CN106754961B

CN106754961B - Gene for regulating plant response to drought induction signal calcium ions and hormone abscisic acid

Info

Publication number: CN106754961B
Application number: CN201611223170.1A
Authority: CN
Inventors: 王召明; 张�林; 祁智; 杨红艳; 杨佳; 苑峰; 刘亚玲; 金悦
Original assignee: Inner Mongolia Mengcao Life Community Big Data Co ltd; Inner Mongolia M Grass Ecological Environment Group Co Ltd
Current assignee: Inner Mongolia Xiaocao Digital Ecological Industry Co ltd; Mengcao Ecological Environment Group Co Ltd
Priority date: 2016-12-27
Filing date: 2016-12-27
Publication date: 2020-08-04
Anticipated expiration: 2036-12-27
Also published as: CN106754961A

Abstract

The invention discloses a gene for regulating the reaction of plants to drought induction signal calcium ions and hormone abscisic acid, and particularly relates to a cloning and functional expression mode of a drought-resistant related gene Sv4317, wherein the drought-resistant Sv4317 gene is cloned by using PCR (polymerase chain reaction) through preparation and treatment of materials, extraction of RNA (ribonucleic acid) and reverse transcription of cDNA (complementary deoxyribonucleic acid), the gene is introduced into an arabidopsis wild plant by a transgenic means, and finally T is obtained by screening resistant seedlings₃The generation pure seedlings are subjected to different element phenotype analysis on the transgenic plants and wild control plants, and the phenotype of the transgenic plants, which is more sensitive to mineral element calcium and drought hormone ABA, can be found.

Description

Gene for regulating plant response to drought induction signal calcium ions and hormone abscisic acid

Technical Field

The invention belongs to the technical field of plant biology. The sabina vulgaris belongs to strong-drought and soil nutrient-tolerant barren plants, and is widely applied to wind prevention and sand fixation ecological engineering in northern desertification areas of China. The invention firstly utilizes an Illumina Solexa technology-based platform to carry out high-throughput transcriptome sequencing on the root RNA of sabina vulgaris. Because calcium is the most important signal molecule for plant sensing and stress resistance, we have performed combing of the gene encoding the calcium binding protein in sabina vulgaris transcriptome. Further, the gene in which the sequence code is Sv4317 was further analyzed and studied. The bioinformatics found that Sv4317 may encode a Ca transporter. In order to clarify the function of the gene, the gene is expressed in a model plant Arabidopsis thaliana by a genetic engineering method. Phenotypic analysis of transgenic arabidopsis thaliana shows that the arabidopsis thaliana plant containing the Sv4317 gene shows a phenomenon of hypersensitiveness to low-calcium and low-concentration drought-sensing hormone abscisic acid (ABA) compared with a normal plant not containing the gene. The result shows that Sv4317 has the function of regulating the response of plants to drought-sensitive signals calcium ions and drought-sensitive hormone abscisic acid, and has the potential of being applied to genetic breeding work for regulating the response of crops to the two drought-related signals.

Background

Sabina chinensis (Sabina vu L garis.), also called Sabina chinensis and Xinjiang juniper, are mainly distributed in inner Mongolia, Shaanxi, Xinjiang, Ningxia, Gansu, Qinghai and other places.

The invention relates to a method for sequencing the transcription group of the first choice tree species juniper desertorum in the desert area of China by utilizing the high-throughput sequencing technology of the company I LL minina, and obtains a drought-resistant related gene Sv4317 screened out in the natural environment for a long time.

Disclosure of Invention

The invention aims to clone an drought-resistant gene Sv4317 by utilizing a molecular cloning technology to finally obtain an expression vector for gene expression, so that the gene is introduced into an arabidopsis wild plant and expressed in the wild plant, and the function of the gene is further verified by phenotypic analysis.

