CN106834301A - Sabina vulgaris adjusts plant nitrogen nutrition and alkaline stress sensing gene CML9（Q6‑1）And its application - Google Patents
Sabina vulgaris adjusts plant nitrogen nutrition and alkaline stress sensing gene CML9（Q6‑1）And its application Download PDFInfo
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- 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
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Abstract
Plant nitrogen nutrition and alkaline stress sensing gene CML9 (Q6 1) can be adjusted the present invention relates to a kind of strong xerophyte Sabina vulgaris (Sabina vulgaris.) from northwestern Inner Mongolia.The gene coded protein has calcium binding sites.By the structure of the preparation of material, gene cloning and follow-up plant expression vector, the gene is further passed through into agriculture bacillus mediated titbit infestation method, in introduction model plant Arabidopsis thaliana wild type, many plants of single-genes insertions, pure and mild T are finally given by screening resistance seedling3For genetically modified plants.Genetically modified plants grow good under the conditions of low nitrogen than wild type, and genetically modified plants grow poor than wild-type plant under the conditions of alkaline stress, and the albumen of the bright gene code has the function of regulation plant nitrogen nutrition and alkaline stress.This cultivates drought-resistant plant there is provided new direction to using technique for gene engineering, significant to the molecular breeding work under drought stress.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, it is related to a kind of northern China desertificated area windproof solid
Husky seeds Sabina vulgaris (Sabina vulgaris.) relevant nitrogen nutrition and alkaline stress sensing gene CML9 (Q6-1).The present invention is selected
The seeds Sabina vulgaris of Inner Mongol Meng Cao drought resistings limited company nursery stock base is experiment material, with Illumina Solexa
The method of transcript profile high-flux sequence, obtains the nitrogen nutrition and alkaline stress sensing gene of the seeds, and candidate's nitrogen nutrition and alkali are coerced
The gene C ML9 (Q6-1) for compeling sensing carries out bioinformatic analysis, while the phenotypic analysis of different elements is carried out to it, more directly
The physiologic function of the understanding gene of sight.
Background technology
Sabina vulgaris (Sabina vulgaris.), also known as savin juniper, savin, be distributed mainly on the Inner Mongol, Shaanxi,
The ground such as Xinjiang, Ningxia, Gansu, Qinghai.There are the ground such as Jiangsu, Zhejiang, Anhui, Hunan main cultivation base.Sabina vulgaris can stand wind
Erosion sand is buried, the long-term desert Environment for adapting to arid, is the fine tree species that arid, semiarid zone are checked winds and fixed drifting sand with water and soil conservation.
Light, happiness feel nice and cool dry weather, it is cold-resistant, drought-enduring, barren-resistant, to soil requirement not sternly, intolerant to waterlogging, fertile penetrating soil into
It is long very fast.Strong adaptability, cuttage is preferably lived, and cultivation management is simple, and the strong aithullium of this vitality is in north greening and makes
Have in woods and lift the light effect of lumping weight, so research Sabina vulgaris, for the development and utilization tool of wild plant gene related to drought tolerance
It is significant.Physiology and ecosystem characterization aspect are concentrated mainly on for the research of Sabina vulgaris at present, on its anti-drought gene
Clone and using aspect without report.
China's plant molecular breeding ability is huge with developed country's gap, and the adversity gene for possessing independent intellectual property right is generation
The focus of boundary plant molecular breeding field competition.For the exploitation and utilization of plant stress-resistance gene, current research is mainly concentrated
In the crop such as model plant arabidopsis or paddy rice, wheat, lack the excavation to the adversity gene enriched in wild plant body.This
Invention is exactly using the high throughput sequencing technologies of Illumina companies, first-selected seeds of being checked winds and fixed drifting sand to northern China desertificated area
Sabina vulgaris carries out transcript profile sequencing, obtains a kind of gene for filtering out nitrogen nutrition and alkaline stress sensing for a long time through natural environment
CML9(Q6-1)。
The content of the invention
The utilization molecule clone technology that the purpose of the present invention is, to relevant nitrogen nutrition and alkaline stress sensing gene CML9 (Q6-
1) cloned, finally given the expression vector for gene expression, so that the channel genes are in arabidopsis wild plant
And expressed in wild plant body, then the function that the gene is further verified by phenotypic analysis.
