 | 100uL |
| Water | It supplies as 100mL |
GUS staining procedure: it reports the homozygote for merging gus gene to strain seed first, is cleaned by surface sterilization, spread
Onto MS ware.Seed is taken to sprout period, seedling two panels, four, six, eight leaves and seedling are soaked in GUS dye liquor, are placed in
37 DEG C of incubator dark processings, moment observe the coloration result of material, prevent the dye liquor change blue time from contaminating, terminate dyeing in time.
It after terminating dyeing, needs to decolourize step by step, outwells dye liquor (if dye liquor uses unchanged blue recyclable next time), washed one time with clear water,
Then 30% alcohol is added into pipe, after decoloration 30 minutes, then changes 70% alcohol into, decolourizes 30 minutes, finally change 100% wine into
It is clean until eluting leaf color that essence continues decoloration.Rehydration step by step and dehydration are needed on the contrary, first with 70% after the completion of decoloration
Alcohol impregnates 30 minutes, reuses 30% alcohol, impregnates 30 minutes, is finally placed in water observation and takes pictures, long-term preservation needs
It is placed in 30% alcohol.
The subcellular localization of 7 arabidopsis AGL16 albumen of embodiment
First design with full connector (attB) connection the code area AGL16 and without terminator codon primer LP and
RP obtains the AGL16-GFP target fragment (figure that size is 750bp or so using the KOD enzyme PCR amplification system in embodiment 2
7A), and glue recycling is carried out.Utilize GatewayR BP ClonaseTMII Enzyme Mix kit (is purchased from
Invitrogen AGL16-GFP) is passed through into 42 DEG C of heat-shock transformed recombinant clones into the shuttle vector of pDONR207, again by
GatewayTMThe attR1 for the pGWB5 end carrier that clone technology drives intermediate shuttle vector recombinant clone to 35S promoter and
Between attR2, correct pGWB5-AGL16 carrier (Fig. 7 B, C) is obtained by digestion identification.Again through sequencing determine whole carrier without
Mutation.Successful pGWB5-AGL16 carrier electricity will be constructed again to be transferred in Agrobacterium C58C1 competence, is converted by dip-flower, is obtained
Transgenic seed.The T0 received is taped against kanamycins containing 50mg/L (being purchased from Beijing Suo Laibao Science and Technology Ltd) for seed
The report strain containing resistant fusion GFP gene is filtered out on MS culture medium.By the T1 positive seed of anti-kanamycins in soil
Maturation is grown in earth, then single plant sowing.T1 is taped against on the MS culture medium containing kanamycins for seed again, selection is isolated
The positive seedling single plant sowing that continues kind to go down obtain T2 for seed.Take the homozygosis sprouted entirely on MS culture medium containing kanamycin
Body strain carries out root Fluirescence observation under OLYMPUSIX-710 laser confocal microscope, and setting excitation wavelength is 488nm,
Launch wavelength is 510nm, as a result as illustrated in fig. 7d, it can be seen that AGL16 green fluorescent protein is located in nucleus, explanation
AGL16 albumen is the transcription factor of a nuclear location.
PCR primer is as follows:
Upstream primer (LR) (lowercase indicates attB1 connector):
5’-ggggacaagtttgtacaaaaaagcaggctATGGGAAGGGGCAAGATCG-3’
Downstream primer (RP) (lowercase indicates attB2 connector):
5’-ggggaccactttgtacaagaaagctgggtGCTGCAATGAAGGAAAAATAG-3’
Phenotypic analysis of the 8 rice Os agl16 afunction mutant of embodiment under salt and treatment with mannitol
Firstly, (address: Hangzhou, Zhejiang province city Binjiang District day and high-tech produce for commission hundred lattice Bioisystech Co., Ltd of Hangzhou
2 building, the industry garden building C) use the knockout technology of CRISPR/Cas9 to knock out the homologous gene of the arabidopsis AGL16 in rice, it obtains multiple
Rice Os agl16 afunction mutant strain.By Osagl16 afunction mutant strain Osagl16-1, Osagl16-2,
Osagl16-4 and WT (11 are spent in wild type) rice paddy seed (need to remove crust) are dispensed into the EP pipe of 2mL, are guaranteed a small amount of more
Pipe, (i.e. the alcohol of 100mL 75% is by 75mL absolute alcohol and 25mL pure water group for first 75% alcohol that 1.5mL is added into pipe
At) use sterile water wash 3 times after cleaning 1 minute, then (i.e. 100mL 10%Bleach is by 10mL blue moon 84 with 10%Bleach
Thimerosal and 90mL pure water composition), the revolving speed for being placed in shaking table 230rpm cleans 30 minutes, then after superclean bench sterilizing
Pure water clean again 5 times, that is, complete.It chooses full rice paddy seed point and is being added with 0mM, 100mM NaCl, 200mM respectively
In the MS culture bottle of mannitol (MS is formulated such as embodiment 3), which completes in superclean bench.After the completion, it will train