The embodiment of the invention selects sabina vulgaris in a seedling base as an experimental material, adopts a high-throughput sequencing method of an I LL uma So L exa transcriptome to obtain a nucleotide sequence of a sabina vulgaris drought-resistant related gene Sv4317, designs a primer through sequence analysis, obtains the gene through a molecular cloning technology, introduces the gene into arabidopsis thaliana, finally obtains a transgenic plant, and learns the biological function of the gene through phenotypic analysis of the transgenic plant.

One purpose of the invention is to provide a new sabina chinensis drought-resistant related gene, which is named as Sv4317 and has a sequence shown as SEQ No: 1.

The invention relates to a gene with higher expression in sabina vulgaris roots, which is named as Sv4317, and the nucleotide sequence of the gene is shown as SEQ NO: 1 or SEQ NO: 2, and 728 encoded amino acid sequences thereof are shown as SEQ NO: 3, respectively.

In particular, the invention provides isolated polynucleotides comprising one of the following sequences:

(1) SEQ NO in the sequence listing: 1 or SEQ NO: 2

(2) DNA sequence which has more than 90% of homology with the DNA sequence limited by SEQ NO.1 or SEQ NO.2 in the sequence table and codes the same functional protein;

the above mentioned related polynucleotides also include substitution, deletion and insertion mutants as well as allelic variants, splice variants, fragments, derivatives, etc.

It will be appreciated by those skilled in the art that the isolated polynucleotides described above also include those polynucleotides that are identical to SEQ NOs: 1 or SEQ NO: 2, e.g., sequences having greater than 95%, or 90%, or even 85% homology;

also included are those that are hybridizable to SEQ NOs: 1 or SEQ NO: 2; or can be identical to SEQ NO: 1 or SEQ NO: 2, or a sequence complementary to the sequence of 2.

The invention also provides a technical scheme for applying the novel gene Sv4317 to plant drought-resistant gene engineering, and the plant can specifically comprise sabina vulgaris and arabidopsis thaliana.

The invention successfully separates and obtains the anti-related gene Sv4317 screened out in the natural environment for a long time from the sabina vulgaris, which provides a new direction for cultivating drought-resistant plants by utilizing a genetic engineering technology and has important significance for molecular breeding work under drought stress. Specifically, one of the embodiments of the present invention is to apply the Sv4317 gene to plant genetic engineering to improve the viability of plants under drought stress.

Having generally described the invention, the same may be further understood by reference to certain specific examples provided herein which are intended to be illustrative only and not limiting.

Drawings

FIG. 1: general flow chart of experiment

FIG. 2: growth phenotype of transgenic Arabidopsis thaliana C6-6 containing the Sv4317 gene in the presence of low concentrations of calcium ions and drought hormone ABA.

FIG. 3 is a statistical graph of fresh leaf weight of transgenic Arabidopsis thaliana C6-6 containing Sv4317 gene in the presence of low-concentration calcium ion and drought hormone ABA.

FIG. 4: transgenic Arabidopsis C6-6 containing the Sv4317 gene has a growth phenotype in the absence of other mineral ions.

FIG. 5: statistical analysis of root length of transgenic Arabidopsis thaliana C6-6 containing the Sv4317 gene in the absence of other mineral ions.

FIG. 6: culture dish planting mode in phenotype analysis

Detailed Description

Example 1 Sabina sample Collection

The invention selects the tree species of the nursery stock base of Menggong grass drought-resistant shares GmbH as the experimental material. In order to maximize the abundance of RNA associated with stress resistance in vivo, the root of sabina vulgaris was sampled in 12 months (2013) in relatively cold weather, and the samples were rapidly stored in liquid nitrogen for later use after collection.

Example 2 construction of expression vector

a. Extraction of Total RNA

Extraction of RNA by TRIzol method

1) Placing a proper amount of liquid nitrogen into a mortar, fully pre-cooling the mortar until the liquid nitrogen does not boil and splash, placing the collected plants into the liquid nitrogen (ensuring that the liquid nitrogen submerges roots or powder)

2) And after adding liquid nitrogen, smashing the plants into fragments by using a pestle, quickly grinding when the liquid nitrogen is volatilized to cover the bottom of the mortar, replenishing the liquid nitrogen in time, and repeating the grinding process for 2-3 times.