Embodiment of the present invention is the Sabina vulgaris from Inner Mongol Meng Cao drought resistings Co., Ltd nursery stock base to test material
Material, using the method for Illumina Solexa transcript profile high-flux sequences, analyzes its transcript profile Sequence Identification, determines Ca2+
Associated proteins type and quantity, so as to obtain Sabina vulgaris about nitrogen nutrition and the nucleotides of alkaline stress sensing gene CML9 (Q6-1)
Sequence, the gene is obtained by molecule clone technology, and is conducted into arabidopsis, finally obtains transfer-gen plant, is passed through
The biological function that phenotypic analysis understands gene is carried out to transfer-gen plant.
It is an object of the present invention to provide new Sabina vulgaris gene related to drought tolerance, it is CML9 (Q6-1), its sequence to name
It is classified as the sequence of SEQ No.1.
The present invention relates to a kind of expression quantity gene higher in Sabina vulgaris root, entitled CML9 (Q6-1), its nucleotides
Sequence as shown in SEQ NO.1 or SEQ NO.2 in sequence table, its coding 67 amino acid sequences, such as SEQ in sequence table
Shown in NO.3.
Specifically, the invention provides the polynucleotides of the separation containing one of following sequences：
(1) SEQ NO.1 or the SEQ NO.2 in sequence table；
(2) DNA sequence dna that SEQ NO.1 or the SEQ NO.2 and in sequence table are limited has more than 90% homology, and compiles
Code-phase congenerous protein DNA sequence；
The above-mentioned polynucleotides being related to also include substitution, missing and insertion mutation body and allelic variant, splice variant, piece
Section, derivative etc..
It will be appreciated by persons skilled in the art that the polynucleotides of above-mentioned separation also include those with SEQ NO.1 or
Sequence shown in SEQ NO.2 has the sequence compared with high homology, the sequence that for example homology is more than 95%, or 90%, or even 85%
Row；Can also be with the sequence of sequence hybridization shown in SEQ NO.1 or SEQ NO.2 under high stringency conditions including those；Or can be with SEQ
The complementary sequence of the sequence of NO.1 or SEQ NO.2.
Invention also provides the skill that new gene CML9 (Q6-1) of the invention is applied to Studies of Gene Engineering on Plant Drought-resistance
Art scheme.
The present invention has successfully been isolated from Sabina vulgaris and obtains a kind of decorrelation gene filtered out for a long time through natural environment
CML9 (Q6-1), this cultivates drought-resistant plant there is provided new direction to using technique for gene engineering, to the molecule under drought stress
Breeding work is significant.Specifically, one of specific embodiments of the present invention are by CML9 (Q6-1) gene application
In plant genetic engineering improving survival ability of the plant under drought stress.
Outlined above describes the present invention, can by referring to provided herein is some specific embodiments further understand this
Invention, these embodiments are merely to illustrate and not limit the present invention.
Brief description of the drawings
Fig. 1：Experiment general flow chart.
Fig. 2：Five kinds of T of CML9 (Q6-1)3For pure and mild plant respectively in CK, 0 μM of NO3 -, the life of pH 7.0 these three concentration gradients
The phenotypic map (A) and fresh weight figure (B) of leaf under elongate member, can be with the apparent blade for seeing genetically modified plants in 0 μ from figure
M NO3 -Upper growing way is better than wild type Col-0, and transfer-gen plant growing way is not so good as WT lines on pH 7.0.
Fig. 3：Five kinds of T of CML9 (Q6-1)3For pure and mild plant respectively in 0 μM of Ca2+, 1 μM of ABA, 100mM NaCl these three
The phenotypic map (A) and fresh weight figure (B) of leaf, are clear that transfer-gen plant from figure under concentration gradient growth conditions
Blade does not have difference with WT lines.
Fig. 4：The planting patterns of culture dish in phenotypic analysis.
Specific embodiment
Embodiment 1, Sabina vulgaris sample collection
In order to increase the richness of its RNA related to resistance in vivo to greatest extent, select relatively cold in weather
December (2013) carry out the sampling of Sabina vulgaris root, sample is stored in rapidly standby in liquid nitrogen after collection.
The structure of embodiment 2, expression vector
1st, due to the more difficult extractions of the RNA of Sabina vulgaris plant, so the genes of interest of our early stages is public by Nanjing Jin Sirui
Department's synthesis, the structure of follow-up expression vector is completed with the synthetic gene of company.Clone used by synthetic gene carries
Body is that pUC57 resistances are Amp.
2nd, the connection of purpose carrier and the completion of structure
The expression vector used in the present invention is pRI 101AN, and its resistance is Kan.