Feeding bottle, which is placed between 16-26 DEG C of temperature and 16h illumination and the culture of 8h dark condition, observes culture.Since the same day after seed
It calculates, after two weeks, photographs to record and count the main root length and aerial part height of different strains.From the point of view of Fig. 8 A result, just
Under the conditions of often, 3 afunction mutant strain above and below ground parts of WT and Osagl16 are without apparent difference；When in 100mM
Under conditions of NaCl processing, the root system and aerial part ratio WT of 3 strains of Osagl16 are more flourishing；When at 200mM mannitol
Under conditions of reason, 3 strains of Osagl16 have more flourishing root system, but their aerial part is suppressed, only
The aerial part ratio WT of Osagl16-4 is more strong.The main root length under each treatment conditions has been counted simultaneously, the results showed that, just
Without significant difference between each strain under the conditions of often；Under conditions of 100mM NaCl processing, Osagl16-1 and Osagl16-
2 main root length is considerably longer than WT；Simultaneously when treatment with mannitol, 3 Osagl16 strains longer than compare WT (Fig. 8 B).These
As a result the function that Osagl16 material has salt tolerant and the osmotic stress of resistance to mannitol simulation is all further demonstrated.
It should be understood that particularly shown and description is carried out to the present invention although with reference to its illustrative embodiment, but
It is it will be apparent to an ordinarily skilled person in the art that without departing substantially from spirit and model of the invention as defined in appended claims
Under conditions of enclosing, any combination of various embodiments can be carried out in the variation for wherein carrying out various forms and details.
Claims (10)
1. a kind of relevant albumen of stress resistance of plant, selected from following:
(a) albumen that the amino acid sequence shown in SEQ ID NOs.1-8 forms；
(b) amino acid sequence shown in SEQ ID NOs.1-8 is passed through to the substitution and missing of one or several amino acid residues
And/or it adds and there is active derivative protein identical as SEQ ID NOs.1-8；Or
(c) other genes coding with the composition of amino acid sequence shown in SEQ ID NOs.1-8 have similitude reach 50% with
On albumen；
Wherein the resistance refers to salt tolerant, drought-enduring and resistance to osmotic stress characteristic.
2. encoding the gene of albumen described in claim 1.
3. gene as claimed in claim 2, wherein the gene be it is following any one:
(1) DNA sequence dna shown in SEQ ID NO.9-16；Or
(2) nucleotide sequence that can hybridize with DNA sequence dna shown in SEQ ID NO.9-16 under high high stringency conditions；
The stringent condition are as follows: in 6 × SSC, the solution of 0.5%SDS, hybridize at 65 DEG C, then with 2 × SSC, 0.1%
It is primary that SDS and 1 × SSC, 0.1%SDS respectively wash film.
4. containing the plant recombinant vector or expression cassette of gene described in claim 2 or 3.
5. the host comprising gene described in claim 2 or 3 or plant recombinant vector as claimed in claim 4 or expression cassette is thin
Born of the same parents, wherein the host cell is microbial cell, preferably agrobatcerium cell or Bacillus coli cells.
6. albumen described in claim 1, gene described in claim 2 or 3, recombinant vector as claimed in claim 4 or table
Up to host cell described in box or claim 5 cultivate salt tolerant, the application in drought-enduring and resistance to osmotic stress plant.
7. a kind of method for cultivating salt tolerant, drought-enduring and resistance to osmotic stress plant, the method includes the following steps:
(1) selection coding SEQ ID NOs.1-8 shown in amino acid sequence nucleotide sequence or target plant in SEQ ID
The highest gene of amino acid alignment similarity shown in NO.1 is as homologous gene, by the nucleotide sequence or homologous base
The segment of cause is building up in gene knockout carrier；
(2) it with the cell or tissue of the gene knockout carrier conversion target plant built in step (1), obtains transgenosis and plants
Strain；
(3) identification that the transgenic plant of acquisition is carried out to expression quantity on resistance screening and transcriptional level, determines target gene
It is knocked, so that obtaining transgenosis knocks out strain；
Obtained transgenosis knocks out strain in the culture medium with 150mM NaCl and 250mM treatment with mannitol with higher
Seed germination rate and more flourishing root system, there is higher survival rate in Osmotic treatment, have good salt tolerant, drought-enduring and resistance to infiltration
Stress ability thoroughly.