3) Precooling a centrifugal tube prepared for containing grinding powder in liquid nitrogen: the sequence is tube bottom-tube wall-tube mouth (the connecting part of the tube body and the tube cover is not extended into liquid nitrogen so as not to be broken). A small hole is drilled in the middle of the centrifuge tube cover by using tweezers to prevent the centrifuge tube from bursting after liquid nitrogen is added.

4) The powder was scooped from a mortar with a centrifuge tube fully cooled with liquid nitrogen (1.5m L centrifuge tube powder volume less than 50 μ L), 1m L TRIzol extract was added quickly, the centrifuge tube bottom powder was impacted while the extract was added and mixed quickly on a shaker (the extract dissolved RNA within 30 seconds to protect RNA.) it was ensured that the powder did not melt before melting into TRIzol (the volume ratio of powder to extract was about 1: 10 to 1: 15). mixing was done thoroughly, and left at room temperature for no less than 5 minutes.

5) 10000x g, centrifugation at 4 ℃ for 20 minutes. Longer centrifugation times may result in better RNA precipitation with less contamination of the RNA phase.

6) About 80% of the supernatant was added to a new centrifuge tube, 1/4 volumes of chloroform was added, and Vortex was performed sufficiently

7) 10000x g, centrifuge at 4 ℃ for 15 minutes. This is a key step in determining whether RNA extraction is successful. If the front was successful, then two layers of completely clear but clearly separated solution were seen, without any material visible in the middle. This is the most perfect. This condition is not difficult to achieve if the previous steps are strictly followed. Relatively imperfect, but it is also acceptable to have a slight, slightly transparent white substance in the middle; if at this time the intermediate layer had a clear opaque milky white precipitate, the experiment had essentially failed. Repeated extraction with chloroform did not solve the problem. Moreover, there is a loss of solution for each extraction and not enough RNA is obtained even if the protein precipitate is finally removed.

8) And taking about 80% of supernatant to a new centrifugal tube. Rather, some supernatant may be sacrificed, as well as avoiding any intermediate layer material being aspirated completely. Equal volume of isopropanol stored at-20 ℃ was added, sufficient for Vortex. Standing at-20 deg.C for 1 hr, and continuing to work if necessary. If due to time problems, it is also possible to stay overnight, at which time the RNA is essentially stable.

9) 10000x g, centrifuge at 4 ℃ for 20 minutes, and discard the supernatant.

10) 1m L ethanol (DEPC water) and mixing thoroughly to break the RNA precipitate completely, so long as the purpose of ethanol salt dissolution can be achieved, 10000x g, 4 ℃ centrifugation for 10 minutes (washing twice) (so as to go all in ultra-clean)

11) Most of the supernatant was slowly decanted, the tube was inverted on sterile filter paper and the residue was aspirated off. High speed rapid centrifugation for 1 minute. The residual supernatant liquid was aspirated with a minimum pipette tip. Drying at room temperature for 5-10 min

12) Adding about 20-30 mu L DEPC water for redissolving.

13) The band pattern was observed by electrophoresis at 1. mu. L, and the voltage was high for a short time and controlled to be within 10 minutes.

Reverse transcription of cDNA

RNA7μL

Anchored OLigo(dT)20Primer(0.5μg/μL) 1μL

DEPC 1μL

Incubating at 65 ℃ for 5 min, ice-cooling for 2 min, and adding

2Xts II Reaction Mix 10μL

TransScript II RT/RI Enzyme Mix 1μL

gDNA Remover 1μL

Reverse transcription was incubated at 50 ℃ for 30 minutes and TransScript II RT/RI and gDNAROMVER were inactivated by heating at 85 ℃ for 5 seconds.

c. Construction of sabina vulgaris Sv4317 gene expression vector

Primer for amplifying Sv4317 gene coding sequence

A forward primer: 5'-ATTTGCGGCCGCATG GAG GAT CCG CTG ATC ATG AAT-3'