The connection of purpose carrier is simultaneously by 5 ' by synthetic genetic fragment and purpose carrier：Sal I 3′：
The digestion of EcoR I, by DNA glue reclaims, then DNA ligase connection converts Escherichia coli, by bacterium colony PCR and extraction matter
Grain, the method for digestion verification finally obtains the plasmid of the vector gene being connected to.
Double digestion reaction system：
The connection of a, purpose fragment and expression vector：
Principle：It is attached according to the ratio that purpose fragment and expression vector mol ratio are 3: 1 or 1: 3；
T4DNA ligases (1 μ l)+buffer (2 μ l)+17 μ l (purpose fragment+expression vector)；
16 DEG C overnight connect；
B, conversion：Take E. coli competent to be thawed on ice, the product of purpose fragment and expression vector will be connected with again
It is transformed into E. coli competent, mixes, ice bath 30 minutes, 42 DEG C of heat shocks 90 seconds is immediately placed on 2 minutes on ice；Add 500
μ l non-resistant LB fluid nutrient mediums, 200rpm or 37 DEG C of constant incubator quiescent culture after 60 minutes on 37 DEG C of shaking tables, by bacterium solution
In coating and the solid LB flat boards containing corresponding antibiotic (Kan 50mg/ml)；Its result is observed after 37 DEG C of inversion overnight incubations.
Single bacterium colony on c, picking bacterium plate is added in the 5ml LB nutrient solutions containing Kan (50mg/ml) antibiotic, 37 DEG C
Incubator overnight culture.
D, plasmid extraction：Plasmid extraction kit from TRAN extracts plasmid.
1) bacterium solution of 2ml incubated overnights, is taken, 10000x g are centrifuged 1 minute, remove supernatant.As bacterium solution amount is big, can several times from
The heart is collected.
2) 250 μ l colourless solutions RB (containing RnaseA), are added, concussion suspended bacterial precipitation should not leave small bacterium block.
3) 250 μ l blue solution LB, are added, mixing 4-6 times is leniently spun upside down, thalline is fully cracked, form blue
The bright solution of color, color from half it is bright be changed into bright blueness, indicate cracking completely (no more than 5 minutes).
4) 350 μ l yellow solution NB, are added, (color indicates mixing by blue yellowing completely to be gently mixed 5-6 times
Uniformly, neutralize complete), until forming the yellow aggegation block of consolidation, it is stored at room temperature 2 minutes.
5), 12000x g are centrifuged 5 minutes, and careful absorption supernatant is added in centrifugal column.12000x g are centrifuged 1 minute, abandoned stream
Go out liquid.As supernatant volume is more than 800 μ l, in being segmented into repeatedly adding post, and ibid it is centrifuged, abandons efflux.
6), add 650 μ l solution Ws B, 12000x g to be centrifuged 1 minute, abandon efflux.
7), 12000x g are centrifuged 2 minutes, thoroughly the WB of removal residual.
8), centrifugal column is placed in a clean centrifuge tube, 30-50 μ l EB or deionized water (PH is added in the center of post
＞ 7.0) it is stored at room temperature 1 minute.
9), 10000x g are centrifuged 1 minute, and the DNA for eluting is in -20 DEG C of preservations.
E, digestion verification：Continue to select restriction enzyme 5 ' above used：Sal I 3′：EcoR I are to positive colony
Plasmid is verified.
Double digestion reaction system
37 DEG C of constant incubators, warm bath 30 minutes.1% agarose gel electrophoresis detects, chooses banding pattern and correctly protected
Bacterium (40% yellow lid glycerine).
10th, Agrobacterium-mediated Transformation：Electrotransformation converts Agrobacterium.
1) Agrobacterium competence, is taken, thawed on ice is placed in, the μ l of plasmid about 5 are added, is placed on ice after mixing 30 minutes；
2), while preparing clean dry electric revolving cup, on ice precooling；
3), electric revolving cup surface is dried, electroporation is put into after the Agrobacterium added with plasmid all being added in electric revolving cups, electricity
Swash conversion；
4), electric revolving cup adds the LB nutrient solutions of lml antibiotic-frees, repeatedly pressure-vaccum, in suction 2ml centrifuge tubes, 28 DEG C of shaking tables
200rpm or 28 DEG C of constant incubator renewal cultivation is after 1.5 hours；
5) 500 μ l bacterium solutions, are taken and coats the solid LB media containing antibiotic (Rif50mg/ml and Kan50mg/ml)
On, 28 DEG C of incubated carton upside down cultures 1-2 days are until monoclonal occurs；
6), choosing positive Agrobacterium single bacterium colony is carried out shaking bacterium, and 28 DEG C of shaking tables, 250rpm is cultivated 1-2 days；
11st, Agrobacterium plasmid is extracted：Plasmid extraction kit from TRAN extracts plasmid；
1) bacterium solution of 2ml incubated overnights, is taken, 10000x g are centrifuged 1 minute, remove supernatant.As bacterium solution amount is big, can several times from
The heart is collected.