8. method of claim 7, wherein gene knockout carrier used in step (1) is CRISPR/Cas9 carrier.
9. method of claim 7, wherein the plant is monocotyledon or dicotyledon.
10. method as claimed in claim 9, wherein the plant is selected from arabidopsis, tobacco, rice, wheat, corn, cotton, oil
Dish or soybean.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810242690.XA CN110305201B (en) | 2018-03-22 | 2018-03-22 | Plant stress resistance related gene and its coded protein and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810242690.XA CN110305201B (en) | 2018-03-22 | 2018-03-22 | Plant stress resistance related gene and its coded protein and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110305201A true CN110305201A (en) | 2019-10-08 |
| CN110305201B CN110305201B (en) | 2022-07-22 |
Family
ID=68073965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810242690.XA Active CN110305201B (en) | 2018-03-22 | 2018-03-22 | Plant stress resistance related gene and its coded protein and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110305201B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760527A (en) * | 2019-12-05 | 2020-02-07 | 山东大学 | Lotus bean No. 12 GmYLD1 gene related to drought stress and allelic mutant gene and application thereof |
| CN111187784A (en) * | 2020-02-12 | 2020-05-22 | 青岛农业大学 | Use of aminoacylase-1 |
| CN112778406A (en) * | 2021-01-29 | 2021-05-11 | 浙江省农业科学院 | Watermelon auxin initial response protein ClSAUR1, gene, expression vector, transformant and method thereof |
| CN114561420A (en) * | 2020-11-27 | 2022-05-31 | 中国科学技术大学 | Plant drought resistance related protein AGL27 and application of coding gene thereof |
| CN114686488A (en) * | 2020-12-25 | 2022-07-01 | 中国科学技术大学 | Rice salt-tolerant stress gene OsAGL16 and application of encoding protein thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1715411A (en) * | 2005-07-12 | 2006-01-04 | 中国科学技术大学 | Salt tolerant, anti-drought gene and proteins encoded thereof of salt mustard and application |
| CN102002101A (en) * | 2010-11-24 | 2011-04-06 | 中国农业大学 | Plant root development related protein ZmNR1 and coding gene thereof |
| KR20110121784A (en) * | 2010-05-03 | 2011-11-09 | 수원대학교 산학협력단 | Osabf1 gene from oryza sativa and uses thereof |
-
2018
- 2018-03-22 CN CN201810242690.XA patent/CN110305201B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1715411A (en) * | 2005-07-12 | 2006-01-04 | 中国科学技术大学 | Salt tolerant, anti-drought gene and proteins encoded thereof of salt mustard and application |
| KR20110121784A (en) * | 2010-05-03 | 2011-11-09 | 수원대학교 산학협력단 | Osabf1 gene from oryza sativa and uses thereof |
| CN102002101A (en) * | 2010-11-24 | 2011-04-06 | 中国农业大学 | Plant root development related protein ZmNR1 and coding gene thereof |
Non-Patent Citations (7)
| Title |
|---|
| CLAUDIA KUTTER: "MicroRNA-mediated regulation of stomatal development in Arabidopsis", 《PLANT CELL》 * |
| GAN YB: "The effects of fluctuations in the nutrient supply on the expression of ANR1 and 11 other MADS box genes in shoots and roots of Arabidopsis thaliana", 《BOTANY-BOTANIQUE》 * |
| JIANG,C.-Z: "Sequence 1420 from patent US 7196245，Accession NO：ABP33036.1", 《GENEBANK DATABASE》 * |
| KEZHEN YANGY: "A new loss-of-function allele 28y reveals a role of ARGONAUTE1 in limiting asymmetric division of stomatal lineage ground cell", 《JOURNAL OF INTEGRATIVE PLANT BIOLOGY》 * |
| SALANOUBAT,M: "Arabidopsis thaliana，AGAMOUS-like 16，NP_001326287.1", 《GENEBANK DATABASE》 * |
| YU,J: "Oryza sativa Indica Group，hypothetical protein OsI_07801，Accession NO：EEC73475.1", 《GENEBANK DATABASE》 * |
| ZHANG,J: "Sequence 566 from patent US 7956242，NO：AEH75290.1", 《GENEBANK DATABASE》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760527A (en) * | 2019-12-05 | 2020-02-07 | 山东大学 | Lotus bean No. 12 GmYLD1 gene related to drought stress and allelic mutant gene and application thereof |