Reverse primer: 5'-CCCTCGAGCTAAGC ACA ATT TTC TTC CAA CTC-3'

Selecting a high-fidelity DNA polymerase Primer Star pair C of TaKaRa_6-6Cloning of the target gene was performed. The concrete scheme is as follows

1. Reaction system of target gene (50. mu. L)

PCR cycling system

98℃ 5min

26 cycles

2. Target gene recovery by using TRAN glue recovery kit

① the DNA band of interest in the agarose gel is cut out and weighed in a clean centrifuge tube, and if the gel weight is 100mg, it is regarded as 100. mu. L (100 mg-100. mu. L), and so on.

② adding 3 times volume of GSB solution, dissolving in 55 deg.C water bath for 6-10 min, intermittently mixing (2-3 min) to ensure complete melting of the gel block, observing the color of the solution when the gel is completely melted, such as purple, adding appropriate amount of 3M sodium acetate (pH5.2), adjusting the color to be the same as GSB (yellow), and adding 1 volume of isopropanol into the melted gel solution to increase DNA recovery, and adding 100 μ L isopropanol if the weight of the gel is 100 mg.

③ cooling the gel solution to be thawed to room temperature (the capacity of the centrifugal column to bind DNA is weak at high temperature), adding into the centrifugal column, standing for 1 min, centrifuging for 1 min at 10000x g, and discarding the effluent.

④ adding 650 μ L solution WB (adding absolute ethanol in advance), 10000x g centrifuging for 1 min, and discarding the effluent.

⑤ 10000x g, and removing the residual WB by centrifugation for 1-2 minutes.

⑥ placing the centrifugal column in a clean centrifugal tube, opening the cover, standing for 1 min to volatilize the residual ethanol, adding 30-50 μ L EB or deionized water (pH > 7.0) (EB or deionized water is preheated in 60-70 deg.C water bath, and has good use effect), and standing at room temperature for 1 min.

⑦ 10000x g, and eluting DNA, storing the eluted DNA at-20 ℃.

3. The target fragment obtained by glue recovery is connected with a cloning vector pEASY-B L un Simp L e (Kan) according to a certain proportion at 25 ℃ for one hour.

4. And (3) converting the escherichia coli competence by a heat shock method, namely unfreezing the escherichia coli competence on ice, adding a ligation reaction product, uniformly mixing, carrying out ice bath for 30 minutes, carrying out heat shock at 42 ℃ for 90 seconds, immediately placing on the ice for 2 minutes, adding 500 mu L nonresistant L B liquid culture medium, carrying out standing culture on the mixture for 60 minutes in a constant temperature incubator at 37 ℃ or 200rpm on a shaking bed at 37 ℃, coating a bacterial liquid on a solid L B plate containing corresponding antibiotics (Kan 50mg/m L), and carrying out inverted culture at 37 ℃ overnight to observe the result.

5. Colony PCR identification

In order to obtain the correct positive clones, single colonies growing on the medium were first randomly picked for PCR identification

(typically about 12) the template was replaced with a monoclonal colony, which was picked with a sterilized toothpick.

Colony PCR system (one DNA polymerase EasyTaqMix of TRAN is selected)

Mixing, centrifuging, and performing amplification reaction according to the following cycle under the following reaction conditions:

94℃ 5min

35 cycles

The PCR amplification results were analyzed by 1% agarose gel electrophoresis, and the correctly banded mycelia were added to 5m L L B medium containing kan (50mg/m L) antibiotic and incubated overnight at 37 ℃ with shaking.

6. Plasmid extraction: plasmid extraction kit for TRAN (transrat's plasmid extraction)

1) And centrifuging the bacterial liquid cultured by 2m L overnight for 1 minute at 10000x g, removing supernatant, and centrifuging and collecting for multiple times if the bacterial liquid is large in quantity.

2) Adding 250 mu L colorless solution RB (containing RnaseA), and shaking to suspend the bacterial sediment without leaving small bacterial block.

3) And adding 250 mu L blue solution L B, and gently mixing the mixture up and down for 4 to 6 times in an overturning way to ensure that the thalli are fully cracked to form a blue bright solution, wherein the color is changed from semi-bright to bright blue, which indicates complete cracking (not longer than 5 minutes).

4) Add 350. mu. L yellow NB and mix gently 5-6 times (color changed from blue to yellow indicating uniform mixing and complete neutralization) until a compact yellow aggregate was formed and let stand at room temperature for 2 minutes.

5) 12000x g for 5 minutes, carefully sucking the supernatant into the centrifugal column, 12000Xg for 1 minute, discarding the effluent, if the volume of the supernatant is more than 800. mu. L, adding the supernatant into the column several times, and centrifuging the column and discarding the effluent.

6) Then, 650. mu. L solution WB was added thereto, and the mixture was centrifuged at 12000Xg for 1 minute, and the effluent was discarded.

7) 12000x g was centrifuged for 2 min to completely remove the residual WB.

8) The column was placed in a clean centrifuge tube and 30-50. mu. L EB or deionized water (pH > 7.0) was added to the center of the column and allowed to stand at room temperature for 1 minute.

9) 10000x g for 1 min, and the eluted DNA is stored at-20 ℃.

7. And (3) enzyme digestion verification, namely verifying the positive clone plasmid by selecting restriction enzymes 5 ', Sa L I3' and EcoR I (double enzyme digestion is generally selected) according to the plasmid map and the enzyme digestion map of the DNA specific fragment to be cloned.

Double enzyme digestion reaction system

After mixing, the temperature is 37 ℃ according to the enzyme activity shown in the catalogue provided by the company, the mixture is subjected to warm bath for 30 minutes, and 1% agarose gel electrophoresis analysis is carried out, so as to judge whether the fragment is correct or not according to the size of an electrophoresis band.

8. Sequencing: the correct clone is verified by colony PCR and plasmid enzyme digestion, and the sequence correctness is verified by sequencing, which is generally handed over to Nanjing Kingsry company to complete. And (4) comparing and analyzing the sequencing result with the known gene sequence, and selecting correct clones from the sequencing result for the next step of constructing a target vector.

9. Completion of ligation and construction of the vector of interest

The connection of the target vector is that the gene segment with correct sequencing and the target vector are simultaneously subjected to enzyme digestion of 5 ': Sa L I3': EcoR I, DNA gel recovery and DNA ligase connection, then escherichia coli is transformed, and the plasmid of the connected vector gene is finally obtained by colony PCR, plasmid extraction and enzyme digestion verification.

a. And (3) connecting the target fragment with an expression vector:

principle: the target fragment and the expression vector are connected according to the molar ratio of 3: 1 or 1: 3

T4DNA ligase (1. mu. L) + buffer (2. mu. L) + 17. mu. L (fragment of interest + expression vector)

Overnight ligation at 16 ℃

b. And (3) carrying out transformation, namely unfreezing escherichia coli competence on ice, transforming a product connected with the target fragment and the expression vector into the escherichia coli competence again, mixing the escherichia coli competence uniformly, carrying out ice bath for 30 minutes, carrying out heat shock for 90 seconds at 42 ℃, immediately placing the mixture on the ice for 2 minutes, adding 500 mu L nonresistant L B liquid culture medium, carrying out standing culture on the mixture for 60 minutes in a constant-temperature incubator with the temperature of 37 ℃ and the speed of 200rpm on a shaker at 37 ℃ or in a constant-temperature incubator with the temperature of 37 ℃, coating a bacterial liquid on a solid L B plate containing a corresponding antibiotic (Spe 50mg/m L), and carrying out inverted culture.

c. Colony PCR identification

Colony PCR system (one DNA polymerase EasyTaqMix of TRAN is selected)

94℃ 5min

35 cycles

The PCR amplification results were analyzed by 1% agarose gel electrophoresis, and the correctly banded mycelia were added to 5m L L B medium containing Spe (50mg/m L) antibiotic and incubated overnight at 37 ℃ with shaking.

d. Plasmid extraction: plasmid extraction kit for TRAN (transrat's plasmid extraction)

5) 12000x g for 5 minutes, carefully sucking the supernatant into the centrifugal column, 12000x g for 1 minute, discarding the effluent, if the volume of the supernatant is more than 800. mu. L, adding the supernatant into the column several times, and centrifuging the column and discarding the effluent.

6) Adding 650 mu L solution WB, centrifuging for 1 minute at 12000x g, and discarding the effluent.

7) 12000x g was centrifuged for 2 min to completely remove the residual WB.

9) 10000x g for 1 min, and storing the eluted DNA at-20 deg.C

e. And (3) enzyme digestion verification, namely, continuously selecting the restriction enzyme 5 ', Sa L I3', EcoR I used in the previous step to verify the positive clone plasmid.

Double enzyme digestion reaction system

The incubator is kept at the constant temperature of 37 ℃ and is used for 30 minutes of warm bath. Detecting with 1% agarose gel electrophoresis, selecting the correct band type for bacteria preservation (40% yellow cover glycerol)

10. And (3) agrobacterium transformation: transformation of agrobacterium by electric transformation method

1) And melting the agrobacterium infection state on ice, adding the plasmid with the diameter of about 5 mu L, mixing uniformly, and placing on ice for 30 minutes.

2) Meanwhile, preparing a clean and dry electric rotating cup, and precooling on ice;

3) wiping the surface of the electric rotating cup, adding the agrobacterium with the plasmid into the electric rotating cup, and then putting the electric rotating cup into an electric rotating instrument for electric excitation transformation;

4) adding 1m of L antibiotic-free L B culture solution into an electric rotating cup, repeatedly blowing and sucking the mixture into a 2m L centrifuge tube, and after the mixture is recovered and cultured for 1.5 hours in a shaking table at the temperature of 28 ℃ at 200rpm or a constant-temperature incubator at the temperature of 28 ℃;

5) spreading 500 μ L bacterial solution on solid L B culture medium containing antibiotics (Rif50mg/m L and Spe 50mg/m L), and culturing in a constant temperature incubator at 28 deg.C for 1-2 days until single clone appears;

6) and selecting a positive agrobacterium single colony for colony PCR, and carrying out amplification on the agrobacterium single colony corresponding to the PCR result containing the correct target band (the system and reaction conditions of the colony PCR are the same as those of the colony PCR of escherichia coli).

11. Extracting agrobacterium tumefaciens plasmids: plasmid extraction kit for TRAN (transrat's plasmid extraction)

7) 12000x g was centrifuged for 2 min to completely remove the residual WB.

9) 10000x g for 1 min, and storing the eluted DNA at-20 deg.C

12. Enzyme digestion verification, namely verifying the extracted plasmid by selecting restriction enzyme 5 ', Sa L I3', EcoR I,

double enzyme digestion reaction system

The incubator is kept at the constant temperature of 28 ℃ and is used for 30 minutes. Detecting by 1% agarose gel electrophoresis, analyzing the result, selecting agrobacterium with correct band to preserve glycerol (40% blue cap) for transforming plants.

Example 3 acquisition of transgenic plants

In this example, the floral infection method was used to obtain the transgenic plant code

1. Activating correctly-preserved Agrobacterium (activation aims to ensure the viability of the strain), adding the activated strain into a culture solution of 5m L L B containing antibiotics (50mg/m L Rif and 50mg/m L Spe), shaking overnight at 28 ℃, putting the shaken strain solution into a centrifuge, carrying out 5000x g and 10min, discarding the supernatant, adding an invasion solution of 1m L (5% of sucrose, 0.02% of silwet suspension in water), and suspending the strain by gentle aspiration with a rubber head dropper.

2. Selecting three boxes of wild Col-0 plants which have good growth vigor and already bloomed, cutting off the pod formed and the bud which has finished pollination on the plants, sucking the bacteria liquid drop on the bud which does not bloom by using a rubber head dropper, labeling and marking, and infecting once every 2-3 days until all the plants bloom (note: watering the plants one day before each infection).

3. Timely collecting T after the plants are mature₁Generating seeds, sowing the seeds on a resistant solid MQA CK culture dish containing antibiotics after the seeds are dried for about one week, firstly placing the seeds in a refrigerator at 4 ℃ for vernalization for three days, then culturing the seeds by illumination for about one week, and carrying out positive seedling T₁And (4) screening generations.

4. Selecting good-growth T on a resistant culture dish₁Plant generation, soil shifting culture and single plant labeling, and timely collecting T from single plant after mature₂Generating seeds, drying for one week, spreading on resistant culture medium, culturing for several days under illumination, and selecting T with survival-to-death ratio of 3: 1₂Replacing plants to carry out soil shifting culture and wait for collecting T₃And (4) generation plants. Will T₃The generation plants are continuously spread on a resistant culture dish, and the selected full-living pure plants are seeds which can be used for phenotypic analysis.

Example 4 phenotypic analysis in different elements

The MQA medium used in this example mainly contains

Macroelements: 1MKNO₃、1MMgSO₄、1MH₃PO₄、1MCaCl₂

Trace elements: MS trace (0.5x),

Fe²⁺Salt: MSFe²⁺Salt (0.5x)

Mn²⁺Salt: MSMn²⁺Salt (0.5X) and 0.5M MES buffer

Carbon source: 1% sucrose

a. The specific medium protocol is shown in Table 1(100m L)

Note: 10 μ M PO43-, 0 μ M K +, 0 μ M Ca2+, 10 μ M MO 3-, 0 μ M Mg2+ were added to 1.0% agarose 1g

1mMCa²⁺1.2% agar 1.2g, 1. mu. MABA, 80mM NaC L were added in several gradients

And ABA is added when the temperature is reduced to the hand temperature after the culture medium is sterilized.

Table 1: the pH of the medium was adjusted to 5.7 by KOH or BTP (7.0 by 80mM Nacl)

b. Planting method is shown in figure 4

Repeating three times for each gradient, sealing with sealing film after finishing planting, vernalizing in a refrigerator at 4 deg.C for three days, culturing in a culture chamber at 22 deg.C and humidity of 40% RH under vertical illumination for twenty days, and observing C_6-6The phenotype of the wild Col-0 is compared, and the data and the picture of the root length and the fresh weight are collected, so that C can be seen_6-6At 0. mu. MCa relative to wild type Col-0²⁺Sensitive phenotype with yellow leaf, small and short root and 1 μ MABA showed small leaf and short root, as shown in figure (figures 2 and 3)

c. Placement of culture dishes

The 8 gradients are divided into three groups

Example (c): suppose eight gradients are each identified by the letter A, B, C, D, E, F, G, H

A、B、C、D、E、F、G、H

B、C、D、E、F、G、H、A

C、D、E、F、G、H、A、B

The foregoing examples further illustrate the present invention and are not to be construed as limiting thereof. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention.

Claims

1. A plant drought-resistant gene Sv4317 has a nucleotide sequence shown as SEQ NO: 1 or SEQ NO: 2, respectively.

2. A drought-resistant related protein of plants has an amino acid sequence shown as SEQ No: 3, respectively.

3. A plant drought resistance gene encoding the plant drought resistance-related protein according to claim 2.

4. An expression vector comprising the gene of claim 1 or 3.

5. Use of the gene of claim 1 or 3 for drought resistance of plants.

6. Use according to claim 5, characterized in that the plant is sabina vulgaris.

7. A method for improving drought resistance of plants, which comprises the step of introducing the drought resistance gene Sv4317 of claim 1 into cells, tissues or individual plants of host plants to obtain plants with drought resistance.