2) 250 μ l colourless solutions RB (containing RnaseA), are added, concussion suspended bacterial precipitation should not leave small bacterium block.
3) 250 μ l blue solution 1B, are added, mixing 4-6 times is leniently spun upside down, thalline is fully cracked, form blue
The bright solution of color, color from half it is bright be changed into bright blueness, indicate cracking completely (no more than 5 minutes).
4) 350 μ l yellow solution NB, are added, (color indicates mixing by blue yellowing completely to be gently mixed 5-6 times
Uniformly, neutralize complete), until forming the yellow aggegation block of consolidation, it is stored at room temperature 2 minutes.
5), 12000x g are centrifuged 5 minutes, and careful absorption supernatant is added in centrifugal column.12000x g are centrifuged 1 minute, abandoned stream
Go out liquid.As supernatant volume is more than 800 μ l, in being segmented into repeatedly adding post, and ibid it is centrifuged, abandons efflux.
6) 650 μ l solution Ws B, 12000xg centrifugation 1 minute, is added, efflux is abandoned.
7), 12000x g are centrifuged 2 minutes, thoroughly the WB of removal residual.
8), centrifugal column is placed in a clean centrifuge tube, 30-50 μ l EB or deionized water (PH is added in the center of post
＞ 7.0) it is stored at room temperature 1 minute.
9), 10000x g are centrifuged 1 minute, and the DNA for eluting is in -20 DEG C of preservations
12nd, digestion verification：From restriction enzyme 5 '：Sal I 3′：EcoR I verify to the plasmid for being extracted,
Double digestion reaction system
28 DEG C of constant incubators, warm bath 30 minutes.1% agarose gel electrophoresis detects that analysis result, selector bar band is correct
Agrobacterium preserve glycerol stock (40% blue lid), for converting plant.
The acquisition of embodiment 3, genetically modified plants
The present embodiment obtains transfer-gen plant code name CML9 (Q6-1) using titbit infestation method.
1st, picking has just ensured that the Agrobacterium of bacterium is activated (vigor that activation purpose is to ensure that bacterium), the bacterium that will have been activated
It is added in the 5ml LB nutrient solutions containing antibiotic (Rif50mg/ml and Kan50mg/ml), 28 DEG C of incubator overnight cultures, will
The bacterium solution shaken is put into centrifuge, 5000x g, 10min, abandoning supernatant, add 1ml infect liquid (5% sucrose,
0.02%silwet77 suspension is dissolved in water), with glue head dropper, gently pressure-vaccum makes bacterium suspend.
2nd, the wild type Col-0 plant for being grown fine and having been bloomed from three boxes, cut off having podded and on plant
The petal of pollination is completed, drawing bacterium solution with glue head dropper drops on the petal do not bloomed, and mark of labelling, and is invaded every 2-3 days
Contaminate once, (the note until all plant all bloom：Infect the previous day every time to water a plant).
3rd, T is collected in time after plant is ripe1For seed, treat that seed drying is sown in for one week or so and contain antibiotic
(Kan50mg/ml) on resistance solid MQACK culture dishes, first it is placed into 4 DEG C of refrigerator vernalization three days, then illumination cultivation one week is left
The right side, carries out positive seedling T1The screening in generation.
4th, the T grown fine on resistance culture ware is chosen1For plant, move earth culture and support and individual plant label, after after its maturation and
When individual plant collect T2For seed, it is dried for a week after, continuation its single tube is sprinkling upon on resistance culture base, after illumination cultivation a couple of days, choosing
Take and meet the dead T than for 3: 1 living2Earth culture is moved for plant to support, timely individual plant collects T after seed maturity3For seed, it is dried for a week after,
By T3It is sprinkling upon on the MQACK culture mediums containing glufosinate resistance for seed, choosing full homozygous plants living carries out phenotypic analysis.
Phenotypic analysis of the embodiment 4, CML9 (Q6-1) in different elements
The MQA medium components used in this example mainly have
A great number of elements：1MKNO3、1MMgSO4、1MCaCl2
Trace element：MS micro (0.5x),
Fe2+Salt：MS Fe2+Salt (0.5x)
Mn2+Salt：MS Mn2+Salt (0.5x) and 0.5M MES buffer solutions
Carbon source：1% sucrose
A. specific culture medium scheme such as table 1 (100ml).
Note：0μM Ca2+、0μM NO3 -, the two gradients add 1.0% agarose 1g；1mM Ca2+、1μM ABA、
100mMNaCl, pH 7.0 this four gradients add 1.2% agar 1.2g；It is 5.7 (notes that KOH or BTP adjusts pH：1mM Ca2+
PH7.0 to be transferred to 7.0,1 μM of ABA of pH with KOH be after autoclaving, when culture medium temperature drops to the temperature that hand can be touched
Add 0.5 μ l).
The culture medium scheme of the various elements of table 1
B. planting patterns is as shown in Figure 4.
The process of program request seed is aseptically carried out, and seed is entered with 75% alcohol first before program request seed
Row sterilization (washing seed time no more than 30 minutes), then instead, then seed is aseptically used 100% alcohol rinse one
Under, and it is dried on aseptic filter paper together with alcohol, after seed dries completely, with sterilized tweezers by seed point
Broadcast on culture medium.
One repetition of every kind of gradient, is sealed after finishing with sealed membrane, first in 4 DEG C of refrigerator vernalization three days, then places temperature
It it is 22 DEG C, humidity is observation CML9 (Q6-1) and wild type Col-0 after the vertical illumination cultivation fortnight of culturing room of 40%RH
Phenotype, and root data acquisition long and fresh weight and photograph taking are carried out to it, it can be seen that CML9 (Q6-1) is relative to wild
Type Col-0 is in 0 μM of NO3 -Leaf grows big；In the basic conditions (pH 7.0) to show as leaf small.Meanwhile, CML9 (Q6-1) is relative
In wild type Col-0 in 100mM NaCl, 0 μM of Ca2+, in pH 7.0 these concentration gradients be no significant difference.Specifically
As shown in the figure (Fig. 2, Fig. 3).
C. culture dish is put
6 gradients are one group, are divided into five groups
Above example further illustrates present disclosure, but should not be construed as limiting the invention.Do not carrying on the back
In the case of spirit of the invention and essence, the modification or replacement made to the inventive method, step or condition belong to this hair
Bright category.
Claims (7)
1. the gene C ML9 (Q6-1) that Sabina vulgaris (Sabina vulgaris.) regulation plant nitrogen nutrition and alkaline stress sense, its sequence
Row are as shown in SEQ No.1 or SEQ No.2.
2. a kind of polynucleotides of separation, its sequence is the nucleotides sequence complementary with nucleotide sequence shown in claim 1
Row.
3. the nucleotide sequence coded polypeptide described in claim 1 or 2.
4. the polypeptide described in claim 3, its sequence is as shown in SEQ No.3.
5. the expression vector of the polynucleotides described in claim 1 or 2 is contained.
6. application of the polynucleotides of the separation described in claim 1 or 2 in plant breeding engineering.
7. application of the polynucleotides of the separation described in claim 1 or 2 in Studies of Gene Engineering on Plant Drought-resistance.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114854767A (en) * | 2022-06-01 | 2022-08-05 | 四川农业大学 | Trifolium repens calmodulin-like protein TrCML6 gene and application thereof in drought resistance |
-
2016
- 2016-12-07 CN CN201611115698.7A patent/CN106834301B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| LIN K等: ""Cnb1 [Rhizophagus irregularis DAOM 197198w]"", 《GENEBANK DATABASE》 * |
| LOUIS-JÉRÔME LEBA等: ""CML9, a multifunctional Arabidopsis thaliana calmodulin-like protein involved in stress responses and plant growth"", 《PLANT SIGNALING & BEHAVIOR》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114854767A (en) * | 2022-06-01 | 2022-08-05 | 四川农业大学 | Trifolium repens calmodulin-like protein TrCML6 gene and application thereof in drought resistance |
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Address after: 010020 Floor 3, Block B, Yutai Business Plaza, Jinqiao Development Zone, Hohhot City, Inner Mongolia Patentee after: Mengcao ecological environment (Group) Co.,Ltd. Address before: 010020 Floor 3, Block B, Yutai Business Plaza, Jinqiao Development Zone, Hohhot City, Inner Mongolia Patentee before: INNER MONGOLIA MONGOLIAN GRASS ECOLOGICAL ENVIRONMENT (GROUP) Ltd. |