| CN110760527B (en) * | 2019-12-05 | 2022-09-23 | 山东大学 | Nelumbo number 12 associated with drought stressGmYLD1Gene, allelic mutant gene and application thereof |
| CN111187784A (en) * | 2020-02-12 | 2020-05-22 | 青岛农业大学 | Use of aminoacylase-1 |
| CN111187784B (en) * | 2020-02-12 | 2022-03-08 | 青岛农业大学 | Use of aminoacylase-1 |
| CN114561420A (en) * | 2020-11-27 | 2022-05-31 | 中国科学技术大学 | Plant drought resistance related protein AGL27 and application of coding gene thereof |
| CN114561420B (en) * | 2020-11-27 | 2024-02-23 | 中国科学技术大学 | Plant drought resistance related protein AGL27 and application of coding gene thereof |
| CN114686488A (en) * | 2020-12-25 | 2022-07-01 | 中国科学技术大学 | Rice salt-tolerant stress gene OsAGL16 and application of encoding protein thereof |
| CN112778406A (en) * | 2021-01-29 | 2021-05-11 | 浙江省农业科学院 | Watermelon auxin initial response protein ClSAUR1, gene, expression vector, transformant and method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110305201B (en) | 2022-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110305201A (en) | Plant stress-resistance related gene and its coding albumen and application | |
| CN102174560B (en) | The delay senility of plant and adverse circumstance endurance | |
| ES2863223T3 (en) | Compositions and procedures to alter flowering and plant architecture to improve yield potential | |
| CN112724214B (en) | Xanthoceras sorbifolia drought induction transcription factor XsMYB308L and application thereof | |
| CN112442506B (en) | Arabidopsis thaliana clubroot disease candidate gene AT2G35930 and application thereof | |
| CN107987141B (en) | Application of corn nuclear factor gene ZmNF-YA1 in plant stress resistance modification | |
| CN110643618A (en) | Jatropha curcas MYB transcription factor JcMYB16 gene and application thereof in improving drought resistance of plants | |
| CN113046360A (en) | Maize genes ZMSPL1 and ZMSPL2 and uses thereof | |
| US20150218581A1 (en) | Use of OXHS4 Gene in Controlling Rice Drought Resistance | |
| CN106967728A (en) | A kind of pumpkin adversity gene CmNAC1 and its application | |
| CN113604480A (en) | Corn transcription factor ZmHsf28 and application thereof | |
| CN104450742B (en) | Maize kernel factor gene ZmNF YB3 and its homologous gene application | |
| CN102453083B (en) | Plant stress tolerance related protein ZmPMP3 and coding gene thereof and application thereof | |
| CN108823220B (en) | Cloning and application of waxy synthesis related gene MdCER1 in apple | |
| CN102965373B (en) | DNA (deoxyribose nucleic acid) fragment and application thereof in preparation of male sterile hot pepper | |
| CN102296084B (en) | Use of auxin efflux carrier PINFORMED1 (PIN1) family genes in breeding corn and broomcorn | |
| CN112410370B (en) | Application of corn 10kDa heat shock protein gene ZmHsp10 in changing stress resistance of plants | |
| CN104673803B (en) | Application of gene methylation in regulation of gene expression | |
| CN104177481B (en) | Plant lodging and development of floral organs modulin ZmLA1 and encoding gene thereof and application | |
| CN114561420A (en) | Plant drought resistance related protein AGL27 and application of coding gene thereof | |
| CN102453084B (en) | Soybean photoreceptor GmPLP1 and coding gene and application thereof | |
| CN111718942A (en) | Rice salt tolerance related gene GT3 and application thereof | |
| CN112521467B (en) | Plant stress resistance related gene AGL103, and coding protein and application thereof | |
| CN115161331B (en) | Rice root morphogenesis regulatory gene CLRD1 and application thereof | |
| US20180223301A1 (en) | Overexpression of plant genes involved in programmed cell death for yield increase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |